‘APOPTOSIS IN CHRONIC LYMPHOCYTIC LEUKAEMIA’ · PDF fileApoptosis in Chronic...

PhD Thesis

‘APOPTOSIS IN CHRONIC

LYMPHOCYTIC LEUKAEMIA’

Diane King BSc (Hons)

Centre for Mechanisms of Human Toxicityand

Department of Pathology

University of Leicester

UMI Number: U124012

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This thesis is dedicated to Barrie, Jennifer and Angela King

and especially to my husband, James Dodd.

ACKNOWLEDGEMENTS

I would like to thank my supervisors, Professor G.M. Cohen and Dr. J.H. Pringle for their

constructive advice throughout the duration of this study, and Dr. R.M. Hutchinson for his

support and guidance.

Many thanks also to Mr. R. Snowden, Dr. M. MacFarlane, Mr. D. Brown, Dr. J. Shaw, Mrs.

L. Primrose, Mrs. A. Gillies, Mrs. L. Potter and Mrs. T. de Haro for their technical advice and

assistance.

PUBLICATIONS

King D, Cohen GM, Pringle JH, Hutchinson RM: Spontaneous apoptosis in chronic

lymphocytic leukaemia as a possible predictor of response to drug therapy. Br. J. Haematol.,

1997, 97, Suppl. 1; Abstract 112.

King D, Pringle JH, Hutchinson RM, Cohen GM: Processing/Activation of caspases -3, -7

and -8 but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia

cells. Leukemia, 1998,12; 1553 - 1560.

King D, Pringle JH, Hutchinson RM, Cohen GM: Processing/Activation of caspases -3, -7

and -8 but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia

cells. J. Pathol., 1998,186 SS; Abstract 31.

ABSTRACT

B cell chronic lymphocytic leukaemia (B-CLL) is the most common adult leukaemia in the

western world. The disease is characterised by the accumulation of a CD5+ B cell clone. Drug

resistance is a major problem in B-CLL and complete remissions are uncommon. The

lymphoaccumulative nature of B-CLL implies that dysregulation of the apoptotic process

may be responsible for the development and progression of the disease. B-CLL cells were

freshly isolated from patients, and an in vitro apoptosis sensitivity assay was developed using

flow cytometric techniques. Initial studies confirmed the existence of ‘spontaneous apoptosis’

when B-CLL cells were cultured in vitro, and demonstrated a strong correlation between

sensitivity to spontaneous apoptosis and sensitivity to apoptosis induced by the

chemotherapeutic drug chlorambucil in vitro. Immunoblotting of chlorambucil and

prednisolone treated B-CLL cells demonstrated the expression and activation of caspases -3,

-7 and -8 in all samples analysed, whilst activation of caspase-2 was seen in cells from only

one patient. Activation of caspases -3 and -7 was accompanied by the proteolysis of the

DNA repair enzyme, poly (ADP-ribose) polymerase (PARP). Induction of apoptosis and

activation of all the caspases was inhibited by the cell permeable caspase inhibitor,

benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). These results

demonstrated a key role for the activation and processing of caspases in the execution phase

of apoptosis in B-CLL cells. The B cell growth factors interleukin-4 and CD40 were

demonstrated to strongly influence survival of B-CLL cells in in vitro culture, and also to

modulate the response of the cells to chemotherapeutic drugs. Investigations into Fas induced

apoptosis in B-CLL cells demonstrated the expression of the adapter protein, FADD, but no

overexpression of the caspase-8 inhibitory protein, c-FLIP. Additionally, B-CLL cells did not

show rapid assembly of the death inducing signalling complex (DISC) in response to

stimulation of Fas receptor, implying that these cells may preferentially utilise the Bcl-2-

inhibitable Type II (mitochondrial) pathway of apoptosis induction, underlining the important

role that Bcl-2 plays in determining the apoptotic sensitivity of B-CLL cells.

KEYWORDS

Chronic lymphocytic leukaemia; apoptosis; caspases; IL-4; CD40; Fas/CD95

LIST OF ABBREVIATIONS

ATP = Adenosine triphosphate

B-CLL = B cell chronic lymphocytic leukaemia

BSA = bovine serum albumin

CAGE = conventional agarose gel electrophoresis

DISC = death inducing signalling complex

EDTA = ethylenediaminetetraacetic acid

ELISA = enzyme linked immunoabsorbancy assay

FADD = Fas associated death domain protein

FIGE = field inversion gel electrophoresis

FITC = fluorescein isothiocyanate

FLIP = Flice-like inhibitory protein

IL-4 = interleukin-4

ISEL = in situ end labelling

PARP = poly (ADP-ribose) polymerase

PBS = phosphate buffered saline

PI = propidium iodide

PS = phosphatidylserine

RT-PCR = reverse transcriptase PCR

SDS = sodium dodecyl sulphate

TBS = Tris buffered saline

TdT = terminal deoxynucleotide transferase

TNF = Tumour necrosis factor

TRAIL = Tumour necrosis factor-like apoptosis inducing ligand

UP = ultrapure

Z-VAD.fmk = benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone

Apoptosis in Chronic Lymphocytic Leukaemia

CONTENTS

PAGE No1.0 GENERAL INTRODUCTION

Introduction to this thesis 11.0 Chronic Lymphocytic Leukaemia 2

1.1.1 Diagnosis and Prognosis 21.1.2 Clinical staging of CLL 41.1.3 Treatment strategies 5

1.1 Apoptosis 71.2.1 Historical perspectives 71.2.2 Morphological and Biochemical characteristics of

apoptotic cells 71.2.3 The execution phase of apoptosis 81.2.4 Mechanisms of induction of apoptosis 11

1.2.4i Receptor/Ligand signalling pathways 111.2.4ii The mitochondrial pathway of apoptosis

induction - role of the bcl-2 family ofapoptosis regulators 14

1.2.5 Inhibitors of apoptosis 161.2.5i Inhibitors of receptor/ligand induced

apoptosis 161.2.5ii Inhibitor of apoptosis proteins (IAP’s) 17

1.3 Apoptosis and Chronic Lymphocytic Leukaemia 181.3.1 The Bcl-2 family 181.3.2 Caspase expression in CLL 191.3.3 Growth factor dependency of CLL cells 20

1.4 Aims and Objectives 21

2.0 MATERIALS AND METHODS 23

2.0 Drugs, chemicals and stock solutions 242.1 Selection of Patients 242.2 Isolation of B-CLL cells from whole blood 27

2.3.1 Isolation of total lymphocyte fraction 272.3.2 Purification of B lymphocytes 27

2.4 Determination of apoptosis sensitivity 282.5 In situ end labelling (ISEL) 282.6 Annexin V assay for apoptosis 292.7 One-stage DNA fragmentation analysis gels 312.8 Field inversion gel electrophoresis (FIGE) 322.9 Immunoblotting 322.10 Stimulation of B-CLL cells using anti-CD40 monoclonal

antibody and interleukin-4 342.11 Analysis of CD95/Fas receptor expression on B-CLL cells 342.12 Isolation of the death-inducing signalling complex (DISC) 35


3.0 RESULTS 1 - Development of an in vitro apoptosis sensitivity 36assay for CLL cells

3.1 Introduction 363.2 Spontaneous apoptosis of CLL cells 363.3 Apoptotic CLL cells exhibit limited nucleosomal DNA cleavage,

but evidence of large DNA fragmentation can be observed 393.4 The ISEL assay underestimates the percentage of apoptotic CLL

cells 413.5 In vitro sensitivity to spontaneous apoptosis is a predictor of in

vitro sensitivity to chemotherapeutic drug-induced apoptosis 433.6 As patients undergo chlorambucil therapy, the in vivo level of

apoptosis can decrease, but the sensitivity of the cells to spontaneous and chlorambucil-induced apoptosis in in vitroculture increases 45

3.7 Discussion 48

4.0 RESULTS 2 - Processing/activation of caspases -3 , -7 and -8 , but 53not caspase-2 in the induction of apoptosis in CLL

4.1 Inhibition of spontaneous apoptosis in CLL cells by Z-VAD.fmk 534.2 Activation of caspase-3 and caspase-7 in apoptosis of CLL cells 564.3 Caspase-2 processing does not generally accompany apoptosis of

CLL cells 594.4 Activation of the effector caspases results in the cleavage of PARP 614.5 Activation of caspase-8 during apoptosis of CLL cells 624.6 Z-VAD.fmk inhibits the processing of caspases in CLL cells 644.7 Discussion 66

5.0 RESULTS 3 - Studies on survival factors and the Fas signalling 69pathway in B-CLL

5.1 Introduction 695.2 Purified B-CLL lymphocytes are more sensitive to apoptosis in

vitro culture than unpurified populations of total CLL lymphocytes 695.3 Culture of B-CLL cells with interleukin-4 and CD40 stimulation

results in a reduction in spontaneous apoptosis 725.4 Culture of B-CLL cells with interleukin-4 or CD40 stimulation

increases their resistance to chemotherapeutic drugs 755.5 B-CLL cells are not sensitive to CD95-induced apoptosis 785.6 Upregulation of CD95 receptor on B-CLL cells does not increase

their sensitivity to apoptosis induced by CD95 ligation 805.7 B-CLL cells do not over-express the caspase-8 inhibitory protein,

c-FLIP 855.8 B-CLL cells have all the necessary components to form a

death-inducing signalling complex (DISC) upon CD95 ligation,but do not assemble a DISC in response to CD95 stimulation 87

5.9 Discussion 925.9.1 Survival Factors in B-CLL 925.9.2 Investigations into the Fas signalling pathway in B-CLL 95


6.0 GENERAL DISCUSSION 100

REFERENCES 105

APPENDIX 119

LIST OF FIGURES AND TABLES

FIGURE1.1 Mechanism of activation of caspase-3 101.2 Pathways leading to cell death 13

2.1 Annexin V/PI dot plot to illustrate positioning of quadrant 31

3.1 CLL cells were isolated from whole blood samples, labelled usingthe ISEL technique and analysed flow cytometrically 38

3.2 CLL lymphocytes were analysed by agarose gel electrophoreticmethods 40

3.4 A comparison of the ISEL and Annexin V labelling techniques 423.5 In vitro sensitivity to spontaneous apoptosis is a predictor of

sensitivity to in vitro chlorambucil-induced apoptosis 443.6 In vivo apoptosis can decrease and sensitivity to spontaneous

apoptosis can increase as drug treatment is administered 47

4.1 Induction of apoptosis in cells from two patients with CLL assessedby phosphatidylserine extemalisation 54

4.2 Induction of apoptosis in a representative B-CLL patient is accompanied by processing of caspase-3 and caspase-7 57

4.3 Activation of caspase-2 only occured in cells from 1 patient 604.4 Cleavage of PARP accompanies apoptosis in CLL cells

Z-VAD.fmk 614.5 Processing of caspase-8 in CLL cells is inhibited by Z-VAD.fmk 634.6 Processing of caspase-3 in CLL cells is inhibited by the caspase

inhibitor Z-VAD.fmk 65

5.1 A Use of CD3+ magnetic beads enriches for CD19+ B cells incultures of CLL lymphocytes

5.1 B Purified B cells are more sensitive to spontaneous apoptosis than cultures of mixed lymphocytes 71

5.2 Interleukin-4 inhibits the induction of spontaneous apoptosis inpurified B-CLL cell cultures 73

5.3 The effect of stimulation of B-CLL cells with interleukin-4 and antibodies to CD40 is case dependent. 74

5.5 Stimulation of B-CLL cells with anti-CD40 and interleukin-4protects against apoptosis induced by chlorambucil. 77

5.6 B-CLL cells are resitant to apoptosis induced by anti-Fasmonoclonal antibody. 79


5.7 Fas receptor expression is elevated on B-CLL cells following stimulation with antibodies to CD40 and/or interleukin-4.

5.8 Upregulation of Fas receptor on B-CLL cells does not increase their sensitivity to apoptosis induced by Fas stimulation.

5.9 Stimulation of B-CLL cells with CD40 does not confer sensitivity to Fas-induced apoptosis.

5.10 B-CLL cells do not overexpress the Fas signalling pathway inhibitory protein c-FLIP.

5.11 B-CLL cells express the adapter protein, FADD.5.12 Immunoblotting for FADD on immunoprecipitates of Fas

stimulated cells.5.13 Immunoblotting for caspase-8 on lysates of anti-Fas stimulated

cells.

TABLE2.1 List of patients involved in the study

81

83

84

8688

90

91

25/26

4.1 Inhibition of spontaneous apoptosis by Z-VAD.fmk4.2 Clinical information and summary of in vitro apoptosis sensitivity

and incidence of caspase activation

55

58

Chapter 1 Introduction

GENERAL

INTRODUCTION


INTRODUCTION TO THIS THESIS

Described in this thesis is an investigation into the role played by apoptosis in the

etiology and treatment of chronic lymphocytic leukaemia (CLL). This first chapter

serves as an introduction to chronic lymphocytic leukaemia, and as a precis of the

increasingly complex field of apoptosis research. The final section of this chapter

summarises research which has been undertaken to date relating to apoptosis and B-

cell chronic lymphocytic leukaemia (B-CLL), and identifies areas where further

research might be implicated. Subsequent chapters describe the techniques applied

and the results generated during the research project pertaining to this thesis. An

appraisal of the main findings, along with a discussion about future directions for

research in this field, is included at the end.

1.1 CHRONIC LYMPHOCYTIC LEUKAEMIA

1.1.1 Diagnosis and Prognosis

B cell chronic lymphocytic leukaemia (B-CLL) is the most commonly occuring adult

leukaemia in the West and is a result of accumulation of a lymphocyte clone that is

highly resistant to cell death. Since the circulating malignant clone does not undergo

apoptosis, the tumour burden increases in volume and in extent of spread. B-CLL can

therefore be termed a Tymphoaccumulative’ disorder (Dameshek, 1967).

Increasingly B-CLL patients are diagnosed in an asymptomatic phase. This is most

likely attributed to the practice of carrying out blood tests for minor reasons.

Diagnosis is made by the presence of lymphocytosis in peripheral blood and bone

marrow and will include immunophenotypic evaluation to confirm the presence of

characteristic markers. B-CLL lymphocytes express low amounts of surface IgM, or a

combination of slgM and slgD (Rozman & Monserrat, 1995). Other surface markers

commonly expressed on B-CLL lymphocytes include CD20, and CD23, however, the

most common B-CLL marker is CD5 in conjunction with CD 19 (Caligaris-Cappio et

at, 1993).

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CD5 is a 67 kD glycoprotein which was originally described as a T cell antigen. Only

a small subset of normal B cells found at the edge of the germinal centres of human

lymph nodes are believed to carry CD5. Additionally, a substantial number of foetal B

cells express CD5. In total approximately 5 - 10% of normal B cells express CD5, so

finding a normal counterpart to the B-CLL lymphocyte to use experimentally to

examine the lineage of the B-CLL clone has proved to be difficult.

Morphologically, the B-CLL lymphocyte is not distinct from normal B cells, if

slightly smaller in size. B-CLL cells typically have a high nuclearrcytoplasmic ratio,

and nuclear chromatin often appears clumped. Nucleoli are usually indistinct or not

visible. Conflicting reports exist regarding the stage of maturation of the malignant

cells, but they do appear to be predominantly mature. Any minor variations in shape

or size do not appear to correlate with clinical status or progression.

The volume of neoplastic B lymphocytes increases with time, so even if a patient is

diagnosed in an asymptomatic phase, symptoms will eventually appear. These

typically include lymphadenopathy, splenomegaly and hepatomegaly. The white cell

count, an important monitor of disease progression, will increase, and anaemia and

thrombocytopenia often occur as a result of bone marrow infiltration. Marrow

infiltration can be classified into three types, diffuse, where marrow fat and

interstitium are replaced by extensive lymphocytosis, focal, characterised by distinct,

randomly distributed aggregates of lymphocytes, and interstitial, where the overall

marrow architecture is preserved.

The median survival is approximately nine years, depending on clinical stage at

diagnosis. Good predictors of survival include low clinical stage, low blood

lymphocyte counts, and positive bone marrow histopathological findings (low levels

of lymphocyte infiltration). Positive response to therapy is also a good prognostic

indicator. An indicator of poor prognosis is transformation of B-CLL into large cell

lymphoma (Richters syndrome) (Sawitsky & Rai, 1992).

Cytogenetic abnormalities are also screened for in B-CLL and found in around 50%

of cases (O’Brien et al, 1995). An abnormal karyotype can indicate poor prognosis.

Karyotypic evolution as the disease progresses is rare. The most commonly found

3


cytogenetic abnormality is a deletion at 13ql4. Trisomy of chromosome 12, alone or

with abnormalities of chromosomes 11 and 14 are also found. No ras mutations (chr

12) have been found to date, and translocation of bcl-2 (chr 14) is relatively rare.

However, increased mRNA and protein expression of bcl-2 is found in a majority of

B-CLL cases, although the mechanism by which this is made possible is unclear,

although it has been postulated that hypomethylation of the bcl-2 gene may be

involved (Hanada et al, 1993).

The retinoblastoma (Rb) gene on the long arm of chromosome 13 is rarely deleted or

translocated in B-CLL. When the chromosome 13 breakpoint region was investigated,

a high frequency of deletions was discovered at a locus 530 kb away from the Rb

gene. These deletions are often homozygous, and could indicate the presence of a new

tumour suppressor gene.

1.1.2 Clinical Staging of B-CLL

Two staging systems exist to define disease status, and therefore aid diagnosis, and

choice of treatment strategy. That developed by Rai (Rai et al, 1975) , distinguishes

between five stages of disease :-

Rai staging system

Stage 0 Lymphocytosis in blood and BM.

Stage I + lymph node involvement.

Stage II + organ involvement.

Stage III + anaemia.

Stage IV + thrombocytopenia.

The other commonly used system, is that developed by Binet (Binet et al, 1981),

which classifies patients into three stages of disease depending on the extent of spread

of lymphocytosis (This is the staging system employed in the present study). :-

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Binet staging system

Stage A Lymphocytosis and < 3 areas of lymphoid

enlargement

Lymphocytosis and > 3 areas of lymphoid

involvement

Lymphocytosis and < lOg/dL Hb,

or < 100 x 109/ L platelets.

Stage B

Stage C

Clinical progression is subsequently assessed by monitoring the peripheral blood

lymphocyte count, platelet count, lymph node appearance and response to treatment.

1.1.3 Treatment Strategies

The alkylating agent chlorambucil has long been the drug of choice in B-CLL. It is

still the most common first-line therapy, and although complete remissions are rare,

chlorambucil does reduce the overall tumour burden in the majority of cases.

In the asymptomatic phase of the disorder it has been demonstrated that treatment

with chemotherapy can actually be detrimental to the patient. For example, patients

with early stage B-CLL treated with chlorambucil have a greater risk of developing

epithelial cancers, and thus have a reduced survival rate when compared with those

patients not receiving early treatment, (Monserrat & Rozman, 1994).

As the disease progresses, and symptoms begin to appear, it is usual to treat with

chlorambucil, at a dose of 0.4 - 0.8 mg/kg every 2-3 weeks. Chlorambucil acts by

alkylating the N7 position of guanine nucleotides producing adducts in the DNA.

Corticosteroids such as prednisolone are often given in conjunction with chlorambucil

to counter immune haemolysis. The dose given is in the range 30 - 60 mg/m2 /day

orally. Prednisolone cytotoxicity is mediated via nuclear receptor interaction.

Combination therapies have been the subject of many clinical trials. These include

COP (cyclophosphamide, vincristine, prednisone), CHOP (as COP but with

doxorubicin), and MOPP (nitrogen mustard, vincristine, procarbazine, prednisone),

5


amongst others. However, results indicate no great improvement on the response rates

for chlorambucil alone (Monserrat & Rozman, 1994).

Recently, much research has centered on the new breed of chemotherapeutic drugs,

the purine analogues, one of which is fludarabine monophosphate (Astrow, 1996).

Fludarabine is transported into the cell by nucleoside transporter proteins. Once inside

the cell, fludarabine is phosphorylated by deoxycytidine kinase to F-ara-adenine

triphosphate (F-ara-ATP). Because of its resistance to deamination by adenosine

deaminase (an enzyme which, incidentally, is naturally depleted in many B-CLL

patients (Sawitsky & Rai, 1992)), F-ara-ATP accumulates in the cell. The result of

this accumulation is suppression of DNA synthesis by inhibition of DNA

polymerase a (O’Brien et al, 1995). Other groups have proposed that incorporation of

F-ara-ATP into replicating DNA is vital to the activity of this drug (Huang &

Plunkett, 1995). The dose given is in the range 25-30mg/m2 for five days, repeated

every four weeks for 4-6 cycles. Since fludarabine is a relatively expensive drug to

administer clinicians are eager to introduce methods of predicting which patients will

respond favourably before treatment commences.

Drug resistance is a major problem in B-CLL and may be linked to an increased

resistance to apoptosis. Several studies have investigated the expression levels of

MDR1 (P-glycoprotein) and glutathione-S-transferases following treatment of B-CLL

patients with chlorambucil. One study showed that MDR1 expression was increased

following treatment (Perri et al, 1989), and GST expression has been shown to be

approximately doubled in a proportion of chlorambucil-treated patients (Schisselbauer

et al, 1990).

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1.2 APOPTOSIS

1.2.1 Historical Perspectives

Apoptosis, or programmed cell death, was first recognised as being distinct from

necrosis by Kerr and co-workers in the early 1970’s. Electron microscopy was

employed to study some novel histopathological events which had been observed in

lysosomes in hepatic ischemia. These events consisted of the formation of discrete,

rounded bodies, some of which contained chromatin fragments. Electron microscopy

revealed that these bodies contained intact organelles, and that they had arisen by

condensation and disruption of an original hepatocyte. This phenomenon , originally

termed ‘shrinkage necrosis’ (Kerr, 1971), was subsequently observed in many

different tissues. The name ‘apoptosis’ was proposed in 1972 (Kerr et al, 1972) and is

derived from the Greek word meaning ‘falling o ff, as in leaves from trees. Apoptosis

has since been shown to occur throughout many physiological events, from limb

development in the foetus, to cell killing by cytotoxic T lymphocytes.

1.2.2 Morphological and Biochemical Characteristics of Apoptotic Cells

Necrotic cell death is characterised by swelling of the cell and organelles followed by

loss of membrane integrity. When this occurs in tissue, inflammation is inevitable.

Conversely, apoptosis is characterised by compaction of chromatin into crescent

shapes which lie against the nuclear membrane followed by condensation of the

cytoplasm which is not accompanied by swelling of the organelles as occurs in

necrosis. The cell membrane becomes crenellated or ‘blebbed’, and the cell detaches

from its neighbours. Eventually, the cell divides up into multiple membrane bound

‘apoptotic bodies’, some of which contain chromatin. In tissues, these apoptotic

bodies are rapidly cleared, due to the expression of signalling molecules on the cell

surface of the apoptotic cell which are recognised by phagocytic cells. As a result of

the rapid clearance of dead cells from the tissue, inflammation does not occur.

Occuring synchronously with condensation of chromatin is cleavage of double

stranded DNA at nucleosomal intervals resulting in the formation of fragments of

7


DNA in multiples of approximately 200 base pairs (Bortner et al, 1995). An

associated biochemical marker is the production of large fragments of DNA (50-700

kbp), prior to or in the absence of intemucleosomal DNA cleavage. (Oberhammer et

al, 1993). A number of techniques for quantifying apoptosis are based upon

recognition of DNA cleavage. The in situ end labelling (ISEL) and TUNEL methods

can be used on tissue sections or as a flow cytometry method on cell suspensions.

Both techniques use the enzyme terminal deoxynucleotide transferase (TdT) to label

the cleaved DNA fragments with a ‘tail’ of digoxygenin-labelled nuclotides. Anti-

digoxygenin Fab fragments incorporating an colourimetric or fluorescent marker are

used to label the cleaved DNA. Other methods of observing apoptotic cells include

agarose gel electrophoretic methods. The classic ‘ladder’ of nucleosomal fragments

can be visualised using conventional agarose gel electrophoresis (CAGE), and the

formation of the larger DNA fragments can be observed using pulsed field or field

inversion gel electrophoresis (FIGE). Since the early 1990’s techniques have been

developed using other hallmarks of the apoptotic process in order to quantify cell

death, usually flow cytometrically. One of the most widely used techniques is that of

Annexin V / propidium iodide labelling (Koopman et al, 1994). One of the molecules

on the cell surface of an apoptotic cell, which acts as a signalling marker to

phagocytic cells, is phosphatidylserine (PS). PS is a phospholipid usually resident on

the inner leaflet of the cell membrane. During apoptosis PS is flipped onto the outer

surface of the cell. Use of fluorescein isothiocyanate (FITC)-labelled Annexin V, a

protein with high affinity for PS, in conjunction with the red fluorescent dye,

propidium iodide (PI) in a dye exclusion role, results in an extremely efficient flow

cytometric method for analysing apoptosis and for distinguishing apoptotic from

necrotic cells.

1.2.3 The Execution Phase of Apoptosis

Apoptosis can be divided into two phases: an initial condemned phase where cells are

committed to die, without any morphological changes, followed by an execution

phase when the characteristic biochemical and morphological changes of apoptosis

occur, as described above. Apoptosis can occur in response to a variety of stimuli.

Chemotherapeutic drugs, irradiation, and triggering of cell surface ‘death receptors’

8


such as Fas (CD95 / Apo-1) and Tumour necrosis factor receptor (TNF-R1) can all

result in induction of apoptosis. Whatever the nature of the death signal, or the

mechanism by which it is received in the cell, there appears to be a common

‘execution’ pathway, which results in cell death. Much of our present knowledge

regarding this phase of apoptosis comes as a result of studies performed on the

nematode worm Caenorhabditis elegans (Yuan et al, 1993). It is known exactly how

many cells are produced, and how many die during development of this organism, and

the genes which must be mutated in order for the organism to deviate from normal

development. The genes ced-3 and ced-4 were demonstrated to be essential for

developmental cell death in C. elegans since ced-3 or ced-4 null worms have 131

surplus cells at birth.

The mammalian ced 3 homologues are cysteine proteases called ‘caspases’

(previously ICE-like proteases) for cysteine-aspartate proteases. Caspases are

produced as inactive zymogens that require cleavage at the PI position of an aspartate

residue in order to attain their active form. They in turn specifically cleave target

substrates at the PI of an aspartate residue in a defined amino acid sequence. The

caspases are composed of pro-domains followed by two shorter domains, the large

and small subunits. Typically activation involves cleavage at the pro-domain/large

subunit junction, followed by cleavage at the large/small subunit junction and

heterodimerisation of the large and small subunits (Han et al, 1997). A complex

consisting of two heterodimers is thought to make up the active enzyme (figure 1.1).

Caspases can be divided into two groups, initiator caspases and effector caspases.

Initiator caspases preferentially cleave at a (IVL)ExD sequence and typically have

long pro-domains. Initiator caspases such as caspase-8 (FLICE/MACH) (Boldin et al,

1996; Scaffidi et al, 1997) and caspase-2 (Ich-1) (Harvey et al, 1997) are involved in

apoptosis directed through the membrane signalling molecules Fas (CD95/Apo-l) and

TNF-R1. Effector caspases, which include caspase-3 (CPP32) (Nicholson et al, 1995)

and caspase-7 (Mch-3) (Fernandes-Alnemri et al, 1995), cleave at a DEVD target

sequence and are responsible for cleaving substrates which result in cell death. These

targets include poly ADP-ribose polymerase (PARP) (Kaufinann et al, 1993;

Casciola-Rosen et al, 1996), nuclear lamins (Rao et al, 1996) and ICAD, the inhibitor

of the caspase-dependent DNAse (CAD) which is responsible for cleaving DNA into

the characteristic intemucleosomal pattern (Enari et al, 1998).

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32 kD

PRO 17 kD 12 kD

17 kD12 kD

Caspase-3 zymogen

IStage 1 : Cleavage at 12 kD/17 kD domain junction

PRO 17 kD12 kD

Stage 2 : Cleavage at pro-domain/17 kD domain junction

17 kD12 kD

Stage 3 : Dimerisation o f two active caspase-3 enzymes

17 kD12 kD

Active caspase-312 kD17 kD

Figure 1.1 Mechanism of activation of caspase-3

10


Until recently it was considered that caspase activation was essential for the execution

phase of apoptosis. However, certain instances of apoptotic cell death exist where no

evidence of caspase activation or intemucleosomal DNA cleavage is seen. Caspase-

independent apoptosis in yeast (Green & Reed, 1998), sperm, and

cyclohexamide/staurosporine stimulated chicken erythrocytes (Weil et al, 1998) has

been described, although the molecules involved in this type of apoptosis are yet to be

described.

1.2.4 Mechanisms of Induction of Apoptosis

1.2.4i Receptor-Ligand Signalling Pathways

Triggering of apoptosis can occur via two mechanisms which are not mutually

exclusive. One route for apoptosis initiation is via triggering of the cell surface death

receptors Fas (CD95/Apo-l), TNF-R1 or the TRAIL (tumour necrosis-like apoptosis

initiating ligand) receptors. Binding of Fas ligand to the Fas receptor initiates

trimerisation of the receptor, bring into close contact three intracellular death domains

(DD), a region of homology shared with members of the TNF-family, and some of

the initiator caspases such as caspase-8 (also known as ‘FLICE’). The receptor death

domains are recognised and bound by a death domain containing adapter molecule

called FADD (Chinnaiyan et al, 1996). Other adapter molecules which are involved in

apoptosis signalling through TNF and TRAIL receptors include RAIDD/CRADD

(Duan & Dixit, 1997) and Daxx (Yang et al, 1997). Activation of the receptor/ligand

apoptosis initiating pathways triggers a series of events which result in activation of

the effector caspases leading to cell death. Activation of caspases via Fas signalling

seems to occur by two distinct mechanisms (Scaffidi et al, 1998). Type I is extremely

rapid (within seconds) and requires formation of a ‘death inducing signalling

complex’ (DISC). The DISC consists of caspase-8 which is activated in response to

Fas signalling by binding to the death effector domain (DED) of the adapter molecule

FADD (figure 1.2). Activated caspase-8 then triggers cleavage and activation of

caspase-3 and caspase-7, leading to degradation of the cell. Type II Fas signalling

does not seem to require the formation of a DISC, but appears to be dependent on

mitochondrial activity, in that it can be blocked by Bcl-2 overexpression. Type II

11


cells appear to activate caspase-3 via the formation of an apoptosome consisting of

caspase-9, Apaf-1 and cytochrome c, which is released from mitochondria (figure

1.2). Type II cells show activation of caspase-3 after approximately 60 minutes of Fas

stimulation. Activation of the TNF receptor by TNF binding can promote survival or

apoptosis depending on the adapter and accessory proteins which bind to the

intracellular domain of TNF-R1. TRADD is the primary adapter molecule for TNF

signalling and can recruit FADD and caspase-8 to induce apoptosis (Yuan, 1997).

Alternatively, the adapter molecule RAIDD/CRADD (Duan & Dixit, 1997) can

mediate an NFkB signal in response to binding of the TNF receptor by the kinase RIP

(TNF-receptor interacting protein) (Ahmad et al, 1997; Kelliher et al, 1889). Other

molecules involved in receptor-mediated apoptosis induction include CARDIAK,

which is a RIP-like kinase that can bind and activate caspase-1 via its DED/CARD

domain, and is also involved in triggering of Jun kinase and NFkB (Thome et al,

1999). Daxx is a second adapter molecule in the CD95 system which triggers Jun

kinase, via activation of apoptosis signal-regulating kinase-1 (ASK). FLASH is

another protein involved in receptor-mediated apoptosis. FLASH is a large protein of

approximately 220 kb with homology to C. elegans CED-4, which interacts with

caspase-8 through a DED domain, and appears to be another Fas DISC component

(Imai et al, 1999), although its function is unclear.

The Fas and TNF receptor/ligand partemships are just two of a number of related

pairings which when triggered can cause induction of apoptosis. TRAIL (TNF-related

apoptosis initiating ligand) can bind to a number of receptors. Some of these, such as

DR4 and DR5, when bound by TRAIL, lead to induction of apoptosis. Others, termed

‘decoy receptors’ such as DcRl and DcR2, lack the intracellular death domain

necessary for apoptosis induction. Binding of TRAIL to these receptors, therefore,

does not induce apoptosis (French & Tschopp, 1999 for review). TRAIL has been

shown to induce apoptosis in cells from a range of haematological neoplasms,

including chronic lymphocytic leukaemia (Snell et al, 1997).

12

CELL MEMBRANE

CYTOPLASM

Fas Ligand

Fas receptor

Chemotherapeutic drugs, growth factor withdrawal, mitogenic signals

A FADD

p- | Activated Caspase-8

c-FLIP

Ceram ide

ActivatedCaspase-3

Cytochrome c MITOCHONDRION

Activated caspase-9, dATP, Apaf-1 apoptosome

NUCLEUS

Morphological nuclear events

Figure 1.2 Pathways leading to cell death tCell Death

i


1.2.4ii The Mitochondrial Pathway o f Apoptosis Induction - Role o f the Bcl-2

Family o f Apoptosis Regulators

The mitochondrial pathway of apoptosis induction can be triggered via Fas signalling

as described above, or directly via triggering loss of mitochondrial membrane

potential (Susin et al, 1997). Cytochrome c, released from the mitochondria due to the

opening of a permeability transition pore, triggers the mammalian homologue of the

C. elegans ced-4 gene product, Apaf-1, in the presence of dATP, to activate caspase-9

in a complex which is termed the ‘apoptosome’ (Li et al, 1997). Activated caspase-9

subsequently activates the effector caspases such as -3 and -7. Whether or not the

loss of mitochondrial membrane potential is linked to the release of cytochrome c is

unclear at the present time. One mediator of the mitochondrial apoptotic pathway,

ceramide, is produced by a number of apoptotic initiating agents and accumulates at

the mitochondria, where it can directly bind and release cytochrome c (Garcia-Ruiz et

a l 1997).

Members of the Bcl-2 family exert some of their effects at the mitochondia. Bcl-2 is a

26 kD protein located at 18q21, originally identified during analysis of the t(14; 18)

breakpoint in follicular lymphoma (Tsujimoto et al, 1985). It is homologous to the

apoptosis inhibitory ced-9 gene product in C. elegans. A family of related proteins has

since been discovered, some with anti-apoptotic effects like Bcl-2, and others, such as

Bax, with pro-apoptotic activity (Oltvai et al, 1993), all containing regions of

homology termed the BH domains. Since induction of apoptosis by a wide variety of

stimuli, including anti-cancer drugs, UV irradiation and receptor-ligand signalling

pathways can be blocked by overexpression of Bcl-2 (Cory, 1995), it appears that

Bcl-2 must act at a point in the apoptotic process that is common to many pathways.

Investigations into the mechanism of action of Bcl-2 and related proteins has centered

on their activity in mitochondria during triggering of apoptosis. Early studies on Bcl-2

localised the protein to the mitochondrial outer membrane, particularly at points

where the outer and inner membranes formed a pore. The structure of Bcl-2 and a

related protein, anti-apoptotic B c1-Xl (Boise et al, 1993) resembles that of certain

bacterial toxins which can insert into the mitochondrial membrane and may influence

ion channel activity (Kroemer, 1997). Bcl-2 and Bcl-XL overexpression inhibits the

14


change in mitochondrial membrane potential induced by chemotherapeutic agents

(Decaudin et al, 1997) thus contributing to chemoresistance, and more recent studies

have demonstrated that anti-apoptotic Bcl-2 interacts with pro-apoptotic Bax on the

mitochondrial membrane to prevent activation of downstream caspases, and may

remove Bax to the endoplasmic reticulum to prevent Bax-induced release of

cytochrome c (Bomer et al, 1999). The important role that Bax plays at the

mitochochondria is underlined by the finding that overexpression of Bax can induce

mitochondrial permeability transition (Pastorino et al, 1998). The relevance of

permeability transition to the release of cytochrome c is a matter under much debate,

the two events may be closely related, but reports of cytochrome c release prior to loss

of mitochondrial membrane potential have been made (Reed et al, 1998).

Members of the Bcl-2 family are often involved in amplification or propagation of the

apoptotic signal. In 1998, two pathways for Fas induced apoptosis were reported

(Scaffidi et al, 1998) (as described in section 1.2.4i of this chapter). In Type I

signalling, characterised by the rapid formation of a DISC leading to activation of

significant amounts of caspase-8 and subsequently caspase-3, Bcl-2 is poorly

effective as an inhibitor. In Type II signalling, characterised by activation of small

amounts of caspase-8, Bcl-2 can act as a potent inhibitory factor, indicating that

mitochondria may be involved in this type of apoptosis induction. The link between

activation of caspase-8 and mitochondrial events has been shown to be due to the

activation of the Bcl-2 family member, BID. BID is a 24 kD, BH3 domain containing

protein which has cleavage sites for caspase-8 and granzyme B. Cleavage of BID at

Asp 59 by caspase-8 removes an amino terminus inhibitory domain, to yield a

potently pro-apoptotic protein (Li et al, 1998, Luo et al, 1998). Truncated BID (tBID)

was shown to translocate from the cytoplasm to the mitochondrial and nuclear

membranes where it could induce clustering of mitochondria, loss of mitochondrial

membrane potential and release of cytochrome c, and eventually cell shrinkage and

nuclear condensation (Li et al, 1998). In this way, the Fas induced apoptotic signal

can be amplified by release of mitochondrial factors. A second way in which an

apoptotic signal can be amplified was revealed by Kirsch and co-workers (Kirsch et

al, 1999). Earlier reports had demonstrated that when recombinant anti-apoptotic Bcl-

2 was cleaved at Asp 34 by caspase-3, a 23 kD pro-apoptotic fragment of Bcl-2 was

produced (Cheng et al, 1997). Later studies by the same group identified caspase-3 as

15


the factor required for Bcl-2 cleavage in Fas treated Jurkat cells, and demonstrated the

co-localisation of intact and cleaved Bcl-2 to the mitochondria. There they

demonstrated that cleaved Bcl-2 could promote release of cytochrome c from the

mitochondria in a manner similar to Bax (Kirsch et al, 1999). The authors postulate

that cleavage of Bcl-2 at Asp 34, removing the amino terminal BH4 domain, may

expose the pro-apoptotic BH3 domain, similar to the effect of caspase-8 cleavage of

BID. Thus cleavage of Bcl-2 may act as part of a positive feedback loop, amplifying

the apoptotic signal at a mitochondrial level, by promoting release of cytochrome c

and subsequent activation of caspase-9 and caspase-3.

The release of cytochrome c from mitochondria following an apoptotic stimulus has

been well established. However, it has since been reported that a number of other

factors are released into the cytosol from mitochondria following induction of

apoptosis. These factors include caspase-2 and caspase-9, and a 50 kD ‘apoptosis

inducing factor’ (AIF) (Susin et al, 1999). Bcl-2 may therefore also be involved in

preventing the release of caspase-9 and cytochrome c from mitochondria in the

absence of an apoptotic stimulus, thereby inhibiting apoptosome formation and

activation of downstream caspases such as caspase-3.

1.2.5 Inhibitors of Apoptosis

Since cancer can be viewed as a situation in which the tumour cells are resistant to

cell death, molecules which inhibit apoptosis may play an important role in

development of malignancies. Accordingly, much work has been done to identify and

characterise the expression of molecules such as these.

1.2.5i Inhibitors o f Receptor/Ligand -induced Apoptosis

The identity of several proteins which may act at the proximal stage of apoptosis

induction have been identified. The first of these was c-FLIP (Irmler et al, 1997;

Rasper et al, 1998) (also called Casper, I-FLICE, FLAME-1, CLARP, MRIT and

Usurpin-a), a ‘FLICE-like inhibitory protease’ which can block apoptosis signalling

16


through the CD95/Fas system. c-FLIP has two death effector or caspase-recruitment

domains (DED/CARD’s) at its amino terminus, which enable binding of c-FLIP with

caspase -8 and FADD in the Fas-induced DISC. Interaction of c-FLIP with activated

caspase-8 causes cleavage and activation of c-FLIP and results in the c-FLIP/caspase-

8 interaction becoming more inhibitive. c-FLIP was found to be predominantly

expressed in lymphoid and muscle tissues, and high levels of the protein were

discovered in melanoma cells (Irmler et al, 1997). The extent of expression of c-FLIP

in B-CLL cells has not previously been reported.

The ‘silencer of death domain’ proteins (SODD’s) are a second class of inhibitor

proteins (Jiang et al, 1999). When complexed with the death domain of TNF-R1,

SODD keeps the receptor in a monomeric form. Ligation of TNF-R1 by TNF, causes

SODD to dissociate, allowing formation of the TNF death inducing signalling

complex consisting of the adapter molecules TRADD and TRAF-2 (Yuan, 1997), and

the kinase RIP (Kelliher et al, 1998). A situation could therefore exist where a

dominant negative mutation of a SODD could leave the receptor trapped in a non

functional state. The existence of SODD’s in the TNF receptor-ligand system may

indicate that similar proteins exist in the Fas system, although none have been

described to date.

1.2.5ii Inhibitor o f Apoptosis Proteins (IAPfs)

IAP’s are a family of ‘inhibitor of apoptosis proteins’ (Liston et al, 1996) originally

identified in baculovirus systems, but with mammalian homologues. Mammalian

IAP’s typically contain one or more baculovirus IAP repeat sequence, and have a

carboxy terminus RING finger domain. The IAP’s, which in humans include c-IAP 1,

c-IAP 2, x-IAP and n-IAP, bind to and block the activity of TRAF-2, a protein

involved in TNF receptor-mediated activation of NF-xp.

Survivin, another IAP (Ambrosini et al, 1997) differs from the original IAP’s in that it

contains only one baculovirus IAP repeat, and has no carboxy terminal RING finger.

Survivin is expressed during foetal development, but is not present in terminally

differentiated adult tissue. However, abundant survivin has been detected in several

17


common human cancers, including breast, colon, prostate and lung, in vivo (Kawasaki

et al, 1998). Survivin was also expressed in approximately 50% of high grade non-

Hodgkin’s lymphomas, but not in low grade cases, indicating that this inhibitor may

be important in regulating apoptosis sensitivity in lymphoid malignancies.

1.3 APOPTOSIS AND CHRONIC LYMPHOCYTIC LEUKAEMIA

1.3.1 The Bcl-2 Family

Due to the discovery that Bcl-2 is overexpressed in a majority of B-CLL cases, many

groups have performed investigations in this area. Methods of assessing Bcl-2

expression in B-CLL by different groups range from western blotting of protein

samples, to RT-PCR and flow cytometric techniques. It is frequently reported that

Bcl-2 gene translocations are extremely rare events in B-CLL, and yet mRNA and

protein levels of this factor are elevated in the majority of cases. No satisfactory

explanation exists, as yet, to explain this phenomenon.

Since Bcl-2 is overexpressed in the B-CLL lymphocyte, this may be a possible reason

for accumulation of the malignant B-CLL clone. Several groups have investigated this

possibility. Gottardi et al (1995) confirmed overexpression of Bcl-2 in seven B-CLL

cases by northern blotting and RT-PCR, and postulated that defective apoptosis due to

this overexpression of Bcl-2 may be one reason why B-CLL lymphocytes appear as a

quiescent cell population, accumulating in the Go phase of the cell cycle. In a later

report, the same group analysed twenty-three CLL cases for expression levels of Bcl-

2, Bax, and the two Bcl-X splice variants (Gottardi et al, 1996). They again found

high level expression of Bcl-2 in the majority of cases, and in addition discovered

elevated Bax protein expression in a proportion of cases. Regarding the expression of

B cI-X l and Bcl-Xs, the pattern appears more sketchy, with Bcl-XL more frequently

expressed at a higher level than Bcl-Xs. In all, they found that levels of the apoptosis

inhibitors, Bcl-2 and Bcl-XL, were raised in a significantly higher proportion of cases

than were the apoptosis potentiators, Bax and Bcl-Xs, thus pushing the cells towards

an apoptotic block.

18


A second study examined the expression of Bcl-2 and Bax in relation to in vitro

survival of B-CLL lymphocytes, and clinical progression of the patient. They

concluded that the ratio of Bcl-2 to Bax was an important factor in determining

apoptotic sensitivity in B-CLL (Aguilar-Santelises et al, 1996). This finding was

mirrored by that of Thomas and co-workers (1996) who also looked at the importance

of this ratio in determining apoptotic sensitivity to chemotherapeutic drugs. Of a panel

of eighteen patients, 88% of those with an intermediate to high ratio of Bcl-2 to Bax

were in vitro drug resistant, indicating the importance of these factors in B-CLL.

More recent work has indicated that drug resistance in B-CLL may be due to selection

of subclones which express high levels of Bcl-2 relative to their Bax content (Pepper

etal, 1999).

1.3.2 Caspase expression in B-CLL

The cellular and tissue distribution of different caspases and their substrate specificity

is generally not known, although this is clearly important as exemplified by the

inability of some neuronal but not other cells to undergo apoptosis in caspase-3 7'

mice (Kuida et al, 1996). Multiple species of caspases-3 and -6 appear to be the major

pool of activated caspases in various tumor cell lines induced to undergo apoptosis

(Faliero et al, 1997). In addition to caspases-3 and -6, caspases-2 and -7 are also

activated in human monocytic tumor cells induced to undergo apoptosis by various

stimuli (MacFarlane et al, 1997). Recently it has been shown that peripheral blood

mononuclear cells from B-CLL patients are caspase-3 immunopositive (Krajewski et

al, 1997) and that glucocorticoid-induced apoptosis of B-CLL lymphocytes requires

protease activation and is accompanied by cleavage of PARP and lamin Bi together

with the loss of caspase-3 (Bellosillo et al, 1997; Chandra et al, 1997). The

importance of caspase activation in spontaneous and chemotherapy-induced apoptosis

in leukaemic cells from patients with B-CLL has not previously been reported.

19


1.3.3 Growth Factor Dependency of B-CLL Cells

The characteristic spontaneous apoptosis which occurs when B-CLL cells are cultured

in vitro has been reported previously (Collins et al, 1989). Since removal of B-CLL

cells from their normal environment appears to trigger the cells into apoptosis, it

would seem that the cells are stimulated in vivo by one or more survival factors,

which are not provided by in vitro culture in standard media.

CD40 is a 45-50kD glycoprotein which is expressed on the surface of B cells at all

stages of development, with the exception of terminally differentiated plasma cells.

CD40 is a member of the TNF/NGF family of receptors, which also includes

Fas/CD95. CD40 ligand (CD40L) is a 35kD glycoprotein, expressed on activated T

lymphocytes, follicular and dendritic cells within the germinal centres. Ligation of

CD40 receptor by CD40L requires receptor trimerisation, and activates a pathway that

results in isotype switching and clonal expansion during the B cell maturation

process. CD40 ligation has been demonstrated to rescue isolated germinal centre B

cells from apoptosis and has been reported to rescue B-CLL cells from spontaneous

apoptosis in in vitro culture. One way in which CD40 might increase protection from

apoptosis is by upregulating B cI-X l expression (Ghia et al, 1996). Planken and

collegues (Planken et al, 1996), used the ‘CD40 system’ to induce proliferation of B-

CLL cells in vitro. Using CD40-expressing CD32L cells as a feeder layer, and IL4 as

a media supplement, they demonstrated proliferation of cells from B-CLL patients.

CD40 activation of B-CLL cells has also been used as a method of inducing

upregulation of Fas receptor (Wang et al, 1997).

Interleukin-4 (B cell growth factor) sources include T cells, monocytes and bone

marrow stroma. It is known to stimulate B cell growth and differentiation and antigen

presentation. It has been demonstrated that B-CLL cells express either high or low

affinity receptors for IL4 at levels comparable to normal B cells (Gileece et al, 1993)

Addition of IL4 to cultures of B-CLL cells has been shown to reduce the level of

spontaneous apoptosis and to protect against hydrocortisone-induced apoptosis,

possibly by upregulating Bcl-2 expression (Danescu et al, 1992). Other techniques for

stimulating B-CLL cell growth in in vitro culture include interleukin-2 in combination

with mitogens such as Staphylococcus aureus Cowan I (DeFrance et al, 1991), or

20


pokeweed mitogen (Mainou-Fowler et al, 1995). In both of these studies, IL2 was

shown to have a proliferative effect, whilst IL4 had an ‘anti-apoptotic’ effect. Other

growth factors may be important in B-CLL, some of which will be discussed later in

this thesis.

1.4 Aims and Objectives

The overall purpose of this study was to examine the significance of apoptosis in B

cell chronic lymphocytic leukaemia. The fact that B-CLL cells accumulate due an

inability to undergo cell death (Dameshek, 1967) points to the importance of

understanding the nature of the apoptotic block in B-CLL cells. In addition, since

drug resistance is a major problem in B-CLL, this may also be related to the inability

of the cells to undergo apoptosis. Research into apoptosis, and the causes and

mechanisms of resistance to apoptosis, encompasses a wide range of controlling

factors to be investigated. Regarding B-CLL, much research had been done on the

role of the Bcl-2 family of apoptosis regulating proteins. This work had been done in

response to the finding that B-CLL cells express increased amounts of anti-apoptotic

bcl-2. Work in this area has linked the ratio of Bcl-2:Bax to apoptosis sensitivity

(Thomas et al, 1996; Aguilar-Santelises et al, 1996, Pepper et al, 1996), but only

partially explains the resistance of B-CLL cells to apoptosis induction by

chemotherapeutic drugs.

In order to contribute to research in this field, it was decided to investigate apoptosis

in B-CLL from a variety of different directions. The initial aim of this study was to

successfully apply current methods for analysing apoptosis in cell lines to clinical

samples with a view to developing a method by which cells from B-CLL patients

could be analysed for their sensitivity to apoptosis induced by chemotherapeutic

drugs. To then define what factors could influence the apoptosis sensitivity of B-CLL

cells was the aim of further research in this study. The identification of caspases as the

machinery proteins involved in the execution phase of apoptosis led to a series of

experiments being performed to examine the nature of caspase expression and

activation in B-CLL cells. The objective of this research was to discover whether B-

CLL cells possessed the caspases necessary for apoptosis, and whether these caspases

21


could be activated in response to stimulation of B-CLL cells with chemotherapeutic

drugs. In response to findings produced by this work, the study progressed onto

investigating the Fas receptor/ligand signalling pathway of apoptosis induction in B-

CLL cells. The aim of this research was to analyse the nature of Fas resistance in B-

CLL, by investigating the expression and activation of the various caspases, adapter

molecules and inhibitor proteins involved in Fas-induced apoptosis. In conjunction

with this work, analysis of the dependency of B-CLL cells on stimulation from

survival factors, and the effect that the such stimulation might have on resistance to

chemotherapy was investigated.

In summary, the main objectives of this study were :-

• To develop an assay for analysing apoptosis in freshly isolated B-CLL cells.

• To determine the significance of apoptosis sensitivity in B-CLL and to analyse

apoptosis following treatment of B-CLL cells with chemotherapeutic drugs.

• To investigate the nature of apoptotic resistance in B-CLL, by analysing

expression of proteins involved in the apoptotic pathway.

• To determine whether survival factor dependency plays a significant role in

determining the sensitivity of B-CLL cells to apoptosis.

22

Chapter 2 Materials and Methods

MATF.RIAT.S

AND

METHODS

23


2.0 MATERIALS AND METHODS

2.1 Drugs, chemicals and stock solutions

Laboratory stock solutions of phosphate buffered saline (PBS) (0.15 M NaCl (Fisher

Scientific, Loughborough, UK), 0.01 M Na2HP0 4 (Fisher Scientific), 2.5 mM

NaH2P0 4 2 H2O (Hopkins & Williams, Essex, UK), and Tris-buffered saline (TBS)

(0.05 M Tris (GibcoBRL, Paisley, Scotland), 0.15 M NaCl (Fisher Scientific)) were

used as indicated.

Unless stated otherwise all drugs and chemicals were obtained from Sigma Chemical

Co. (Poole, Dorset, UK). Prednisolone-21-hemisuccinate was stored as a 0.1 M stock

solution in PBS, chlorambucil as a 3 mM solution in ethanol, staurosporine as a 0.2

mM solution in dimethylsulphoxide (DMSO) and fludarabine as a 35 mM solution in

dimethyl formamide (DMF). All drugs were stored at “ 20°C in small aliquots to avoid

repeated freezing and thawing. The recommended maximum storage periods at this

temperature were not exceeded. Dilutions prior to use were made in 1 x PBS, so that

the final concentration of diluent never exceeded 0.1 %.

2.2 Selection of Patients

Local ethical committee approval was obtained prior to this study commencing.

Patients were assessed according to the Binet staging system, and were sourced from

Haematology outpatients clinics. Patients who were undergoing a course of treatment

and those who had completed a course within the previous 4 weeks were excluded

from the study. Table 1 lists the patients from whom blood samples were obtained for

analysis during the study. Of the 44 patients analysed, 55% were male and 45% were

female. The ages of the patients ranged from 40 to 94 years, the average age being

70.1 years. The majority (63%) of patients had received no previous treatment for

their condition at the time of sample collection. Of the patients who had been treated

with chemotherapeutic drugs nine had received chlorambucil alone, four had received

24


chlorambucil and prednisolone in combination, two had received fludarabine alone,

and one had received fludarabine in combination with prednisolone.

Patient no. Sex/Age Binet stage WBC count (x 109/L)

Previoustreatment

11 M/94 A 15.3 none

21 M/85 A 42.0 none

31 M/77 C 3.7 FLD

41 F/73 A 38.2 CHL

51 M/72 A 8.5 PD/FLD

61 F/66 A 51.8 none

71 M/76 C 8.1 PD/CHL

81 F/60 A 68 none

91 F/62 B 35.8 CHL

101 F/84 ND ND none

111 M/67 A 41.7 none

121 M/44 A 15.3 CHL

1 A F/73 A 38.2 CHL

2 A F/80 A 17.1 none

3 A M/74 A 23.1 none

4 A M/72 A 49.7 CHL

5 A M/75 A 20.7 none

6 A F/54 A 29.0 none

7A M/48 A 38.9 none

8 A M/72 A 30.2 none

9 A M/52 A 9.1 FLD

10 A M/91 C 24.5 none

11 A F/74 B 14.2 CHL

12 A F/67 B 17.5 none

13 A M/62 A 34.3 none

25


Patient no. Sex/Age Binet stage WBC count (x 109/L)

Previoustreatment

14 A F/60 A 8.0 none

15 A M/51 A 24.6 none

16 A M/81 A 20.5 CHL

17 A M/71 A 12.7 none

18 A F/84 B 22.7 PD/CHL

19 A M/77 C 7.2 PD/CHL

20 A F/85 A 47.7 none

21 A M/50 B 23.7 none

22 A M/81 C 11.3 PD/CHL

23 A F/75 A ND CHL

24 A F/72 A 10.9 none

25 A F/82 A 66.1 none

26 A M/59 A 22.2 none

27 A F/63 A 29.9 none

28 A F/74 A 39.1 CHL

29 A F/87 A 42.4 none

30 A F/40 A 9.6 none

31 A M/79 A 35.9 none

32 A M/60 A 12.9 none

Table 2.1. List of patients involved in the study (PD = prednisolone, CHL = chlorambucil, FLD = fludarabine). White blood cell counts (WBC) and Binet stage refer to patients’ status at time of sampling. ND indicates not determined.

26


2.3 Purification of B-CLL cells from whole blood

2.3.1 Isolation of whole lymphocyte fraction

Whole blood was collected into K+/ EDTA or Heparin tubes (Sarstedt, Leicester, UK),

and layered over Ficoll (Pharmacia Biotech, Sweden). Following centrifugation for 20

minutes at 400g, the lymphocyte layer was aspirated off, and cells were washed twice

in culture media (as described below), in order to remove any remaining Ficoll.

Freshly isolated lymphocytes were suspended in culture medium (RPMI 1640

(GibcoBRL, Paisley, Scotland), 10% foetal calf serum (FCS) (Sigma), 1%

penicillin/streptomycin (GibcoBRL)) and incubated at 37 °C for 20 minutes in plastic

culture flasks in order to remove adherent cells (macrophages, monocytes). Cells were

resuspended at an average density of 1 x 106 cells /ml. (Cells were counted using a

haemocytometer, and viability was assessed by Trypan Blue exclusion).

2.3.2 Purification of B lymphocytes

Lymphocytes isolated as described above were washed in 1 x PBS/2% FCS, and

resuspended in PBS / FCS at an average density of 10 x 106 cells/ml. Dynabeads M-

450 CD3 (Dynal, Oslo, Norway) were added to achieve a final ratio of dynabeads:— • 7target T cells of at least 4:1, and a minimum concentration of dynabeads of 2 x 10 /

ml. The cells and dynabeads were incubated at 4°C for 30 minutes with rolling action.

The magnetic beads (and bound CD3+ T cells) were removed by placing the tube of

cells into a magnetic particle concentrator (Dynal MPC-1). Unbound cells (CD3* B

cells) were pipetted off, washed and resuspended in media (as previously described) at

an average density of 1 x 106 cells/ml. Purity of the resulting cell population was

determined flow cytometrically using antibodies against the B cell marker CD 19

(CD19-FITC, Dako, High Wycombe, Bucks), and the T cell marker CD3 (CD3-RPE,

Dako) at 5pi per 106 cells. Flow cytometry is the measurement of cells in a flow

system which has been designed to deliver cells in single file past a laser beam.

Fluorescence and scattered light are recorded for each cell and data is depicted

27


graphically in the form of dot-plots and histograms. From these plots, populations of

cells with similar characteristics can be identified and quantified (see figure 5.1, page

71, for an example dot-plot resulting from use of the antibodies described above).

2.4 Determination of Apoptosis Sensitivity

Freshly isolated cells were incubated in six-well plates at 37°C in an atmosphere

containing 5% CO2. Samples of the cells were incubated in the presence or absence of

chlorambucil ( 3 - 1 5 pM), prednisolone (20 - 200 pM), fludarabine (3 pM),

staurosporine (0.2 pM), or anti-Fas monoclonal antibody (CH-11) (0.5 pg/ml)

(Immunotech, Marseilles, France) (Yonehara et al, 1989) as indicated in the text. For

analysis of caspase activation, duplicate samples of untreated and drug-treated cells

were pre-incubated for 30 min with 100 pM Benzyloxycarbonyl-Val-Ala-Asp (OMe)

fluoromethyl ketone (Z-VAD.fmk), a cell permeable caspase inhibitor (Enzyme

Systems Products, Dublin, CA). At specified times samples were analysed for

apoptosis, initially using in situ end labelling (ISEL) and agarose gel techniques for

visualising DNA fragmentation, and later by Annexin V labelling. 1 x 106 cells from

each culture were washed in ice-cold PBS in order to remove media and serum, and

were stored at ”80 °C as cell pellets for immunoblotting.

2.5 In situ end labelling (ISEL)

General Principles

ISEL incorporates digoxygenin-labelled nucleotides to the ends of DNA strand breaks

produced by endonuclease cleavage of DNA during the final stages of apoptosis. The

enzyme utilised is terminal deoxynucleotide transferase (TdT), which adds

nucleotides to the 3' hydroxyl group of a DNA strand, without the need for a template.

The strand breaks are visualised by the addition of an anti-digoxygenin-Fab fragments

with a fluorescein isothiocyanate (FITC) label. The cells are also labelled with

propidium iodide (PI), a red fluorescent DNA staining dye in order to quantify DNA

content. Flow cytometry is used to analyse the resulting cell populations.

28


Method

1 x 106 cells were fixed in 1ml 1% formaldehyde/PBS for 15 minutes on ice, washed

in two changes of ice-cold PBS, and resuspended in 1ml 70% ethanol/PBS for storage

at 4°C until required. For the labelling reaction, the cells were washed first in 1 x

TBS, resuspended in 100 pi of reaction mixture (80 pi UP water, 20 pi 5x TdT buffer,

2 pM digoxygenin-ll-dUTP (Boehringer Mannheim, Germany), and 10 U TdT

(GibcoBRL, Paisley, Scotland)), and incubated for 35 mins at 37°C. To stop the

labelling reaction the samples were removed immediately to ice for 3 mins. The cells

were then washed in staining buffer (1% of 10% Triton X-100, 20% 20x standard

saline citrate (SSC), plus 5% w/v bovine serum albumin (BSA)) and resuspended in

250 pi labelling mixture (250 pi staining buffer and 0.1 pg anti-digoxygenin-FITC

antibody (Boehringer Mannheim)) in which they were incubated for a further 35 mins

at 37°C. Following centrifugation at 400g for 5 minutes the samples were resuspended

in 1ml propidium iodide (PI) (50 pg/ml), and incubated overnight at 4°C. Flow

cytometric analysis was performed the following day on a Becton-Dickinson

FACScan, using Lysys II software. A dot-plot of FL2-Area (DNA content) vs FL1-

Height (FITC-labelled apoptotic cells) was produced for each sample. Regions were

set on FITC-positive and FITC-negative cells for the freshly isolated cell sample in

each case, and remained in place for all further analysis on that patient (see figure 3.1,

page 38, for an example dot-plot which demonstrates the positioning of the quadrant).

Values for the percentage of FITC-positive apoptotic cells in the sample were

generated from these regions. Typically 5000 events were recorded per sample.

2.6 Annexin V assay for apoptosis

General Principles

The Annexin V assay measures a cell surface alteration which occurs during

apoptosis. Phosphatidylserine (PS), a membrane phospholipid, is translocated from

the inner leaflet to the outer leaflet of the cell membrane. The externalised PS assists

recognition of the apoptotic cell by phagocytic cells to aid rapid clearance by

engulfment. Annexin V is a Ca2+ -dependent phospholipid binding protein with high

affinity for PS. When conjugated with a FITC label, this protein can be used as a

29


probe for externalised PS by flow cytometry. The cells can be dual labelled with

propidium iodide, which acts as a dye exclusion assay for necrotic and secondary

necrotic cells. This combination enables a distinction to be made between apoptotic

and secondary necrotic cells. An ideal result using this technique gives three

populations of cells. Viable cells have minimal staining with both fluorochromes

(Pr / FITC ), apoptotic cells are positive for Annexin V-FITC (PI7 FITC+) and

secondary necrotic cells (late stage apoptotic cells) stain highly with both

fluorochromes (PI+ / FITC+).

Method

1 x 106 cells were micro-centrifuged at 400g and then re-suspended in 1ml Annexin V

buffer (10 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 5 mM KC1, 1 mM MgCl2, 1.8

mM CaCh). Annexin V-FITC (Bender MedSystems, Vienna, Austria) was added to a

final concentration of 1.5 pg/ml. After incubation at room temperature for 8 min,

propidium iodide was added (50 pi of 50 pg/ml stock in PBS), and the cells incubated

for a further 2 min at room temperature before being placed on ice prior to flow

cytometric analysis on a Becton-Dickinson FACStar Plus, using Lysys II software. A

quadrant was set on each FL1-H vs FL2-H (Annexin V vs PI) dot plot as illustrated in

figure 2.1. Apoptotic cells were identified as Annexin V+ / PI" cells. Secondary

necrotic (Annexin V+/ PI*) cells were included in the dead cell count as these were

identified as late stage apoptotic cells. Typically 5000 events were recorded per

sample.

30


ANNEXIN V-FITC (3) v s PI (4)

<D’3oHHg.23'3.2

Oh

<N-JOh

&

Secondary necrotic cells A nnexin V+/PI+

Viable cells

r :

■■ ■ ■

■ •

. .. j \ . .

=:• : A poptotic cells l ^ ’iJ l^ ^ ' . - A r m e x m V+/PI-

10“FL1-H (Annexin V-FITC)

Figure 2.1 Annexin V FITC/Propidium iodide dot plot to illustrate positioning of the quadrant used to

quantify cell death in the cell population.

2.7 One-stage DNA fragment analysis gels.

Essentially as described by Sorensen et al (1988), this technique enables visualisation

of oligonucleosomal DNA cleavage without the need to extract DNA using organic

solvents. Briefly, 1 x 106 cells were resuspended in 15 pi of sterile ultrapure (UP)

water. 9 pi loading buffer (0.02% bromophenol blue, 40% glycerol in 1 x TBE) and 6

pi RNase A (50 mg/ml) was added and the samples were incubated at room

temperature for 30 minutes. A 1.8% agarose gel (UltraPure Agarose, GibcoBRL) (in

1 x TBE) was prepared, and a slice cut from the area above the sample wells. A

‘digesting geF of 0.8% agarose (in 1 x TBE, 2 % SDS), supplemented with proteinase

K (50 pl/ml) was poured into the gap above the wells. Once the samples were loaded,

the gel was run in 1 x TBE, and bands were visualised by staining for 30 minutes in

ethidium bromide (50 pg/100 ml) followed by a 60 minutes de-staining in UP water.

31


2.8 Field inversion gel electrophoresis.

Essentially as described by Brown et al (1993) and Anand & Southern (1990). Field

inversion gel electrophoresis (FIGE) is a form of pulsed field gel electrophoresis.

FIGE allows large fragments of DNA up to 700 kb to be resolved on an agarose gel,

by alternating the orientation of the electrical field by 180°, whilst ensuring forward

mobility by increasing the length of the forward pulse over the reverse pulse. In order

to protect the DNA from shear stresses, and to ensure support of the DNA fragments

in an agarose matrix at all times, the cells were embedded in low melting point

agarose plugs (Agarose L, Pharmacia). The plugs were incubated in NDS (1% Lauryl

sarcosine, 0.5 M EDTA, 10 mM Tris) and Pronase (1 mg/ml) (Sigma) for 48 hours at

50 °C, rinsed in NDS followed by three changes of Tris-HCl pH 8.0, before being

loaded on a vertical 1.5 mm thick 1% agarose gel (NA Agarose, Pharmacia). The gel

was run overnight using a pulse controller (PC 750, Hoeffer Scientific Instruments),

before being stained with ethidium bromide and viewed under UV light.

2.9 Immunoblotting

Frozen cell samples were lysed in a 10% SDS sample loading buffer containing

bromophenol blue. Electrophoresis was performed on either a 20 cm x 20 cm Flowgen

apparatus (Flowgen, Ashby de la Zouch, Leics, UK) using 7 % or 13 % resolving gels

with a 4 % stacking gel (Sambrook et al, 1989), or on a 10 cm x 10 cm Novex Mini-

Cell II apparatus (Novex, Germany), using 4 - 12 % pre-cast gradient gels. Samples

were run on SDS-polyacrylamide gels as described before being blotted onto

nitrocellulose membranes (Hybond C-extra, Amersham, Bucks). Membranes were

blocked for 60 min in 5% non-fat dried milk in Tris buffered saline containing 0.1%

Tween 20. The membranes were incubated with the primary antibody for 1 h at room

temperature followed by washing with Tris buffered saline containing 1% Tween-20

and then incubated with horseradish-peroxidase conjugated secondary antibody (rabbit

anti-mouse IgG or goat anti-rabbit IgG, Dako, High Wycombe, Bucks) for a further

hour. Rabbit polyclonal antibodies directed to the 17 kD large subunit of caspase-3

(kindly provided by Dr D Nicholson, Merck-Frosst, Quebec, Canada), which

recognize the proform of caspase-3 and its 17-20 kD large subunit(s), and to the

32


carboxy terminus of caspase-2, which recognize both pro-caspase-2 and the 12 kD

subunits, were used (Santa Cruz Biotechnology, CA). A mouse monoclonal antibody

to poly (ADP-ribose) polymerase (PARP) was used, which recognizes both intact

PARP (116 kD) and a cleavage product of 89 kD (kindly provided by Dr G Poirier,

Laval University, Quebec, Canada). A polyclonal antibody to caspase-7 (kindly

provided by X-M Sun, MRC Toxicology Unit, University of Leicester, Leicester,

UK), which recognizes the pro-caspase-7 and the catalytically active 19 kD large

subunit, was also used as described. A rabbit polyclonal antibody to caspase-8 was

raised against the large subunit of caspase-8 (amino acids 210 - 374) and kindly

supplied by Dr X-M Sun (MRC Toxicology Unit, University of Leicester, Leicester,

UK). The antibody obtained was characterized by ELISA and Western blot analysis,

which verified that it recognized intact procaspase-8 and the 43 kD and 18 kD

subunits. A rabbit polyclonal antibody to c-FLIP (kindly provided by Dr. D

Nicholson, Merck-Frosst) which recognises the intact form of c-F L IP l and the intact

and clipped forms of c-FLIPs (Rasper et al, 1998), and a monoclonal antibody against

FADD which recognises a 26 kD band corresponding to FADD in Jurkat cell lysates

(BD Transduction Labs, Kentucky, USA) were also used. All antibodies were checked

for specificity using lysates prepared from control cell lines. A sample of apoptotic

cells from a cell line was also included on each blot of experimental samples. The cell

lines used were THP-1 (human monocytic) and Jurkat (human T lymphoblastoid)

treated with staurosporine (0.2 pM) for 4 h in order to induce caspase

activation/substrate cleavage. The blots were developed using enhanced

chemiluminescence (ECL, Amersham, Bucks, UK) according to the manufacturer’s

instructions. Blots were exposed to X-OMAT LS film (Kodak, Rochester, NY) for

appropriate time intervals, and were developed using a Kodak X-OMAT film

processor (Kodak, Rochester, NY), in order to visualize caspase activation / substrate

cleavage.

33


2.10 Stimulation of B-CLL cells using anti-CD40 monoclonal antibody and

interleukin-4

Culture flasks were prepared as described by Walker et al (1997), by incubating

overnight with goat-anti-mouse immunoglobulins (Dako, High Wycombe, Bucks) in

TBS at a concentration of 5 pg/ml. 3.4 ml of this solution was used per 25cm3 volume

of flask. Flasks were blocked for lh at room temperature using a solution of TBS/1%

FCS. B-CLL cells were resuspended in culture medium (as previously described) and

dispensed into the prepared flasks. Anti-human CD40 monoclonal antibody (R&D

systems, Abingdon, UK) was added to a final concentration of 1.5 pg/ml. The cells

were cultured overnight at 37°C.

For stimulation with interleukin-4, cells were dispensed into culture flasks, and the

media was supplemented with recombinant human interleukin-4 (R&D Systems) at a

concentration of 10 ng/ml. Cells were then cultured for the time periods indicated in

the text.

2.11 Analysis of CD95/Fas receptor expression on B-CLL cells

1 x 106 cells were resuspended in 100 pi of PBS/1 % FCS. 1 pi of anti-human Fas

monoclonal antibody (CH11, Immunotech, Marseilles) and 5 pi of goat-anti-mouse-

FITC antibody (Dako) were added. As a negative control, 1 x 106 cells were incubated

with 1 pi control (non-human specific) IgM antibody (Dako) and 5 pi goat-anti-

mouse-FITC. The cells were incubated on ice for 30 minutes prior to centrifugation at

6,000 rpm for 2 minutes. The cells were resuspended in 1 ml PBS for analysis by flow

cytometry. Histogram plots of FL1-H (FITC) staining were obtained, the

photomultiplier tube (PMT) voltage settings being determined by the level of staining

in the negative control sample.

34


2.12 Isolation of the death inducing signalling complex (DISC)

Technique courtesy of Dr. M.E. Peter, German Cancer Research Center, Heidelberg,

Germany.

1 x 10 SKW6.4 cells (murine B lymphoblastoid) were resuspended in 5 ml cell

culture media (RPMI 1640, 10% FCS, 1% penicillin/streptomycin, 1% non-essential

amino acids (GibcoBRL)). Anti-Apo-1 (IgG (Bender MedSystems, Vienna, Austria))

was added at 1 pg/ml, and the cells were cultured for 10 minutes at 37°C. Following

stimulation, the cells were washed in ice-cold PBS and resuspended in 1 ml ice-cold

lysis buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X 100, 10%

glycerol, 1 pg/ml each aprotinin, leupeptin and pepstatin A). As negative controls, two

aliquots of unstimulated cells were also lysed. The lysate from one of these samples

was supplemented with 1 pg/ml anti-Apo-1. The samples were vortexed and

incubated on ice for 15 minutes. The samples were then spun at 13,000 rpm at 4°C for

15 minutes. 30 pi of a 50% solution of Protein-A Sepharose CL-4B (Sigma) was

added to each lysate. The samples were then incubated at 4°C with rolling action for 1

h 30 mins. Following this incubation period, the samples were spun at 6,500rpm for 2

minutes, the supernatant removed, and the sepharose washed in 4 changes of lysis

buffer. Sample loading buffer containing 10% SDS, 1% p-mercaptoethanol and

bromophenol blue was then added, the samples were boiled and SDS-PAGE and

immunoblotting was performed as previously described. A sample of whole Jurkat

cells which had been incubated with anti-Fas monoclonal antibody (clone CH11,

Immunotech, Marseille, France) for 4 h was included on the gels as a control for

antibody specificity. The membranes were probed with antibodies against FADD and

caspase-8 (all as previously described). The experiment was repeated using Jurkat

cells and B-CLL lymphocytes, where the initial stimulation period was increased to 60

minutes.

35

Chapter Three Results 1

Chapter 3

Development of an in vitro apoptosis sensitivity assay for CLL cells

3.1 Introduction

For analysing the effects of chemotherapeutic drugs on ex vivo cells, toxicity assays

such as the differential staining cytotoxicity assay (Bosanquet et al, 1993) and the

MTT assay (Campling et al, 1988) have traditionally been used. Apoptosis has been

shown to be the primary form of cell death induced by chemotherapeutic drugs. Many

techniques for observing and quantifying apoptosis have been developed, which

recognise various features of an apoptotic cell. The aim of this study was to develop

an in vitro apoptosis sensitivity assay to measure susceptibility of B-CLL cells to the

chemotherapeutic drugs prednisolone and chlorambucil. Initial experiments in this

project used as a measure of apoptosis the extent of DNA fragmentation in the cells,

using agarose gel electrophoresis to assess nucleosomal DNA cleavage, and the

quantitative technique of in situ end labelling (ISEL), where analysis is performed

flow cytometrically. As the study progressed and the need for an earlier marker of

apoptosis became evident, the Annexin V assay was employed which measures the

extent of extemalisation of phosphatidylserine.

3.2 CLL cells undergo spontaneous apoptosis in in vitro culture - measurement

using the ISEL method

In order to make a valid estimation of the in vivo apoptosis sensitivity of a malignant

cell type in an in vitro system, it must be taken into account that removal of the

malignant cells from their normal cellular environment may alter their innate

susceptibility to induction of apoptosis. This most likely occurs due to the loss of one

or more survival stimuli which are present in vivo, but which are not present in the

cell culture environment. When estimating the sensitivity of ex vivo cells to

chemotherapeutic drugs, this altered sensitivity must be taken into account. The term

‘spontaneous apoptosis’ refers to the apoptosis level induced following culture of the

CLL cells an in vitro culture system, without the addition of any apoptosis-inducing

36


stimuli such as chemotherapeutic drugs. The existence of spontaneous apoptosis has

been reported previously (Collins et al, 1989) and was evident from the first few

experiments performed during this study, and will be referred to throughout this

thesis. In order to determine whether cells from different patients were sensitive to

spontaneous apoptosis to varying degrees, samples from nine patients were analysed

as described below.

The total lymphocyte fraction was purified from blood samples obtained from nine

patients with B-CLL. The cells were labelled using the ISEL method to measure DNA

fragmentation and analysed flow cytometrically (figure 3.1 A). In all nine patient

samples, the level of apoptosis in the freshly isolated cells was less than 3% indicating

a low rate of apoptotic cell death in vivo (figure 3.1 B). Lymphocytes purified from

the same B-CLL patients were also cultured for 24 h. Subsequently, an aliquot of

cells (1 x 106 cells) from each culture was labelled using the ISEL method and

analysed flow cytometrically to measure the level of apoptosis in the culture. These

cells analysed following culture in vitro without the addition of anti-cancer drugs

showed a wide variation in spontaneous apoptosis (figure 3.1 B), indicating variable

sensitivity to apoptosis induction between B-CLL cells from individual patients.

37


<uX

Pm

Freshly isolated cells

O FL2-Area (6) v s FL1 -Height (3)

Control cells at 24 h

o FL2-Area (6) v s FL1 -Height (3)

3%£97%

"I 1 T 1 1 I—1 1 1 1- 1

/ ' k 93%

90.7%1023 1023

FL2-AreaB

30

_ 25-I LU2 20<0oaa. 10 <* 5

0

□ 24 h

1 I4 J _ □ J J J _

2 1 3 1 4 1 5 1 6 1 7 1 8 1 91 101 11 I 121 Patient Number

Figure 3.1 CLL cells were isolated from whole blood samples, and were labelled using the ISEL

method and analysed flow cytometrically. Samples of the cells were also cultured for 24 h in vitro prior

to being labelled and analysed using ISEL. A- Dot plots showing typical of results obtained from use of

the ISEL method on freshly isolated and cultured CLL cells. On the X-axis, FL2-Area is a measure of

the DNA content of the cells, and on the Y-axis, FL1-Height is a measure of green (FITC) flourescence.

Cells expressing high green fluorescence are apoptotic. B- Analysis of freshly isolated cells revealed a

low ex vivo rate of apoptosis (0 h). After 24 h culture in vitro, the level of spontaneous apoptosis varied

widely between patient samples.

38


3.3 Apoptotic CLL cells exhibit limited intermicleosomal DNA cleavage, but

evidence of large DNA fragmentation can be observed

In situ end labelling measures the extent of DNA fragmentation in apoptotic cells. In

order to visualise the extent of this DNA fragmentation in apoptotic CLL cells,

agarose gel DNA fragmentation analysis was performed on freshly isolated CLL cells,

and those which had been cultured in the presence of chemotherapeutic drugs.

Following the culture of CLL lymphocytes for 24 h alone or in the presence of

prednisolone (0.1 - 10 mM), chlorambucil (15 pM), fludarabine (3 pM) or etoposide

(2 pM), samples of cells were analysed by agarose gel electrophoresis for evidence of

intemucleosomal DNA fragmentation. In all of the patient samples, after 24 h of

culture, there was no clear evidence of intemucleosomal DNA cleavage (figure 3.2

A). Samples from five of the patients were analysed using field inversion gel

electrophoresis (FIGE). This analysis revealed the presence of large fragments of

DNA of 300 kb and 50 kb produced following induction of apoptosis by

chemotherapeutic dmgs (figure 3.2 B).

From the agarose gel electrophoresis analysis it appears that within 24 h of removal

from the in vivo environment, with or without treatment with chemotherapeutic dmgs,

CLL cells do not exhibit significant amounts of nucleosomal DNA cleavage, but do

undergo cleavage of the DNA into large (50 - 700 kb) fragments. This finding raised

significant questions as to the accuracy of using DNA fragmentation as a measure of

apoptosis in CLL cells. It was decided to employ an alternative flow cytometric

method, the Annexin V assay, which measures the extemalisation of

phosphatidylserine, a common feature of apoptotic cells (Koopman et al, 1994)

39


4 h 24 h( \ ( 'i

M M 0 10 1 0.1 0 10 1 0.1 mM Pd

B1 2 3 4 5 6 7 8 9 10 11

300 k B > 5 0 k B >

< 700 kB

Figure 3.2 B-CLL lymphocytes were analysed by agarose gel electrophoretic methods in order to

determine the extent of DNA fragmentation occuring during apoptosis in these cells. A. Cells were

analysed for nucleosomal DNA cleavage after 4h and 24h culture alone or with prednisolone (Pd). B.

Cells were analysed for formation of large (>50kB) DNA fragments in response to culture alone (lanes

2,3,c,d) or with prednisolone (10 mM, lanes 4 and 5; 1 mM, lanes 6 and 7; 0.1 mM, lanes 8 and 9) or

chlorambucil (15 pM, lanes e and f) or etoposide (2 pM, lanes 10 and 11). (Lanes 1 and a are lambda

phage genome concatamers, and lane b is S. cerevisiae chromosome marker).

40


3.4 The ISEL assay underestimates the percentage of apoptotic CLL cells

In situ end labelling measures the extent of apoptotic DNA fragmentation in a

population of cells. Previous results had demonstrated that CLL cells undergoing

apoptosis exhibited only a limited amount of intemucleosomal DNA fragmentation

(section 3.3), and so an alternative method of assessing apoptosis was chosen, the

Annexin V assay. In the hope that results obtained using ISEL would relate to results

gained using the Annexin V assay, comparative experiments were performed to

establish the relationship between results from the two techniques

CLL cells from six patients were cultured for 24 h alone (control) or in the presence of

prednisolone (20 pM) or chlorambucil (7 pM) prior to being analysed using both the

Annexin V and ISEL labelling techniques. The results obtained from use of the ISEL

method were plotted against the results obtained from using the Annexin V assay

(figure 3.4). Regression analysis confirmed that the two sets of data had a linear

relationship which was highly significant (P < 0.0001) at the 99.5% confidence level.

The equation for the regression line was calculated (y = 2.0lx), the coefficient of 2.01

indicating that the Annexin V values are double those recorded using the ISEL

method. As a result of this finding, it was deemed unsuitable to continue using DNA

fragmentation as a marker for apoptosis in CLL cells, and the Annexin V assay was

used in all subsequent analysis in this thesis.

41


y = 2.0185x80

ao< 50

c 30

10 ^

20 25 30 350 5 10 15 40ISEL % Apoptosis

Figure 3.4 A comparison of the ISEL and Annexin V labelling techniques

demonstrates that measuring DNA fragmentation using ISEL underestimates the

number of apoptotic CLL cells in a population by approximately 50 %. Cells were

isolated from six CLL patients. At 0 h and after 24 h of culture alone or in the

presence of 20 pM prednisolone or 7 pM chlorambucil, duplicate samples were

labelled using ISEL and the Annexin V assay. The flow cytometry results obtained

using each technique were plotted against each other. Regression analysis was

performed to check for a linear relationship, and the regression line was plotted. The

equation for the line is stated on the graph.

42


3.5 In vitro sensitivity to spontaneous apoptosis is a predictor of in vitro

sensitivity to chlorambucil-induced apoptosis

Previous results had demonstrated the variability between patient samples in

spontaneous apoptosis sensitivity. To determine whether this sensitivity was reflected

in the response of the cells to chemotherapeutic drug-induced apoptosis, CLL cells

from 30 patients were tested for chlorambucil sensitivity. For each case, duplicate

cultures were set up. One of these cultures was left untreated (control), and the

chlorambucil was added to the second culture (7 pM). The cells were incubated at

37°C for 24 h. Apoptosis sensitivity in the two cultures was assessed flow

cytometrically using the Annexin V assay to measure externalised phosphatidylserine.

Control cultures were scored as sensitive or resistant to spontaneous apoptosis.

(Sensitive samples were given the value 1, and resistant values were scored 0). The

value chosen to delineate between the two groups was the median value of 20.75 %

spontaneous apoptosis. The chlorambucil-treated samples were then split into two

groups based on the sensitivity to spontaneous apoptosis of their corresponding

control cultures and the results were plotted (figure 3.5). A one-tailed T-test

(assuming unequal variances) was performed in order to determine if the means of the

two groups were significantly different. A significant P-value of < 0.0001 confirmed

that the patients which were scored as sensitive to spontaneous apoptosis were most

likely to be highly sensitive to chlorambucil-induced apoptosis. This implies that the

relationship between sensitivity to spontaneous apoptosis and sensitivity to in vitro

drug-induced apoptosis is significant, a finding which would greatly simplify any

predictive testing technique based upon apoptosis sensitivity.

43


(0

CO

80

70

60

50

■g 40■T

l ^E| 206̂

10

t

I

♦♦♦♦♦♦

0 1

Sensitivity to spontaneous apoptosis

Figure 3.5 Cells from thirty CLL patients were cultured for 24 h alone (control) in

order to determine sensitivity to spontaneous apoptosis, or in the presence of 7 pM

chlorambucil. After 24 h, flow cytometric analysis of the cells was performed using

the Annexin V labelling technique to measure externalised phoshatidylserine. The

control cultures were scored as sensitive (1) (n = 15) or resistant (0) (n = 15) to

spontaneous apoptosis. The value obtained for chlorambucil-induced apoptosis for

each sample was plotted against the spontaneous apoptosis score in each case.

44


3.6 As patients undergo chlorambucil therapy, the in vivo level of apoptosis can

decrease, but the sensitivity of the cells to spontaneous and chlorambucil-induced

apoptosis in in vitro culture increases

In order to attempt to determine how closely the in vitro system of apoptosis induction

by chemotherapeutic drugs was related to the in vivo reaction of CLL cells to drug

therapy, a small in vivo study was performed. Blood samples were taken from two

CLL patients (8 A and 12 1) immediately prior to them beginning a 14-day course of

chlorambucil. This was to be the first course of treatment for patient 8 A, but patient

12 I had received chlorambucil therapy previously. In addition to the pre-treatment

blood sample, further samples were taken from the same patients at day 7 of the

course of treatment, and at day 30, two weeks post-treatment. On each sample day, the

in vivo level of apoptosis was analysed by labelling the freshly isolated lymphocytes

using the Annexin V technique. Flow cytometric analysis was performed immediately.

This method attempts to give as accurate a measure of in vivo apoptosis rate as is

possible, the time lapse from phlebotomy to flow cytometric analysis being less than

60 minutes. This analysis revealed that the in vivo apoptosis rate for patient 8 A

remained relatively stable, whereas the in vivo apoptosis level for patient 12 I

decreased as the course of treatment was administered (figure 3.6).

A proportion of cells from each isolate were resuspended in culture medium and

cultured for 24 h. Assessment of the level of spontaneous apoptosis induced in the

cultures was made usisng the Annexin V assay. This analysis revealed that the in vitro

apoptosis sensitivity of the ex vivo CLL cells was elevated during administration of

chemotherapy. In both cases, the level of spontaneous apoptosis increased during and

after the course of treatment when compared with the level of spontaneous apoptosis

obtained pre-treatment (figure 3.6).

In order to further compare the in vivo and in vitro responses of CLL cells to

chlorambucil, the remaining cells from each sample were cultured in vitro in the

presence of 7 pM chlorambucil. After 24 h culture, the sensitivity of these cells to

chlorambucil-induced apoptosis was assessed flow cytometrically using the Annexin

V technique. The cells from patient 12 I showed a decrease in sensitivity to

45


chlorambucil as treatment commenced, which increased again by the 30 day sample

point (figure 3.6). However, the sensitivity of the cells did not recover to the levels

observed prior to the treatment beginning, possibly indicating the loss of a particularly

chlorambucil-sensitive clone. Since this patient had in the past had one course of

chlorambucil therapy, this result may indicate some degree of resistance of the

remaining clone to apoptosis induction by chlorambucil. Additionally, the level of

apoptosis induced by chlorambucil at the 30 day sample point was not significantly

higher than the level of spontaneous apoptosis induced at the same timepoint. When

compared with the greater difference in the cells’ sensitivity to spontaneous and

chlorambucil-induced apoptosis at the pre-treatment sample point, this may indicate

that the cells are becoming increasingly resistant to chlorambucil as a result of the

therapy.

Cells from patient 8 A showed a steady increase in sensitivity to chlorambucil as the

course of treatment progressed, mirroring the increase in sensitivity to spontaneous

apoptosis. Of interest is the observation that the post-treatment level of chlorambucil-

induced apoptosis in this patient was 37% higher than the pre-treatment level (figure

3.6), which implies that a number of chlorambucil-sensitive CLL cells remain in

circulation following the end of the course of treatment.

46


C/5cooQ.OQ.<

100

80

o 60

40

20

Patient 12 I

Re-treatment (Day 0) Day 7

Sample Day

□ Freshly isolated cells ■ Control□ 7 uM Chlorambucil

Fbst-treatment (Day 30)

100

Patient 8 A

<0'(02Q.OQ.<

80

60

40

20

Re-treatment (Day 0) Day 7

Sample DayFbst-treatment (Day 30)

Figure 3.6 In vivo apoptosis can decrease and sensitivity to spontaneous apoptosis

can increase as drug treatment is administered. Blood samples were taken from

patients 121 and 8 A prior to them starting a two week course of chlorambucil (day 0).

Blood samples were also taken at day 7 of their courses of treatment, and at day 30

(post-treatment). At each sample point, freshly isolated lymphocytes were labelled and

analysed using the Annexin V technique. Other cells from the same isolate were

cultured for 24 h in culture medium alone (control) or with 7 pM chlorambucil before

being analysed using the Annexin V assay. For flow cytometry histograms of freshly

isolated and control cells at each of the sample points for both patients, please refer to

the Appendix, figure A1.

47


3.7 Discussion

The initial aim of this reseach project was to investigate the relationship between

chemotherapy and apoptosis in chronic lymphocytic leukaemia (CLL). CLL is often

diagnosed in an asymptomatic phase, and patients can live with the disease for several

years before in some cases the tumour burden and its accompanying effects on the

body mean that chemotherapeutic treatment is necessary. The treatment of choice has

been the alkylating agent, chlorambucil with or without the glucocorticoid,

prednisolone. More recently, the purine analogues, of which fludarabine is one, have

shown increasing promise and a higher rate of remissions when used as first line

therapy, when compared with the remission rate obtained using chlorambucil.

However, drug resistance is a major problem in CLL, and second or third treatments

with chlorambucil do not usually produce high remission rates. Since the advent of

chemotherapy, it has been a goal for many researchers to develop a means of

analysing the toxicity or killing efficacy of the drugs on the target cells in order to

reduce the incidence of unsuccessful treatment (Hanson et al, 1991; Bosanquet, 1993).

In addition the effect of the drugs on other cells in the body needs to monitored, a

process which plays a crucial role in the development and testing of new drug

therapies. Toxicity tests have been in existence for many years, and have been used

extensively for purposes such as those outlined above. Examples include the MTT

assay which assesses the amount of insoluble formazan produced by living cells

(Campling et al, 1988) and the differential staining cytotoxicity assay (Bosanquet et

al, 1983). In the 1970’s apoptosis was identified as a mode of cell death distinct from

necrosis (Kerr, 1972). Since then, investigations into the effects of chemotherapy on

mammalian cells have revealed that apoptosis is the primary method by which cells

die as a result of exposure to these drugs.

This study began with the development of a method to analyse apoptosis sensitivity of

lymphocytes sourced from CLL patients attending outpatient clinics, some untreated,

and some who were receiving chemotherapy. At the time that this study commenced

there were numerous techniques available for analysing or quantifying apoptotic cells

(Carbonari et al, 1995), several of which were based upon the characteristic

48


intemucleosomal DNA cleavage that occurs in an apoptotic cell. Qualitative agarose

gel electrophoresis techniques and flow cytometric methods, such as in situ end

labelling, appeared to be the ideal techniques to apply to CLL samples. In particular,

in situ end labelling (ISEL) (Wolfe et al, 1996) was chosen over some newer flow

cytometric labelling techniques (such as the Hoerscht 33342 ‘high blue’, method

(Brown et al, 1996)), because of the inclusion of a fixation step in the procedure. This

enabled the samples to be stored until a sufficient number were available to be

analysed, and also decreased the risks asscociated with handling unfixed clinical

specimens. The data obtained from the flow cytometric analysis of the CLL cells were

supplemented using agarose gel electrophoretic methods to enable visualisation of

intemucleosomal and larger DNA fragmentation (Figure 3.2 A and B).

Initial results using ISEL analysis revealed a low level of in vivo apoptosis in the cells

from every patient analysed, demonstrating that the CLL lymphocytes are not

undergoing cell death in the patient to any significant degree (Figure 3.1 B). It is

possible that the in vivo level could be higher than this, but that apoptotic cells are

being cleared effectively from the blood, and so cannot be measured as such.

However, if these figures do accurately represent levels of in vivo apoptosis, then this

supports the lymphoaccumulative model of CLL (Dameshek, 1967), in which CLL

lymphocytes remain for a long period in circulation due to reduced levels of cell

death.

ISEL analysis also confirmed the existence of spontaneous apoptosis, a feature of

cultured ex vivo CLL cells which had been observed previously (Collins et al, 1989).

From a common low level of in vivo apoptosis, CLL cells cultured for 24 h prior to

being labelled using the ISEL method and analysed flow cytometrically varied

dramatically in their sensitivity to spontaneous apoptosis (Figure 3.1 B). This implies

that differences may exist between the CLL lymphocytes of the spontaneous apoptosis

sensitive and resistant patients. Differences in the expression levels of apoptosis-

controlling factors could be one reason for this variability. The relative levels of anti-

apoptotic Bcl-2 and pro-apoptotic Bax in these cells may be instrumental in

determining their apoptosis sensitivity, a theory which has been investigated by a

number of research groups (McConkey et al, 1996; Gottardi et al, 1996; Pepper et al,

49


1996; Thomas et al, 1996). A high Bcl-2:Bax ratio has been shown to correlate with

resistance to chlorambucil in vitro (Thomas et al, 1996), and Bax upregulation

appears to be required for chemotherapeutic drug-induced apoptosis to take place,

whilst a high expression level of Bcl-2 confers survival (Pepper et al, 1999). It is also

likely that CLL cells differ in their sensitivity to growth factors in their surrounding

environment, a subject which will be discussed in later sections of this thesis.

Populations of cells which depend heavily on growth factor stimulation (such as less

mature and less well differentiated CLL clones) are likely to be more susceptible to

cell death upon growth factor withdrawal (such as occurs in in vitro culture).

Use of gel electrophoretic methods to confirm that apoptosis was indeed the

prevailing mode of cell death in CLL cells exposed to chemotherapeutic drugs led to

the observation that, following 24 h of in vitro culture, there was little evidence of the

intemucleosomal cleavage characteristic of apoptosis (Figure 3.2 A). However, large

DNA fragmentation analysis demonstrated that the cells were in the early stages of

DNA fragmentation confirming that apoptosis was occuring (Figures 3.2 B and 3.3).

This phenomenon of little or no intemucleosomal cleavage in apoptotic CLL cells had

been reported previously (Huang & Plunkett, 1995), but the presence of

intemucleosomal DNA fragments may be dependent on the sampling point chosen

following apoptosis induction. Later time points may pick up DNA cleavage more

effectively than earlier time points. However, since the ISEL technique is based upon

labelling the fragmented DNA with digoxygenin-labelled nucleotides in order to

visualise the apoptotic cells, it was of some concern that the cells in question were not

undergoing intemucleosomal DNA fragmentation within the timescale of the assay.

In addition, since the study was moving towards more in-depth analysis of apoptosis

in CLL cells, it became more pmdent to use an earlier marker than DNA

fragmentation in order to quantify apoptosis in these cells.

Annexin V has a high affinity for the membrane phospholipid, phosphatidylserine.

During apoptosis, phosphatidylserine (PS) is flipped from the inner leaflet of the cell

membrane to the outer surface, where it serves as a recognition marker to phogocytic

cells (Koopman et al, 1994), and where it can also be bound by fluorescently labelled

recombinant Annexin V and used as a marker for flow cytometric analysis of

50


apoptosis (Martin et al, 1995). In order to introduce this new technique into the study,

a small series of comparitive experiments were performed. Cells from six CLL

patients were cultured alone or for 24 h in the presence of chemotherapeutic drugs

prior to being labelled and analysed using both the ISEL and Annexin V techniques.

When the results obtained with each technique were plotted against the other, it was

discovered that the proportion of apoptotic cells in the CLL cell population was being

underestimated by ISEL by as much as 50%, confirming the observation that DNA

fragmentation was not taking place in these cells to any great degree (Figure 3.4). All

further analysis of apoptosis levels in this study was made using the Annexin V assay.

Large variation had been observed in the sensitivity of CLL cells from different

patients to spontaneous apoptosis. To determine if this sensitivity to spontaneous

apoptosis was mirrored in the response of the cells to chlorambucil-induced apoptosis,

cells from thirty patients were cultured alone or in the presence of chlorambucil for 24

h, prior to assessment of apoptosis using the Annexin V assay. When the results were

compared, it was discovered that cells which were sensitive to spontaneous apoptosis

were also more likely to be sensitive to apoptosis induced by chlorambucil (p <

0.0001). This implies that there is a relationship between sensitivity to spontaneous

apoptosis and sensitivity to in vitro drug-induced apoptosis, a finding which could

greatly simplify predictive testing techniques based upon apoptosis sensitivity.

In order to assess how closely this in vitro analysis of CLL cells response to

chemotherapeutic agents was related to the in vivo response to chemotherapy, and to

analyse how the apoptosis sensitivity of the cells altered during a course of treatment,

a small in vivo study was performed. Cells were taken from two CLL patients prior to,

during and after they had received a 2-week course of chlorambucil. As the treatment

commenced the in vivo level of apoptosis in one of the patients decreased, whilst the

level in the other patient remained stable. More interesting was the effect on the in

vitro spontaneous apoptosis sensitivity of the cells. As the patients’ course of

treatment progressed, the CLL cells became increasingly sensitive to spontaneous

apoptosis, possibly indicating that the treatment was having the desired effect, in that

the cells were being triggered into apoptosis, removal to the in vitro situation serving

only to hasten their eventual death. The fact that the cells were more sensitive to

51


spontaneous apoptosis two weeks after the end of the course of treatment than they

were prior to the treatment commencing may have some bearing on treatment

decisions for future patients. However, a much larger group of patients would need to

be analysed using this method before this observation could be confirmed.

Samples of cells taken from the treated patients were also cultured for 24 h in the

presence of 7 pM chlorambucil. Analysis of the chlorambucil sensitivity of these cells

using the Annexin V assay revealed differences between the two patients studied.

Patient 12 I had previously undergone treatment with chlorambucil. The cells from

this patient decreased in their sensitivity to in vitro chlorambucil-induced apoptosis as

the course of therapy progressed. Two weeks post-treatment, the cells had recovered

some sensitivity to chlorambucil-induced apoptosis, but not to the level seen at the

pre-treatment sample point. This could signify the loss of a chlorambucil-sensitive

clone of cells and may indicate some degree of resistance of the remaining clone to

apoptosis induction by chlorambucil. Since the patient had been treated with this drug

in the past, it may be conceivable that some degree of chlorambucil-resistance had

developed. In support of this theory, increases in p-glycoprotein (Perri et al, 1989) and

upregulation of glutathione-s-transferase mRNA expression (Schisselbauer et al,

1990) have been demonstrated in some cases of chlorambucil-resistant CLL.

The cells from patient 8 A behaved in a slightly different manner. For this patient, this

was the first course of treatment with chlorambucil or any other chemotherapy. The

cells from this patient showed a steady increase in sensitivity to apoptosis induction in

vitro by chlorambucil as the course of treatment progressed. Of some interest is the

observation that the post-treatment level of chlorambucil-induced apoptosis in this

patient was higher than the pre-treatment level. This may imply that a significant

number of chlorambucil-sensitive CLL cells remain in circulation following the end

of the course of treatment, a finding which also may have relevance to the way in

which CLL patients are treated therapeutically. Again, a larger group of previously

treated and untreated patients would need to be analysed before this observation could

be confirmed.

52

Chapter Four Results 2

Chapter 4 - Processing/Activation of caspases -3 , -7, and -8 , but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia cells

4.1 Introduction

The importance of caspase activation in spontaneous and chemotherapy-induced

apoptosis in leukaemic cells from patients with CLL had not previously been

described. It had been shown that peripheral blood mononuclear cells from CLL

patients were caspase-3 immunopositive (Krajewski et al, 1997) and that

glucocorticoid-induced apoptosis of CLL lymphocytes requires protease activation

and is accompanied by cleavage of PARP and lamin Bi, together with loss of caspase-

3 (Bellosillo et al, 1997; Chandra et al, 1997). The aim of the work described in this

chapter, was to further define the role of caspases in induction of spontaneous and

drug-induced apoptosis in CLL cells.

4.2 Inhibition of spontaneous apoptosis in CLL cells by Z-VAD.fmk

Apoptosis was assessed in CLL cells using Annexin V to measure extemalisation of

phosphatidylserine (figure 4.1). Freshly isolated cells, prior to culture, showed a very

low level of spontaneous apoptosis, as measured by Annexin V (figure 4.1 A). During

culture, the cells underwent spontaneous apoptosis (figure 4.IB), which was increased

in the presence of chemotherapeutic agents, such as chlorambucil (figure 4.1C). A

variable amount of spontaneous apoptosis was observed (Tables 4.1 and 4.2) in

agreement with other studies (Collins et al, 1989). The measurement of

phosphatidylserine exposure allowed a clear quantification of the percentage of

apoptotic cells. In order to assess the role of caspases in the execution phase of

apoptosis in B-CLL cells, Z-VAD.fmk, a cell permeable caspase inhibitor, was used

which inhibits apoptosis in many different model systems (MacFarlane et al, 1997,

Zhu et al, 1995, Feamhead et al, 1995). Z-VAD.fmk (100 jliM ) inhibited spontaneous

apoptosis in all but one of the patient samples examined (Table 4.1) supporting the

involvement of caspases in the spontaneous apoptosis of CLL cells. In order to obtain

more definitive evidence for the involvement of caspases, the processing/activation of

the effector caspases was examined as well as the proteolysis of PARP, which has

been used as a marker of apoptosis ( Kaufmann et al, 1993).

53


54.5%

93.6%

CO66 .2%

M2

33.9%

4

M1 -----145% M2 -----1

55% 16A

Annexin V - FITC

10* 1 0 1 102 1 0 J 10*

►

Figure 4.1 Induction of apoptosis in cells from two patients with CLL assessed by

phosphatidylserine extemalisation. A - Freshly isolated CLL cells, from patients 15 A

and 16 A, were examined for phosphatidylserine exposure by binding of Annexin V as

described in Chapter 2, section 2.6. The percentages of cells with low and high

Annexin V binding representing normal (marker 1) and apoptotic (marker 2) cells are

shown. CLL cells were also cultured for 20 h either B. alone to measure spontaneous

apoptosis or C. in the presence of chlorambucil (7.5 pM).

54


Patient No % Spontaneous apoptosis

- Z-VAD.fmk + Z-VAD.fmk

2 A 37 18

3A 17 8

4A 14 5

11A 21 11

12A 18 11

19A 7 7

Table 4.1 Inhibition of spontaneous apoptosis by Z-VAD.fmk

Freshly isolated CLL cells from patients were cultured for 24 h at 37°C either alone or

in the presence of Z-VAD.fmk (100 pM) as indicated. Apoptosis was assessed by

measuring extemalisation of phosphatidylserine using Annexin V staining.

55


4.3 Activation of caspase-3 and caspase-7 in apoptosis of CLL cells.

The time course of induction of apoptosis was examined in four previously untreated

Binet Stage A cases of B-CLL. In each patient, a time dependent induction of

spontaneous apoptosis was observed (figure 4.2 A). Cells from these patients exposed

to chlorambucil, showed a concentration dependent increase in the induction of

apoptosis compared to control cells (figure 4.2 A). These cells were also analysed by

immunoblotting for activation of caspases -3 and -7. Freshly isolated untreated CLL

cells contained primarily the proform of caspase-3 (figure 4.2 B, lane 1). Processing of

caspase-3 at Asp 175 between the large and small subunits yields a 20 kD subunit,

which is further processed at Asp 9 and Asp 28 to yield 19 kD and 17 kD large

subunits (Fernandez-Alnemri et al, 1996). A time dependent processing of caspase-3

to its catalytically active large subunit(s), 17-20 kD, was observed (figure 4.2 B, lanes

2-5), which was increased in the presence of chlorambucil (figure 4.2 B, lanes 6-9).

Activation of caspase-3 was observed in both spontaneous and drug-induced apoptosis

in all 9 samples examined to date (Table 4.2).

Caspase-7 was also present in freshly isolated CLL cells as a ~35 kD proform without

any detectable large 19 kD subunit (figure 4.2 C, lane 1). Processing of caspase-7

initially occurs at Asp 198 between the large and small subunits, followed by cleavage

at Asp 23 to yield the 19 kD large subunit (MacFarlane et al, 1997). A time dependent

processing of caspase-7 to its 19 kD subunit was observed in spontaneous apoptosis,

which was also increased as a result of exposure to chlorambucil (figure 4.2 C).

Caspase-7 was activated in all 10 samples of spontaneous and drug-induced apoptosis

examined (Table 4.2). Thus induction of apoptosis in CLL cells was accompanied by

the processing of both the effector caspases-3 and -7 to their catalytically active large

subunits.

56


(/>o+•>a.o£ 20

0 4 8 12 16 20 24

Time (h)

B Control 7 pM Chir

Time (h) 0N /"

10 20 2 10 20

32 kD ►

LS ►

Control 7 pM Chir "n r - \

Time (h) 0 2 6 10 20 2 6 10 2035 kD

19 kD ►•xO l '

Figure 4.2 Induction o f apoptosis in cells from a representative CLL patient is accompanied by

processing of caspase-3 and caspase-7. (A) Freshly isolated CLL cells were incubated either alone ( ♦ -

♦ ) or in the presence o f chlorambucil (3 pM) ( ■ - ■ ) or (7.5 pM) (A -A ) and apoptosis assessed at the

indicated times by extemalisation o f phosphatidylserine as shown in Fig 4.1. (B) Processing of

caspase-3 in CLL cells undergoing apoptosis. CLL cells from patient 15 A were cultured for the

indicated times either alone (Con) or in the presence of chlorambucil (Chi, 7.5 pM) and examined by

western blot analysis for caspase-3 as described in Chapter 2, section 2.9. The arrows denote either the

32 kD proform o f caspase-3 or the catalytically active large subunits (LS) of approximately 17 - 20 kD.

(C) Processing of caspase-7 in CLL cells undergoing apoptosis. CLL cells from patient 15 A were

cultured for the indicated times either alone (Con) or in the presence of chlorambucil (Chi, 7.5 pM) and

examined by western blot analysis for caspase-7. The arrows denote either the 35 kD proform of

caspase-7 or the large subunit o f 19 kD.

57


PatientNo

Sex/Age

Binetstage

Previoustherapy

WBC

x 109/1% Apoptosis

Spon Pd Chi -2Caspase -3 -7 -8

PARP

2A M/74 A - 23.1 36 55 57 + + + ND ND3A M/72 A Chi 49.7 17 19 21 - + + ND ND8A M/72 A Chi 30.2 14 71 36 ND + ND ND ND10A M/91 A - 24.5 7 22 18 - + + ND ND11A F/74 B Chi 14.2 20 24 19 - + + ND ND12A F/67 B - 17.5 18 27 20 - + + + +13A M/62 A - 34.3 13 27 22 - + + ND ND14A F/60 A - 8.0 22 ND 42 ND + + ND +15A M/51 A - 24.6 27 ND 48 ND + + + +16A M/81 A Chi 20.5 42 ND 66 ND ND ND “f* +17A M/71 A - 12.7 7 19 25 - ND ND + ND

18A F/84 B Chi, Pd 22.7 41 58 67 - ND + + ND

19A M/77 C Chi, Pd 7.2 29 42 67 - ND + + ND

20A F/85 A - 47.7 20 44 15 ND ND ND + ND

Table 4.2 . Clinical information and summary of in vitro apoptosis sensitivity and

incidence of caspase activation. Peripheral blood lymphocytes from patients

diagnosed with B-CLL were cultured in vitro for 20 h either alone or in the presence

of prednisolone (Pd) and chlorambucil (Chi). Both spontaneous (spon) and drug-

induced apoptosis were then determined by Annexin V binding. Activation of

caspases -2, -3, -7 and -8 as well as proteolysis of PARP was determined by western

blot analysis. Expression of the pro-forms of the caspases was found in all cases

examined. A *+’ indicates activation of the pro-enzyme to an active subunit was

observed following in vitro culture with and without addition of the drugs, whereas a

indicates no such activation was observed. (ND indicates not determined).

58


4.4 Caspase-2 processing does not generally accompany apoptosis of CLL cells.

Several recent studies have shown that caspase-2 (ICH-1, Nedd2) is processed in

some but not all cells during the induction of apoptosis (MacFarlane et al, 1997, Li et

al, 1997, Harvey et al, 1997). The prodomain of caspase-2 binds to the adaptor

molecule RAIDD/CRADD which also binds the receptor-interacting protein RIP and

may thus regulate apoptosis (Duan & Dixit, 1997, Ahmad et al, 1997). It is not known

which if any intracellular substrates are cleaved by caspase-2 in apoptosis. In order to

investigate the possible importance of caspase-2 in apoptosis of CLL cells, an

antibody was used which recognizes both the proform and the small p i2 subunit of

caspase-2. In agreement with previous studies (MacFarlane et al, 1997), induction of

apoptosis in the human monocytic tumor cell line, THP. 1, resulted in the activation of

caspase-2 to its ~ 12 kD small subunit (figure 4.3, lane 1) and was included as a

positive control for the processing of caspase-2. In cells undergoing spontaneous and

drug-induced apoptosis, processing of procaspase-2 was seen in only 1/9 samples

examined to date (Table 4.2). The results from this case are shown in more detail

(figure 4.3). Untreated freshly isolated CLL cells from this patient contained primarily

the ~ 48 kD proform of caspase-2 with no detectable small 12 kD subunit (figure 4.3,

lane 2). These freshly isolated cells also contained an immunologically reactive

protein of ~ 33 kD, which may represent an early processed form of caspase-2 (figure

4.3, lane 2). In cells undergoing spontaneous apoptosis, little or no cleavage of

procaspase-2 to its 12 kD small subunit was observed (figure 4.3, lane 3). Induction of

apoptosis with prednisolone resulted in the processing of procaspase-2 to its 12 kD

subunit (figure 4.3, lane 4), which was more marked with chlorambucil (figure 4.3,

lane 5). Thus although processing of caspase-2 was observed in cells from this patient,

it did not appear to be a general feature accompanying either spontaneous or drug-

induced apoptosis of CLL cells.

59


+ ve Oh Con Pd

+ Z - VAD.fink

Chi Con Pd Chi

48 kD^*

33 kD^*

12 kD

Figure 4.3 Activation of caspase-2 only occurred in cells from one patient. Freshly

isolated CLL cells, from patient 2A, were cultured for 20 h either alone (Con) or with

prednisolone (Pd, 200 pM) or chlorambucil (Chi, 15 pM) in the absence or presence

of the caspase inhibitor Z-VAD.fmk (100 pM) as indicated. Freshly isolated cells (0

h) were included as a control. The cells were examined by western blot analysis for

caspase-2 as described in Chapter 2, section 2.9. The arrows indicate the proform of

caspase-2 and its 12 kD small subunit. Of the 9 patients examined, this was the only

one to show activation of procaspase-2. Human monocytic THP.l cells treated with

etoposide (25 pM) for 4 h were included as a positive control (+ve) as caspase-2 is

processed in these cells to its 12 kD small subunit.

60


4.5 Activation of the effector caspases result in the cleavage of PARP.

As processing of caspases-3 and -7 was observed, CLL cells were also examined for

the cleavage of PARP, a substrate for both these caspases (Nicholson et al, 1995,

Fernandez-Alnemri, 1995). Induction of spontaneous apoptosis was accompanied by a

time dependent cleavage of PARP to a characteristic 89 kD signature fragment (figure

4.4 - Patient 14A, lanes 2-5; Patient 17A, lanes 1-4). The induction of apoptosis and

the cleavage of PARP were both induced following exposure of the CLL cells to

chlorambucil (figure 4.4 - Patient 14A, lanes 6-9; Patient 16A, lanes 5-12), further

supporting the involvement of caspases in the execution phase of apoptosis in CLL

cells.

Patient 14 A

rTime (h) 0

Control 7 pM Chia r

10 20 2 10 20

116kD >

89 k D >

Patient 16 A

Time (h)

Control 3 pM Chi 7 pM Chi1 r

6 10 20 2> r

10 20 2 10 20

116kD>"

8 9 k D >

Figure 4.4 Cleavage o f PARP accompanies apoptosis in CLL cells. Freshly isolated CLL cells, from patients 14 A and 16 A, were cultured for the indicated times either alone (Con) or with chlorambucil (Chi, 3 pM or 7.5 pM) and then analysed by western blot analysis for intact PARP (116 kD) or its cleaved product (89 kD).

61


4.6 Activation of caspase-8 during apoptosis of CLL cells.

Although caspase-8 has been implicated as one of the main “activator” caspases in

receptor-mediated apoptosis (Boldin et al, 1996, Muzio et al, 1996) , its role if any in

drug-induced apoptosis is not clear. In order to examine its possible involvement in

apoptosis of CLL cells, a caspase-8 antibody was utilised. In freshly isolated CLL

cells from 7/7 patients, the antibody recognised two protein(s) of -55 kD (figure 4.6),

most probably corresponding to two different isoforms of caspase-8, MACHal and

MACHa2 (Boldin et al, 1996, Scaffidi et al, 1997). In addition the freshly isolated

cells also contained two immunoreactive proteins of - 43 kD (figure 4.5 A and B, lane

1), which probably resulted from loss of the small 12 kD subunit following cleavage

at Asp 374 (Medema et al, 1997). The cells also contained two immunoreactive

proteins of -28 kD, which may have arisen following removal of the two death

effector domains following cleavage at Asp 216. Culture of the CLL cells resulted in a

time dependent processing of the -55 kD protein(s) to fragments of -43, -28 and a

small amount of an 18 kD fragment (figure 4.5 A, lanes 2-5). Formation of the 18 kD

large subunit most probably occurs following further cleavage of the -43 kD

fragments at Asp 216. Formation of all these fragments was slightly increased

following treatment of CLL cells with chlorambucil (figure 4.5 A, lanes 6-9). The

time course of cleavage of caspase-8 appeared similar to that of caspase-3 and

caspase-7. Some variation in the processing of caspase-8 was noted in the samples

analysed to date. For example in patient 17A, caspase-8 was activated to fragments of

-43 and -28 kD with little or no 18 kD being formed (figure 4.5 B). Thus

interindividual variation was observed in the activation of caspase-8. Further

investigation indicated that this interindividual variation in caspase-8 activation was

apparent in a larger number of cases. Particularly striking in all samples was the

apparently high levels of the proform of caspase-8 and the relatively small amounts of

the proform which were processed during culture.

62


Time (h) 0 2

5 5 k D > 4 3 k D >2 8 k D >

1 8 k D >

Con Chi

6 10 20 2 6 10 20

B + Z-VAD.fmk ( ̂

0 h Con Chi Pd Con Chi Pd

5 5 k D >4 3 k D >

2 8 k D > _______ ________

III1 8 k D >

+ ve

Figure 4.5 Processing of caspase-8 in CLL cells is inhibited by Z-VAD.fmk. (A)

CLL cells from patient 15 A were cultured for the indicated times either alone (Con)

or in the presence of chlorambucil (Chi, 7.5 pM). (B) CLL cells from patient 17 A

were cultured for 20 h either alone (Con) or in the presence of prednisolone (Pd, 20

pM) or chlorambucil (Chi, 7.5 pM) in the absence or presence of Z-VAD.fmk (100

pM) as indicated. Cells from both patients were analyzed for activation of caspase-8

by immunoblotting. Freshly isolated cells (0 h) were included as a control. In both

cases THP.l cells induced to undergo apoptosis by exposure to staurosporine (0.5

pM) were included as a positive control (+ ve) for the processing of caspase-8.

63


4.7 Z-VAD.fmk inhibits the processing of caspases in CLL cells.

In order to determine at what stage of the apoptotic process, Z-VAD.fmk was

inhibiting apoptosis (Table 4.1), its ability to inhibit the processing of different

caspases was examined. Freshly isolated CLL cells from patient 2A contained

primarily the proform of caspase-3 (figure 4.6, lane 2). Spontaneous apoptosis was

accompanied by the processing of caspase-3 to its catalytically active large subunit(s)

(figure 4.6, lane 3), which was increased following treatment with both prednisolone

and chlorambucil (figure 4.6, lanes 4 and 5 respectively), commensurate with their

ability to induce apoptosis. In both spontaneous and drug-induced apoptosis, Z-

VAD.fmk almost completely inhibited the processing of procaspase-3 to its

catalytically active large subunits (figure 4.6, lanes 6-8). Z-VAD.fmk also inhibited

the drug-induced processing of caspase-2 observed in patient 2A (figure 4.3, lanes 7

and 8). In patient 17A, Z-VAD.fmk inhibited the activation/processing of caspase-8 to

a p43 fragment (figure 4.5 B, lanes 5-7). Thus Z-VAD.fmk acts to block drug-induced

apoptosis of CLL cells by blocking the activation/processing of caspases.

64


+ Z - V A D .fm k „ A __________ .r ^

+ ve Oh Con Pd Chi Con Pd Chi

w (mm ■ •|£ 1 1 ^ ^ ‘m m m m m m m m m - -.-■■■ ■■■

LS

Figure 4.6. Processing of caspase-3 in CLL cells is inhibited by the caspase inhibitor,

Z-VAD.fmk. CLL cells from patient 8 I were cultured for 20 h either alone (Con) or in

the presence of prednisolone (Pd, 200 pM) or chlorambucil (Chi, 15 pM) either in the

absence or presence of Z-VAD.fmk (100 pM) as indicated. Freshly isolated cells (0 h)

and THP.l cells treated with etoposide to induce apoptosis were included as controls.

The cells were analyzed for activation of caspase-3 by immunoblotting. The arrows

denote either the 32 kD proform of caspase-3 or the catalytically active large subunits

(LS) of approximately 17-20 kD.

65


4.8 Discussion

In this study it has been demonstrated that induction of apoptosis of CLL cells leads to

the selective induction of some but not all caspases. The processing/activation of at

least three caspases (caspases-3, -7 and -8) has been shown during the execution phase

of apoptosis of CLL lymphocytes whereas caspase-2 does not generally appear to be

activated. Recent studies have implicated a role for the release of mitochondrial

cytochrome c in the activation of procaspase-3 provided Apaf-3, now identified as

caspase-9, and dATP are present (Zou et al, 1997; Li et al, 1997). In all the patients

examined, activation of the effector caspases, -3 and -7, were observed (Table 4.2 and

figures 4.2 and 4.6) suggesting that these caspases may be responsible for the cleavage

of PARP found in CLL cells in this and other studies (Bellosillo et al, 1997; Chandra

et al, 1997). Further support for this hypothesis was provided by the observations that

Z-VAD.fmk, a cell permeable caspase inhibitor, inhibited the activation/processing of

all the caspases studied, as well as the cleavage of PARP and both spontaneous and

drug-induced apoptosis of CLL cells (Table 4.1 and figures 4.2, 4.3, 4.5 and 4.6).

Thus these effector caspases also play a central role in the execution phase of

apoptosis in CLL cells as they do in other cell systems (Cohen, 1997; Nicholson &

Thomberry, 1997).

In 8/9 patients, caspase-2 was not activated (Table 4.2). In only one case was

activation observed (figure 4.3). At the time of sampling this patient was Binet stage

A, but in the last year has progressed to stage C. Activation of caspase-2 during the

execution phase of apoptosis has been observed in different tumor cell lines

(MacFarlane et al, 1997; Li et al, 1997; Harvey et al, 1997). The precise mechanism

by which procaspase-2 is activated in cells is not known. It may involve recruitment

through an adapter such as RAIDD/CRADD (Duan & Dixit, 1997; Ahmad et al,

1997) or based on activities of recombinant enzymes, it has been proposed that

caspase-3 activates pro-caspase-2 (Li et al, 1997; Harvey et al, 1996). No activation of

procaspase-2 was observed even in the presence of activated caspase-3 and caspase-7

demonstrating that neither caspase-3 nor -7 activates procaspase-2 in CLL cells. The

data also demonstrates that activation of caspase-2 is not required for the induction of

apoptosis in CLL cells.

66


The results on the activation/processing of caspase-8 are particularly intriguing.

Triggering of the Fas/CD95 receptor leads to the recruitment and activation of

caspase-8, which may then act as the apical “initiator” caspase responsible for the

activation of other caspases and the execution of apoptosis (Boldin et al, 1996; Muzio

et al, 1996). Whilst caspase-8 is activated early in Fas/CD95-induced apoptosis, little

is known as to its role if any in drug-induced apoptosis. Of particular interest were the

high levels of procaspase-8 present in 7/7 patients (Table 4.2). Procaspase-8 was

present as two main forms of —55 and 53 kD, which correspond to caspase-8/a

(MACHal) and caspase-8/b (MACHa2) (Scaffidi et al, 1997). At the end of the

culture, despite the induction of significant apoptosis (25 - 65 %), most of the caspase-

8 was still present as the proform (figure 4.5), in contrast to Fas/CD95-induced

apoptosis when all the caspase-8 is rapidly processed (Scaffidi et al, 1997). In addition

in 2/4 cases, caspase-8 was activated concurrently with caspases -3 and -7 after 6 to 10

h of in vitro culture. These results would seem to preclude an initiator role for

caspase-8 in drug-induced apoptosis of CLL cells, and suggest that it may be activated

non-specifically by other caspases.

Some cytotoxic agents, such as doxorubicin, induce apoptosis in human leukaemic

cells lines and neuroblastoma cells via the Fas/CD95 receptor/ligand system, a

hypothesis supported by the finding that these cells also display cross-resistance

between the cytotoxic agent and Fas/CD95-induced apoptosis (Freisen et al, 1996;

Fulda et al, 1997; Freisen et al, 1997). However, other studies have suggested that

chemotherapy-induced apoptosis is not dependent on Fas/CD95 receptor/ligand

interaction (Eischen et al, 1997; Gamen et al, 1997). While the reasons for these

discrepancies are not clear, they may be related to the rate of induction of apoptosis.

For example, in those systems where a positive relationship between chemotherapy

and involvement of the Fas/CD95 receptor/ligand system has been implicated (Freisen

et al, 1996; Fulda et al, 1997; Freisen et al, 1997), apoptosis is induced over a long

period of time (24 - 48 h) so allowing the synthesis of new proteins. The possible

involvement of the Fas/CD95 receptor/ligand system in CLL cells remains to be

determined.

67


Interestingly CLL cells have been reported to have undetectable or very low levels of

CD95 expression (Mapara et al, 1993; Moller et al, 1993; Wang et al, 1997). This

expression can be increased following in vitro activation with interleukin-2,

Staphlyococcus aureus 1 or CD40 although in some cases this may lead to

proliferation (Marpara et al, 1993; Wang et al, 1997). Thus CLL cells express high

levels of caspase-8 in conjunction with very low levels of CD95 receptor. These

results highlight the possibility of developing new therapies for CLL based on the

upregulation of CD95- or other death receptors, which would synergize with the high

levels of caspase-8 in CLL cells. Freshly isolated B-CLL cells possess caspase-2, -3, -

7 and -8. Some of these caspases can be activated to cleave protein substrates such as

PARP. These results demonstrate that B-CLL cells possess the complete apoptotic

machinery required to execute the apoptotic programme. Thus the dysregulation of

apoptosis in CLL cells does not appear to be due either to a deletion of pro-caspases or

to point mutations leading to their inactivation. Rather, this study suggests that the

molecular basis of dysregulated apoptosis in vivo may reside in the signalling leading

to the activation of caspases, or the presence of inhibitory proteins at the apoptosis

induction stage.

68

Chapter 5 Results 3

Chapter 5 - Studies on survival factors and the Fas signalling

pathway in B-CLL

5.1 Introduction

Spontaneous apoptosis occurs when B-CLL cells are removed from their normal

environment and placed into in vitro culture (Collins et al, 1989 and section 3.2 of

this thesis). This implies that one or more survival factors needed by the B-CLL cells

are not being provided by the in vitro culture conditions. All previous analysis in this

study had been performed on populations of mixed lymphocytes, both B and T cells.

The T cell fraction can be assumed to be small due to the high proportion of tumour B

cells, but still the presence of T lymphocytes may be reducing the accuracy of the

studies. For experiments investigating the effect of survival factors in chronic

lymphocytic leukaemia it was considered important to be studying a purer population

of what will now be referred to as B-CLL cells. Accordingly, a T cell depletion step

was incorporated into the lymphocyte isolation procedure. Further work in this chapter

describes an investigation into the finding that B-CLL cells have large amounts of

caspase-8 which is not cleaved to any great extent during induction of apoptosis (see

Chapter 4, section 4.5). Since caspase-8 is known to play a major role in the Fas

signalling pathway, the response of B-CLL cells to stimulation of this pathway was

investigated.

5.2 Purified CLL B lymphocytes are more sensitive to apoptosis in in vitro

culture than unpurified populations of total CLL lymphocytes

To further increase specificity when analysing apoptosis sensitivity of CLL cells, it

was decided to purify the B lymphocyte fraction (B-CLL) from the total lymphocytes

obtained from the patients. By incubating a proportion of the isolated lymphocytes

with CD3+ dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s

instructions, depletion of T cells was performed. A determination of the purity of the

pre- and post-depletion fractions was obtained flow cytometrically using antibodies to

CD3 and CD 19 (see Chapter 2, section 2.3.2) (Figure 5.1 A).

69

Chapter 5 Results 3

Blood samples were obtained from six patients and the lymphocyte fraction was

isolated as described in chapter 2. A proportion of these cells were cultured for 24 h

alone or in the presence of chlorambucil (7 pM) or staurosporine (0.2 pM). The

remaining cells from each isolation were further purified by T cell depletion prior to

being cultured for 24 h alone or in the presence of chlorambucil (7 pM) or

staurosporine (0.2 pM). After the 24 h culture period, cells from all of the cultures

were labelled using the Annexin V technique and analysed flow cytometrically. In all

six cases, the level of spontaneous apoptosis in the purified B cell cultures was

slightly increased over that in the total lymphocyte cultures (Figure 5.1 B), which does

indicate that the B-CLL cells may be losing one or more survival stimuli as a result of

the T cell depletion step.

70

Chapter 5 Results 3

WPhIcnQCJ

Total lymphocyte populationo FL1 -Height (3) v s FL2-Height (4)

11.6% -T cell

• - 8 .

. • •83.4%B cells

™l---..........10° 101

Post-T cell depletion

10"

1.3% -T cell

*92%B cells

10° W io2 10J

CD19-FITC

B70

50

WB 602 CLoQ.<w 40 o£ 30 2 § 20

£ 10

\ J

15 A 21A 23A 24A 25APatient number

28A

m B cells ■ Total cells

Figure 5.1 (A) Use of CD3+ magnetic beads enriches for CD 19+ B cells in cultures

of CLL lymphocytes. (B) Purified B cells are more sensitive to spontaneous apoptosis

than cultures of mixed lymphocytes. Lymphocytes were isolated from CLL blood

samples and were split into duplicate cultures. T cells were depleted from one fraction

using CD3+ magnetic beads. Both fractions of cells were cultured for 24 h prior to

being labelled using the Annexin V technique to analyse the level of apoptosis in the

samples.

71

Chapter 5 Results 3

5.3 Culture of B-CLL cells with Interleukin-4 and CD40 stimulation results in a

reduction in spontaneous apoptosis

In order to investigate the requirement of B-CLL cells for stimulation by survival

factors, two B cell growth factors, interleukin-4 and CD40, were incorporated into the

study. Both of these factors had previously been identified as B cell growth stimulants

in normal and malignant cells (Nakanishi et al, 1996; Crawford et al, 1993).

Interleukin-4 has been reported to have anti-apoptotic effects when applied to in vitro

cultures of B cells (Panayiotidis et al, 1993) and acts as an apoptosis inhibitory agent

by maintaining the Bcl-2 expression level in B-CLL cells (Danescu et al, 1992). CD40

stimulation promotes survival by activation of NFkB (Rothe et al, 1995) and protein

tyrosine phosphorylation (Laytragoon-Lewin et al, 1998).

Freshly isolated, purified B-CLL cells from 10 patients were cultured alone or in the

presence of recombinant human interleukin-4 (10 ng/ml). In all instances, the addition

of interleukin-4 reduced the level of spontaneous apoptosis after 24 h of culture

(figure 5.2 A), and increased viability of IL-4-treated cells was maintained to 48 h

(figure 5.2 B). The effect of interleukin-4 on inhibition of spontaneous apoptosis

varied between patient samples, but a reduction in spontaneous apoptosis level was

seen in all of the samples. Addition of a monoclonal antibody directed against CD40

also reduced the level of spontaneous apoptosis in the majority of cases, however, in

the cells from patient 27A, addition of anti-CD40 monoclonal antibody actually

increased the level of spontaneous apoptosis (figure 5.3). Overall, the level of

protection afforded by CD40 stimulation against spontaneous apoptosis was less

dramatic than the effect provided by interleukin-4 stimulation. In order to investigate

the combined effect of these two survival stimuli on B-CLL cell survival, samples of

cells from eight patients were cultured with a combination of interleukin-4 (10 ng/ml)

and anti-CD40 monoclonal antibody (1.5 pg/ml). The level of apoptosis in the

cultures was assessed after 24 h using the Annexin V assay. In cases 23A, 27A, 29A

and 31 A, anti-CD40 increased the protection provided by interleukin-4, in the

remaining 4 cases, anti-CD40 abrogated the protective effect of interleukin-4 (figure

5.3). This trend may indicate a variable dependency on survival factors between

patients.

72

Chapter 5 Results 3

A

15A 23A 27A 28A 29A 30A 31A 32A

Patient Number

a Control B 10ng/ml IL4

B

100

= 60 B .2> /in

20

0 4 ------Oh 24 h 48 h

Time (h)

ControlIL4

Figure 5.2 Interleukin-4 inhibits the induction of spontaneous apoptosis in purified B-

CLL cell cultures. (A) Freshly purified cells were cultured for 24 h alone (control) or

in the presence of 10 ng/ml IL-4. Apoptosis was assessed using the Annexin V assay

to measure externalised phosphatidylserine. (B) B-CLL cells were cultured alone or

in the presence of IL-4 (10 ng/ml) for 48 h. Samples (1 x 106 cells) were taken from

each culture at 24 and 48 h following culture setup, viability was assessed by

propidium iodide exclusion. Results plotted are the mean of six experiments. Standard

error bars are also depicted.

73

Chapter 5 Results 3

70

60

50_co

23A 24A 27A 28A 29A 30A 31A 32A

Patient Number

□ Control ■ IL4H anti-CD40 m anti-CD40 + IL4

Figure 5.3 The effect of stimulation of B-CLL cells with interleukin-4 and antibodies

to CD40 is case dependent. B-CLL cells were cultured for 24 h alone or in the

presence of IL-4 (10 ng/ml), anti-CD40 monoclonal antibody (1.5 pg/ml) or both

stimuli. Following the culture period, 1 x 106 cells from each culture were analysed

for apoptosis level using the Annexin V assay.

74

Chapter 5 Results 3

5.3 Culture of B-CLL cells with Interleukin-4 or CD40 stimulation increases

their resistance to chemotherapeutic drugs

In order to determine the level of protection afforded by interleukin-4 against

apoptosis induced by chemotherapeutic drugs, B-CLL cells from patients 28A and 29

A were pre-incubated for 24 h alone (control) or in the presence of interleukin-4 (10

ng/ml). After 24 h pre-incubation, chlorambucil (7 pM) or staurosporine (0.2 pM)

were added and the cells were cultured for a further 24 h. The protective effect of

interleukin-4 against drug-induced apoptosis was then analysed by assessing the

extent of apoptosis in these cultures compared to apoptosis levels in cultures of cells

incubated for 24 h with chlorambucil or staurosporine alone. In both cases the

addition of interleukin-4 to the cells reduced the level of spontaneous apoptosis. The

level of apoptosis induced by chlorambucil and staurosporine was also reduced in the

cells pre-incubated with interleukin-4, compared to the levels in cultures after 24 h

incubation with the drugs alone. In cells from patient 28 A, the protection from drug-

induced apoptosis was no more than 7%. However, in cells from patient 29A, the

level of apoptosis in the cultures pre-incubated with interleukin-4 were reduced by as

much as 22% (figure 5.4). This finding, although concerned with a limited sample of

B-CLL patients, again indicates that B-CLL cells from different patients vary in their

level of dependence on survival factors, and also demonstrates the important role that

interleukin-4 may play in maintaining survival of the malignant lymphocyte clone and

protecting the cells from chemotherapeutic drug-induced apoptosis in vivo.

In order to determine the protection from drug-induced apoptosis afforded by a

combination of CD40 and interleukin-4 stimulation, B-CLL cells from three patients

were cultured for 24 h in the presence of anti-CD40 mAb (1.5 pg/ml) and IL-4 (10

ng/ml). Following this pre-incubation period, chlorambucil (7 pM) was added to the

cultures. The cells were incubated for a further 24 h, prior to assessment of apoptosis

levels in the cultures by Annexin V labelling. Control cultures were also analysed,

where the cells had been incubated for the full 48 h in the presence of IL-4 and anti-

CD40, without the addition of chlorambucil.

75

Chapter 5 Results 3

Spontaneous apoptosis was inhibited in all the three patient samples analysed, and

chlorambucil-induced apoptosis was also inhibited in the samples which had been pre-

incubated with IL-4 and anti-CD40 (Figure 5.5 A). The average protective effect of

IL-4 + anti-CD40 against spontaneous apoptosis was 21.2%, and the average

protection against chlorambucil-induced apoptosis was 26.8% (Figure 5.5 B).

However, there was wide variation between the patient samples as to the effectiveness

of IL-4 and CD40 stimulation. Cells from patients 21A and 27A were highly receptive

to the protective effects of IL-4 and CD40 against chlorambucil-induced apoptosis,

whereas the cells from patient 24A were not protected to such a great extent (Figure

5.5 A). The extent of protection against chlorambucil-induced apoptosis afforded by

the two stimuli was not always to the same degree as the protective effect against

spontaneous apoptosis. Cells from patient 21A were protected to a similar degree

from spontaneous and chlorambucil-induced apoptosis, cells from patient 24A were

protected to a greater degree from spontaneous apoptosis than from chlorambucil-

induced apoptosis, and, interestingly, cells from patient 27A were protected to a

greater extent against chlorambucil-induced apoptosis than against spontaneous

apoptosis (figure 5.5 A). This again underlines the wide variability in survival factor

dependency between individual B-CLL cases, a dependency which may also have

implications in determining the response of different patients to chemotherapy.

76

Chapter 5 Results 3

A

Patient No. Control IL-4 + CD40 Chi IL-4 + CD40

+ Chi

21 A 39 42 57.9 16.4

24 A 62 43.5 84.7 79.8

27 A 14.1 3.4 72.7 38.7

B

90

Control CD40+IL4 CHL CD40+IL4+CHL

Figure 5.5 Stimulation of B-CLL cells with anti-CD40 and interleukin-4 protects against apoptosis

induced by chlorambucil. B-CLL cells from three patients were cultured for 24 h alone (Control), or in

the presence of chlorambucil (7 pM) (CHL), prior to assessment of apoptosis using the Annexin V

assay. Identical cultures of cells from the same patients were pre-incubated for 24 h in the presence of

anti-CD40 monoclonal antibody (1.5 pg/ml) and interleukin-4 (10 ng/ml) (CD40 + IL-4) prior to

chlorambucil (7 pM) being added (CD40 + IL-4 + CHL) and the cells being cultured for a further 24 h.

A. Table of results obtained from each patient analysed in this way. All numerical values are percentage

apoptosis. B. Plotted mean results from the three patients. Standard error bars are also depicted.

77

Chapter 5 Results 3

5.4 B-CLL cells are not sensitive to Fas-induced apoptosis

High levels of the initator caspase, caspase-8 had previously been observed in B-CLL

cells (see chapter 4, section 4.5). Caspase-8 cleavage in response to induction of

apoptosis by chlorambucil was also observed (figure 4.5 A, lanes 7-9 and 4.5 B, lane

4). The role of caspase-8 activation in drug-induced apoptosis is unclear, but in

proportion to the amount of caspase-8 pro-form present in these cells, there appeared

to be little activation taking place as a result of treating the cells with

chemotherapeutic drugs (figure 4.5). Since caspase-8 is known to play a major role in

the Fas signalling pathway, the response of B-CLL cells to stimulation of this pathway

using an anti-Fas (IgM) monoclonal antibody was investigated.

B-CLL cells isolated from eight patients were cultured alone or in the presence of

anti-Fas monoclonal antibody (clone CH11, 0.5 pg/ml) for 24 h. Following the

incubation period, 1 x 106 cells from each culture were labelled using the Annexin V

assay, and analysed flow cytometrically (figure 5.6). Fas stimulation induced

apoptosis above the level of spontaneous apoptosis in only two instances (patients

22A and 29A). In the other six cases addition of anti-Fas monoclonal antibody

inhibited the induction of spontaneous apoptosis. This finding mirrors that of

Laytragoon-Lewin and co-workers (1998) who also determined that B-CLL cells were

generally not sensitive to Fas stimulation. Further studies were initiated in order to

determine the nature of Fas resistance in B-CLL cells.

78

Chapter 5 Results 3

</)8Q.O*

22A 23A 24A 25A 26A 27A 28A 29A

Patient Number

■ Control 24 h■ Fas 24 h

Figure 5.6 B-CLL cells are resistant to apoptosis induced by anti-Fas monoclonal

antibody. B-CLL cells were cultured for 24 h alone or in the presence of anti-Fas mAb

(0.5 pg/ml). Analysis of apoptosis levels in the cultures was made using the Annexin

V assay.

79

Chapter 5 Results 3

5.5 Upregulation of Fas receptor on B-CLL cells does not increase sensitivity to

apoptosis induced by Fas ligation.

Previous reports have confirmed the low density of expression of the Fas receptor on

B-CLL cells (Mainou-Fowler et al, 1995). Freshly isolated B-CLL cells were labelled

with anti-Fas monoclonal antibody and a FITC-conjugated secondary antibody, as

described in Chapter 2, and analysed flow cytometrically for the level of expression of

Fas receptor. Previous reports had demonstrated that Fas receptor levels on B-CLL

cells could be upregulated by culturing the cells with either CD40 or interleukin-2 and

Staphylococcus aureus Cowan I (Wang et al, 1997). To induce upregulated expression

of Fas receptor, B-CLL cells were cultured for 24 h in the presence of anti-CD40

monoclonal antibody (1.5 pg/ml) alone or in combination with interleukin-4 (10

ng/ml). Since culturing B-CLL cells for 24 h (with or without CD40 stimulation)

results in induction of apoptosis (see figure 5.3), interleukin-4 was added to the

cultures in order to inhibit apoptosis to a level from which analysis of Fas-induced

apoptosis could be made following on from the initial 24 h culture to upregulate Fas

receptor expression. The level of Fas receptor on the freshly isolated cells was

compared with the level of Fas receptor on the CD40 +/- IL-4 -stimulated CLL cells

(figure 5.7 A). For all five patient samples, the level of expression of Fas receptor

increased following stimulation of the cells with CD40, compared to the level of Fas

receptor on the freshly isolated cells (figure 5.7 B). The addition of interleukin-4 did

not appear to alter the effectiveness of CD40-induced Fas receptor upregulation

(figure 5.7 B).

80

Chapter 5 Results 3

A.

Control IgM (MFI = 2.3)

/ Freshly isolated B-CLL cells (MFI = 8.2)

B-CLL cells + anti-CD40 mAb (24 h) ^(M FI = 27.2)

Log green fluorescence (FITC)

B.

21A 23A 24A 27A 29A 30A 31A 32A Patient Number

■ Fresh cells @ CD40 24h■ CD40 + IL4 24h

Figure 5.7 Fas receptor expression is elevated on B-CLL cells following stimulation with antibodies to

CD40 and/or interleukin-4. A. B-CLL cells (from patient 30A) show increased Fas receptor expression

following 24h incubation with anti-CD40 mAb (0.5 fig/ml) when compared with the level of expression

on the freshly isolated cells. B. Culture of B-CLL cells with anti-CD40 alone or in combination with

interleukin-4 results in upregulation of Fas receptor expression. Freshly isolated B-CLL cells were

labelled with anti-Fas monoclonal antibody (as described in Chapter 2, section 2.11) and analysed flow

cytometrically for the amount of expression of the cell surface receptor, Fas. Subsequently, cells from

the same patients were cultured for 24 h in the presence of anti-CD40 monoclonal antibody alone or

with interleukin-4 (10 ng/ml) added, before analysis of Fas receptor expression was made again.

81

Chapter 5 Results 3

To determine if the increased expression of Fas receptor on the B-CLL cells would

confer increased sensitivity to Fas-induced apoptosis, B-CLL cells from three patients

which had been cultured with anti-CD40 (1.5 pg/ml) and IL-4 (10 ng/ml) for a period

of 24 h to enable upregulation of Fas receptor expression as described above, were

exposed to an anti-Fas monoclonal antibody (clone CH-11, 0.5 pg/ml). Following a

further 24 h culture period, the level of apoptosis in the cultures was analysed using

the Annexin V assay. From the three cases analysed using this method, the level of

apoptosis induced by anti-Fas was not increased over that in the control (anti-CD40 +

IL-4 alone) cultures (figure 5.8). To check that the addition of interleukin-4 to the

cultures was not inducing Fas resistance, cells from patient 27A were cultured for 24 h

with anti-CD40 alone prior to addition of anti-Fas and a further 24 h culture period. At

48 h, the level of apoptosis in the CD40 alone culture was 59.7%, and the level of

apoptosis in the culture pre-incubated with anti-CD40 and with anti-Fas added for the

second 24 h, was 57.1%. This analysis demonstrated that CD40 stimulation alone,

whilst effectively upregulating Fas receptor, was not conferring Fas sensitivity on the

B-CLL cells. It also demonstrated that the addition of interleukin-4 to the cultures for

the first 24 h was not contributing to this resistance to Fas-induced apoptosis.

82

Chapter 5 Results 3

91©ao

70Patient 21 A60

50

40

30

20

10

00 12 24 36 48

Time (h)

Patient 27 A60

•23 5 0912 40 ag . 30

20

10

00 12 3624 48

Time (h)

70 T60 .. Patient 29 A

Time (h)

Control

CD95

A — CD40 + IL4

CD40+ IL4 + Fas

Figure 5.8 Upregulation of Fas receptor on B-CLL cells does not increase their

sensitivity to apoptosis induced by Fas stimulation. B-CLL cells from three patients

were cultured for 24 h in the presence of anti-CD40 monoclonal antibody (1.5 fig/ml)

and IL-4 (10 ng/ml). Subsequently, anti-Fas antibody was added (CH11, 0.5 pg/ml).

The cells were cultured for a further 24 h, before the extent of phosphatidylserine

extemalisation in the cultures was assessed using the Annexin V assay. Results were

compared against samples of cells stimulated for the 48 h period with anti-CD40 + IL-

4 alone, control cells which had received no stimulation, and cells which had received

stimulation from anti-Fas antibody for 48 h.

83

Chapter 5 Results 3

80 n70 -

co 60 ■ ® 50

O 40 -Q_< 30 -

20 -

120 24

Time (h)

• — Control ■•—Fas tIc—CD40 ■ m - CD40 + Fas

Figure 5.9 Stimulation of B-CLL cells with CD40 does not confer sensitivity to Fas-

induced apoptosis. Cells from patient 27A were cultured for 24 h in the presence of

anti-CD40 monoclonal antibody (1.5 pg/ml). Fas receptor upregulation was checked

flow cytometrically (see figure 5.7B), anti-Fas monoclonal antibody (clone CHI 1, 0.5

pg/ml) was added and the cells were cultured for a further 24 h (CD40 + Fas).

Samples of cells from the same patient were cultured for 48 h alone (Control), with

Fas stimulation alone (Fas) or with CD40 stimulation alone (CD40). The apoptosis

level at 48 h in each of the cultures was assessed using the Annexin V assay to

measure externalised phosphatidylserine.

84

Chapter 5 Results 3

5.7 B-CLL cells do not overexpress the caspase-8 inhibitory protein, c-FLIP

Several proteins have been identified which can inhibit Fas-induced apoptosis by

blocking the interaction of caspase-8 with the death effector domain (DED) of the

adapter molecule FADD. One such protein is c-FLIP. c-FLIP is present in mammalian

cells as two isoforms, c-FL IP l (-55 kD) and c-FLIPs (—33 kD) (Irmler et al, 1997;

Rasper et al, 1998). High levels of c-FLIP have been detected in some melanoma

tumours, and c-FLIP is predominantly expressed in lymphoid and muscle tissue

(Irmler et al, 1997), indicating that this protein may play a role in regulating apoptosis

of lymphoid malignancies.

The following experiments were performed in order to determine the relative

expression of c-FLIP in B-CLL cells, compared to the expression levels of caspase-8,

and to examine any alterations in expression as the cells were exposed to

chemotherapeutic drugs and Fas stimulation. B-CLL cells from patients 21 A, 31A and

32A which had been cultured alone or in the presence of chlorambucil (7 pM),

interleukin-4 (10 ng/ml) or anti-CD40 (1.5 pg/ml) were labelled using the Annexin V

method, and analysed flow cytometrically to record the level of apoptosis in the

cultures (figure 5.9 A). Samples of cells from these cultures were examined using

immunoblotting for the presence of the caspase-8 inhibitory protein c-FLIP (figure 5.9

B). Blots were subsequently stripped, and re-probed with anti-caspase-8 polyclonal

antibody (figure 5.9 C), allowing a comparison of the relative levels of c-FLIP and

caspase-8 to be made. The immunoblot results demonstrate that B-CLL cells do

express c-FLIP and caspase-8, but the level of expression of c-FLIP does not appear to

be increased over that of caspase-8. This implies that c-FLIP overexpression is not

responsible for the apoptotic block in Fas-induced apoptosis in B-CLL cells. Upon

examination of the immunoblot results, it was noted that in one case (21 A)

chlorambucil had induced clipping of the short form of c-FLIP (figure 5.9 B, lane 3),

which occured concurrently with activation of caspase-8 (figure 5.9 C, lane 3). The

positive control lane for this experiment was THP-1 monocytic cells treated for 4 h

with anti-Fas monoclonal antibody. In the positive control lane, c-FLIP is also

cleaved. This phenomenon may represent a caspase dependent cleavage of c-FLIP.

85

APatient 21A Patient 31A Patient 32A

t

55 k D -^ '« * 4 3 k D -> »

28 k D -> *

18kD->- —

Figure 5.7 B-CLL cells do not overexpress the caspase-8 inhibitory protein c-FLIP. Freshly isolated B-CLL cells (1) from patients 21 A, 31A and 32 A were cultured in vitro alone (2) or in the presence of chlorambucil (7 J»M) (3), interleukin-4 (10 ng/ml) (4) or anti-CD40 monoclonal antibody (1.5 jpg/ml) (5). (A) The level of apoptosis in the cultures was assessed after 24 h using the Annexin V assay. (B) Cells from the cultures were analysed by immunoblotting for the presence of c-FLIP. (C) The blots were stripped and re-probed with an antibody against caspase-8.

Chapter 5 Results 3

5.6 CLL cells have all the necessary components to form a death inducing

signalling complex (DISC) upon Fas ligation but do not assemble a DISC upon

Fas stimulation

The formation of a death-inducing signalling complex (DISC) in response to Fas

ligation on Jurkat cells was reported in 1997 (Medema et al). The DISC consists of

the intracellular ‘death domains’ (DD) of the trimerised Fas receptors, the adapter

molecule FADD, which binds to the Fas receptor DD through its own death domain.

FADD also consists of a region called a ‘death effector domain’ (DED) which recruits

caspase-8 to the DISC. Cells which respond to Fas stimulation by assembling a DISC

can be split into two categories. Type I cells assemble the DISC in a matter of

seconds, whilst type II cells take 15-30 minutes to form a DISC and activate

downstream caspases such as caspase-3 (Scaffidi et al, 1998). In order to investigate

the possible reasons for the resistance of B-CLL cells to Fas-mediated apoptosis,

immunoblotting for FADD was performed on freshly isolated and cultured B-CLL

cells (figure 5.10), the presence of caspase-8 already having been confirmed in these

cells (figures 4.5 and 5.9). The expression level of FADD appeared to increase

slightly after the culture period of 24 h, but no significant alterations in expression of

FADD were seen as a result of culturing the cells with chlorambucil, anti-Fas or anti-

CD40 monoclonal antibodies or interleukin-4 (figure 5.10). In the cells from patient

22 A, there is evidence of a slight decrease in FADD expression following treatment

of the cells with staurosporine (figure 5.10 A, lane 4), which corresponded with

induction of a high level of apoptosis and activation of caspase-8 (figure 5.10 B, lane

4). In samples from patient 22 A, FADD appeared as a single band at approximately

26 kD. In all other patient samples, FADD appeared as a doublet band (Figure 5.10 C

and D), which may correspond to different FADD isoforms, or serine phosphorylation

states (Xerri et al, 1999).

SKW6.4 (murine B cells, Type I) and Jurkat (human T cells, Type II) cell lines were

used as positive controls for DISC formation and isolation. Using the method of Peter

and co-workers (1998) as described in chapter 2, section 2.12, SKW6.4 and Jurkat

cells were stimulated with anti-Fas monoclonal antibody (anti-Apo-1, IgG) for 15

minutes.

87

Chapter 5 Results 3

24 h B ? 4 h

f ^ Oh Con Chi STS FasO h C o n C hi STS Fas

55/53 * ■ * mm mmF A D D > — — - A - — 4 3 k D > mm~ 26 kD 28 kD >■

18 kD >

7.3 13.9 11.8 67.3 27.4 % A po

24 hThp-1+ STS

O h rCon C hi IL 4 C D 40 |

FADD > 9 ~ 26 kD

DPatient Number

Single or double band corresponding to FADD

15 A21 A22 A28 A29 A31 A32 A

DoubleDoubleSingle

DoubleDoubleDoubleDouble

Figure 5.10 B-CLL cells express the adapter protein, FADD. (A) Freshly isolated B-CLL cells from

patient 22 A, and those cultured for 24 h alone (Con) or with chlorambucil (Chi, 7 pM), staurosporine

(STS, 0.2 pM), or anti-Fas monoclonal antibody (Fas, 0.5 pg/ml) as indicated, were run on a 4-12%

gradient polyacrylamide gel, blotted onto nitrocellulose filter and probed with a monoclonal antibody

directed against the DISC adapter molecule, FADD. In patient 22 A, FADD appears as a single band at

approximately 26 kD. (B) Samples of cells from the same patient were run on a gel, blotted and probed

with a polyclonal antibody to caspase-8, as described previously. The percentage apoptosis level in the

cultures is indicated below the blot. (C) Cells from patient 32 A, were cultured alone (Con), or with

chlorambucil (Chi, 7 pM), interleukin-4 (IL4, 10 ng/ml) or anti-CD40 monoclonal antibody (CD40, 1.5

pg/ml), prior to being run on a 12 % polyacrylamide gel, blotted onto nitrocellulose and probed with a

monoclonal antibody directed against FADD. In this patient, FADD appears as a doublet band. (D)

Table showing FADD expression in all 7 cases analysed. FADD appears as a doublet band in the

majority of cases.

88

Chapter 5 Results 3

Samples o f stimulated and unstimulated (control) cells were lysed, and the same

concentration of anti-Fas antibody used for stimulation was added to the control

samples as a negative control. The cells were incubated with Protein A-Sepharose to

immunoprecipitate any complexes which may have formed as a result of stimulating

the Fas receptor with an IgG anti-Apo-1 monoclonal antibody. Immunoblotting of

immunoprecipitates of anti-Fas stimulated cells showed a band corresponding to

FADD (~26 kD), in the lane containing stimulated SKW6.4 cells, which was absent in

the lane containing stimulated Jurkat cells (figure 5.12A, lanes 1 & 3). This

demonstrated that FADD had been recruited to a DISC within 15 minutes in SKW6.4

cells, but not in Jurkat cells. The negative control lanes (lysate supplemented samples,

figure 5.12A, lanes 2 & 4) showed higher levels of FADD than was evident in the

lanes containing stimulated cells. This was most likely due to inefficient cell lysis

leaving functional, membrane bound Fas receptor in the lysate which could be

stimulated to form DISCs. FADD molecules which are also freely available in the

lysate appear to bind more effectively in this situation.

In order to investigate whether or not Fas-stimulated B-CLL cells were capable of

forming a DISC, similar experiments on B-CLL cells were performed which included

SKW6.4 cells as a positive control. B-CLL cells from patients 31A and 32A were

cultured for 24 h in the presence of anti-CD40 in order to upregulate Fas receptor

expression, which was checked flow cytometrically. The cells were subsequently

treated with anti-Fas monoclonal antibody (anti-Apo-1, IgG) for 60 minutes.

Following lysis of the cells the anti-Fas bound complex was immunoprecipitated

using Protein A-Sepharose, subsequently the DISC complex was immunoblotted for

FADD and caspase-8.

SKW6.4 cells assembled FADD to the DISC after only 15 minutes (figure 5.12A),

however, after stimulation with anti-Fas monoclonal antibody for 60 minutes, B-CLL

cells did not show FADD binding to a DISC (figure 5.12B). The blots were stripped

and re-probed with anti-caspase-8 polyclonal antibody, however, due to the presence

of a strong band on the blots corresponding to protein A (~42 kD), the presence of

caspase-8 proform and activated fragments (55 and 43 kD) could not be established

(figure 5.13).

89

Chapter 5 Results 3

c lCL3C/3

S 3 B• ^ C/3 \ g■+T* IX rr>

o.D.3coSC3

£C /3

&i4C /D

c313

c3•i3

(26 kD)

B

C3-j£

a.a.3

S '• - P

C/3

&C3 CO

c CO3 V ©

£ £ £>4 *C /3 C /3 C /3

JVCQ

I3

JuCQ

< IgGI g G >

E .gVP to3 -<1-3 M5£ £*C /1 c / 3

S

13 t ohJ_lu o

CQ CQ

< - FADD (26 kD) - > « * » * *

Patient 32 A Patient 31 A

Figure 5.12. Immunoblotting for FADD on immunoprecipitates of Fas-stimulated cells. (A) SKW6.4

and Jurkat cells were stimulated for 15 minutes with anti-Fas monoclonal antibody (anti-Apo-1, IgG).

Samples of stimulated and unstimulated cells were lysed, and anti-Apo-1 was added to the unstimulated

lysates as a control. The protein complexes formed by Fas stimulation were immunoprecipitated using

Protein-A-Sepharose. The lysates were run on a 4-12% gradient SDS-polyacrylamide gel, and

transferred onto a nitrocellulose filter. The filter was probed with a monoclonal antibody directed

against FADD. (B). B-CLL cells from patients 31A and 32A were stimulated for 60 minutes with anti-

Fas (anti-Apo-1, IgG). Lysates from stimulated and unstimulated cells, along with samples of SKW6.4

cells treated as above, were run on 4-12% polyacrylamide gels, transferred onto nitrocellulose filters

and probed with anti-FADD monoclonal antibody.

IgG

FADD

90

Chapter 5 Results 3

a.a.

55 kD ^ " 43 k D -> »

*

FADD (26 k D )> -. 18 k D >

ts*3i—> JUo

3C/5 • Eg M d on'■&a

c3C/5

C/5on

C3

3 VO J£ £ _ )

VJVu i t

C/5 C/5 C/5 0Q 0Q

Figure 5.13 Immunoblotting for caspase-8 on lysates of anti-Fas stimulated cells.

Cells from patient 32A were stimulated with anti-Apo-1 for 60 minutes, lysed and

incubated with Protein-A-Sepharose for 90 minutes. Samples of stimulated and

unstimulated B-CLL cells were run on a 4 -12 % polyacrylamide gel along with

samples of stimulated (15 minutes), unstimulated and lysate supplemented samples of

SKW6.4 cells. The proteins were transferred onto a nitrocellulose filter, which was

probed with a polyclonal antibody to caspase-8 (as described previously).

91

Chapter 5 Results 3

5.7 Discussion

5.7.1 Survival factors in B-CLL

In order to increase specificity when analysing apoptosis in CLL, it was decided to

purify B-CLL cells from the total lymphocyte fraction, as had been used in previous

experiments. B-CLL patients have a high proportion of tumour B cells in their

peripheral blood compared with the ratio of T to B cells in normal blood. The patients

sampled in this study had white blood cell counts ranging from 3.7 to 68 x 109/L, the

average white cell count being 28 x 109/L. In cases such as these the T cell fraction

can be assumed to be minimal, but nevertheless still present. For studies into the

effect of growth factors of B-CLL cells, the presence of T cells may provide a

contaminating source of survival stimuli and so removal of this fraction was deemed

necessary. Accordingly, a T cell depletion step was incorporated into the B-CLL cell

purification procedure. In order to assess the degree to which T cell-mediated stimuli

could influence B-CLL cell survival, a series of comparitive experiments were

performed. In all six of the cases analysed, the level of spontaneous apoptosis was

elevated in the purer B-CLL culture compared to the culture containing B and T cells

(Figure 5.1 B). The difference in sensitivity was significant in only one case, patient

24A, where the B-CLL cells were 21.9% more sensitive to spontaneous apoptosis than

cells in the unpurified culture. In the remaining cases the difference was slight (4.6%

average), but still demonstrates the effect that T cells may have in stimulating survival

of B-CLL cells.

The effects on cell growth and apoptosis of growth factor additions to cultures of CLL

cells has been investigated by a number of groups, (DeFrance et al, 1991, Mainou-

Fowler et al, 1995) but information regarding variable requirements between cases is

limited. In order to investigate the requirement of B-CLL cells for stimulation by

survival factors, the effects of two B cell growth factors, interleukin-4 and CD40,

were studied. Both of these factors had previously been identified as B cell growth

stimulants in normal and malignant cells (Nakanishi et al, 1996; Crawford et al,

1993).

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Chapter 5 Results 3

Interleukin-4 had previously been reported to have anti-apoptotic effects when applied

to in vitro cultures o f B cells (Panayiotidis et al, 1993) and so the ability of

interleukin-4 to inhibit spontaneous apoptosis of B-CLL cells was investigated.

Interleukin-4 inhibited spontaneous apoptosis in all of the patient samples analysed,

and could significantly improve the survival of the cells (Figure 5.2A and B). The

extent of inhibition of spontaneous apoptosis ranged from 5% to 53.8%, indicating

that B-CLL cells vary in their dependency on interleukin-4 for protection against

apoptosis induction. In order to investigate this further, it would have been interesting

to monitor the Bcl-2 expression levels in these cells before and after culture with

interleukin-4, since this is one of the ways in which IL-4 might protect cells from

apoptosis (Danescu et al, 1992).

An inter-patient variability in sensitivity to interleukin-4 stimulation has been

described above. It was of interest, therefore, to discover if IL-4 sensitive B-CLL cells

could be equally as sensitive to a second survival stimulus, and to determine whether

an additional survival advantage could be provided by stimulation of the cells with a

combination of growth factors. A second B cell survival factor is CD40. CD40

stimulation promotes survival by activation of the anti-apoptotic transcription factor,

NFkP (Rothe et al, 1995) and protein tyrosine phosphorylation (Laytragoon-Lewin et

al, 1998). In this study, samples of B-CLL cells from eight patients were cultured in

the presence of interleukin-4 and a monoclonal antibody to CD40. This culture system

for delivery of the CD40 stimulation was as described by Dive et al, 1998. This

analysis revealed that B-CLL cells were less sensitive to CD40 stimulation than to

interleukin-4 stimulation (Figure 5.3). In fact, in one of the patient samples analysed,

addition of anti-CD40 monoclonal antibody induced apoptosis above the level in the

control culture, a phenomenon which had been reported previously (Wang et al,

1997). Stimulation of B-CLL cells with a combination of interleukin-4 and CD40

stimulation revealed further inter-patient variability. In 50% of the samples, the

combination of survival factors reduced the level of apoptosis below that resulting

from stimulation of the cells with interleukin-4 alone. In the remaining 50% of

samples, the combination of stimuli was less effective at inhibiting spontaneous

apoptosis than interleukin-4 alone.

93

Chapter 5 Results 3

Since B-CLL cells appear to vary in their sensitivity to inhibition of spontaneous

apoptosis by growth factors, it would be interesting to discover if these stimuli could

also promote resistance of the cells to apoptosis induced by chemotherapeutic drugs.

The level of apoptosis in drug-treated cultures pre-incubated with IL-4 was less than

the level of apoptosis in the cultures incubated with the drugs alone (Figure 5.4).

Samples of cells from two patients were analysed for the protective effect of

interleukin-4 against drug-induced apoptosis, and the cells from the two patients

differed in the degree of protection afforded by interleukin-4. A combination of

interleukin-4 and CD40 stimulation also inhibited induction of apoptosis by

chlorambucil (Figure 5.5). Again, the response differed between cells from the three

patient samples analysed, the percentage reduction in apoptosis level in the survival

factor pre-incubated samples, compared to the cultures incubated with the drugs alone,

ranging from 4.9% to 41.5%. Because this reduction in apoptotic rate might be due to

inhibition of spontaneous apoptosis, and not due to inhibition of drug-induced

apoptosis, samples from the same patients were cultured alone and with the survival

stimuli alone as controls. Results from one patient (27A) showed that IL-4 and CD40

stimulation had inhibited drug-induced apoptosis to a much greater extent than

spontaneous apoptosis, indicating that the survival factors were affecting the cells’

response to chemotherapeutic drugs. Of the remaining two cases, one showed the

reverse pattern, indicating that in this patient’s cells the survival stimuli were only

effective in reducing spontaneous apoptosis, and in the third case, spontaneous and

drug-induced apoptosis were inhibited to roughly the same extent.

These findings demonstrate a wide inter-patient variability in dependence of the B-

CLL cells on stimulation from survival stimuli. Spontaneous apoptosis and

chemotherapeutic drug-induced apoptosis can be inhibited by culturing B-CLL cells

with interleukin-4 and/or CD40 stimulation. This raises the question of how relevant

these factors are in determining apoptosis sensitivity in vivo. B-CLL patients have

already been shown to have elevated levels of CD40 ligand in their serum (Younes et

al, 1998), and this, in conjunction with a normal level of expression of CD40

receptors on the CLL cells (Laytragoon-Lewin et al, 1998), could be one mechanism

in which B-CLL cells escape apoptotic cell death. B-CLL cells have also been shown

to express normal levels of the interleukin-4 receptor (Gileece et al, 1993), and so the

94

Chapter 5 Results 3

levels of interleukm-4 in the peripheral blood of B-CLL patients could also be of great

importance. In relation to this, it has been shown that T cells derived from B-CLL

patients have increased levels of cytoplasmic IL-4 compared to normal control T cells

(Mu et al, 1997), although whether or not this IL-4 was secreted was not determined.

Further studies could be performed, possibly using ELISA-based assays, which would

determine whether or not this IL-4 is secreted by the B-CLL T cells, and whether or

not B-CLL patients have normal or elevated levels of serum interleukin-4.

5.7.2 Investigations into the Fas signalling pathway in B-CLL cells

Previous experiments in this study had demonstrated that B-CLL cells have caspase-8

proform present in abundance. However, caspase-8 does not seem to be cleaved to any

great extent during drug-induced apoptosis (Figure 4.5). Caspase-8 is the apical

caspase in the signalling pathway induced following stimulation of the Fas receptor.

B-CLL cells are known to express reduced levels of Fas receptor, and this, in

conjunction with high levels of inactive caspase-8 may be one mechanism whereby B-

CLL cells are resistant to apoptosis. Investigations into the Fas signalling pathway in

B-CLL cells began with a series of experiments to determine whether the cells could

be triggered into apoptosis via Fas signalling. In only 2/8 patients did Fas stimulation

induce apoptosis above the level of spontaneous apoptosis. In the remaining cases, Fas

stimulation inhibited spontaneous apoptosis. One reason for the resistance of B-CLL

cells to Fas-induced apoptosis may be the low level of expression of Fas receptor on

the cells’ surface (Mainou-Fowler et al, 1995; Wang et al, 1997). There are a number

of ways in which Fas receptor expression can be upregulated on B cells. Tonsillar B

cells and Burkitt’s Lymphoma cells can be induced to upregulate Fas receptor

expression by CD40 stimulation (Garrone et al, 1995; Scattner et al, 1995), and B-

CLL cells have been shown to upregulate Fas receptor upon stimulation with a

combination of interleukin-2 and a mitogenic stimulus such as Staphylococcus Aureus

Cowan I (Mapara et al, 1993). In all of these studies the cells became sensitive to Fas

stimulation following upregulation of receptor levels. In this study, B-CLL cells were

cultured for 24 h with a monoclonal antibody to CD40 in order to upregulate Fas

receptor expression. Comparison of the level of Fas receptor on freshly isolated B-

CLL cells with the levels on CD40 stimulated cells demonstrated that CD40

95

Chapter 5 Results 3

stimulation was indeed inducing increased expression of Fas receptor on these cells

(Figure 5.6).

The aim of upregulating Fas receptor levels was to analyse the functionality of the Fas

signalling pathway in B-CLL cells. Following 24 h pre-incubation with CD40

stimulation, the cells were cultured for a further 24 h with anti-Fas monoclonal

antibody in order to assess Fas sensitivity. However, by the time the 48 h time point

was reached, the level of spontaneous apoptosis in the control cultures was very high,

as was the level of apoptosis in the CD40-stimulated samples. This meant that

examination of the Fas signalling pathway would be hampered by a high baseline

level of apoptosis. In order to promote survival of the B-CLL cells over the first 24 h

culture period, interleukin-4 was added to the cultures. The combination of CD40 and

interleukin-4 stimulation also resulted in upregulation of Fas receptor, but successfully

inhibited the induction of high levels of spontaneous apoptosis making analysis of

Fas-induced apoptosis possible.

To determine whether or not the upregulated Fas receptor was functional, cells which

had been cultured with CD40 and interleukin-4 for 24 h were stimulated with anti-Fas

monoclonal antibody for a further 24 h. After this time, apoptosis levels in the cultures

were measured using the Annexin V assay. None of the three patient samples analysed

in this manner were sensitised to Fas-induced apoptosis following CD40 plus

interleukin-4 stimulation (Figure 5.8). Interleukin-4 had previously been shown to

inhibit Fas-induced apoptosis in B-CLL cells (Mainou-Fowler et al, 1995). To

determine whether the addition of interleukin-4 to the cultures for the first 24 h was

causing resistance to Fas-induced apoptosis, cells from one patient were cultured with

CD40 alone prior to fas stimulation. However, no increased sensitivity to Fas-induced

apoptosis was seen (Figure 5.9).

Reports that CD40-upregulated Fas receptor can be functional have been published.

Stimulation of murine B cells with anti-CD40 results in upregulation o f Fas receptor,

and renders the cells sensitive to Fas-induced apoptosis (Nakanishi et al, 1996). When

human tonsillar B cells are stimulated with anti-CD40 to upregulate Fas receptor, they

too become sensitive to Fas-induced apoptosis (Garrone et al, 1995), as do Burkitt’s

96

Chapter 5 Results 3

lymphoma cells (Scattner et al, 1995). However, the situation in B-CLL cells appears

to differ somewhat. Wang and co-workers (1997) reported that, regardless of the

stimulus used to upregulate Fas receptor on B-CLL cells (including CD40 stimulation

or culture with interleukin-2 and pokeweed mitogen), the cells did not become

sensitive to Fas-induced apoptosis. This result conflicts with that of Marpara and co

workers (1995) who demonstrated increased sensitivity to Fas-induced apoptosis in B-

CLL cells pre-incubated with interleukin-2 and S. aureus Cowan I. The sample group

of patients analysed in this study was very small, but this study has demonstrated that

upregulation of Fas receptor by CD40 stimulation does not increase sensitivity to Fas-

induced apoptosis in B-CLL cells.

To further define the nature of Fas resistance in B-CLL cells, components of the

signalling pathway upstream of caspase-8 activation were investigated.

Immunoblotting for proteins involved in the formation of the death inducing

signalling complex (DISC) was performed. This analysis confirmed that B-CLL cells

contain the adapter molecule, FADD (Chinnaiyan et al, 1996), which is required for

caspase-8 activation in response to stimulation of the Fas receptor (Boldin et al,

1996). Since B-CLL cells had now been shown to express both FADD and caspase-8,

and since upregulation of the Fas receptor had been confirmed, it was decided to

analyse expression of a known inhibitor of Fas signalling. c-FLIP (FLICE-like

inhibitory protein) is an inhibitor of the Fas receptor/ligand system, and acts at the

level of FADD/caspase-8 binding. Interaction of c-FLIP with activated caspase-8

causes cleavage and activation of c-FLIP, which results in the c-FLIP/caspase-8

interaction becoming more inhibitive. Since c-FLIP is predominantly expressed in

lymphoid and muscle tissue (Irmler et al, 1997), this indicates that this protein may

play a role in regulating apoptosis of lymphoid malignancies. To determine whether

B-CLL cells were overexpressing c-FLIP, in comparison with the level of caspase-8,

immunoblotting using a polyclonal antibody to c-FLIP (Rasper et al, 1998) was

performed on B-CLL cells which had been stimulated with chlormabucil or anti-Fas

monoclonal antibody. The blots were re-probed with anti-caspase-8 polyclonal

antibody, so that the relative expression of each protein could be compared. This

analysis demonstrated that, while B-CLL cells do express c-FLIP, the level of

expression does not appear to be elevated above that of caspase-8 (Figure 5.9), which

97

Chapter 5 Results 3

means that c-FLIP overexpression is unlikely to be responsible for the block in

apoptosis signalling through Fas in B-CLL cells.

Fas signalling occurs by two distinct mechanisms (Scaffidi et al, 1998). Type I Fas

signalling is extremely rapid, occuring in under 10 minutes, and requires the

formation of a death inducing signalling complex (DISC). Type II Fas signalling is

slower, and requires amplification of the apoptotic signal through release of

cytochrome c from the mitochondria, which activates the apoptosome, a complex

consisting of caspase-9 and Apaf-1. Since B-CLL cells do not undergo apoptosis in

response to Fas signalling, regardless of the level of expression of Fas receptor on the

cells’ surface, it was postulated that the B-CLL cells may be unable to form a DISC to

transmit the apoptotic stimulus into the cell. Accordingly, analysis of the ability of B-

CLL cells to form DISC’S was carried out. SKW 6.4 murine T cells were used as

positive controls for Type I DISC formation, and Jurkat T cells were used as controls

for the Type II Fas response. As expected, SKW 6.4 cells showed binding of FADD to

a Fas-induced DISC after only 10 minutes of stimulation (Figure 5.13 A). B-CLL and

Jurkat cells, however, did not show any evidence of DISC formation even after 60

minutes of Fas stimulation (Figure 5.13 B), confirming that B-CLL cells cannot be

classified as Type I cells. Whilst this evidence does not fully reveal the nature of Fas

resistance in B-CLL cells, it does throw up some interesting possibilities. The Type II

signalling pathway, unlike the Type I pathway, can be inhibited by Bcl-2 (Scaffidi et

al, 1998), most probably at the level of amplification of the apoptotic signal at the

mitochondria leading to apoptosome activation. Several studies have correlated

apoptotic resistance in B-CLL with increased expression of Bcl-2 (Thomas et al,

1996; Aguilar-Santelises et al, 1996; Pepper et al, 1996). This evidence in conjunction

with the discovery that B-CLL cells are most likely to act in a Type II manner in

response to Fas stimulation, may be one reason for apoptotic resistance in B-CLL

cells. In addition, other groups have reported that some cytotoxic drugs, such as

doxorubicin, can induce apoptosis via the Fas signalling pathway (Freisen et al, 1997;

Fulda et al, 1997). If this pathway is inhibited in B-CLL by the overexpression of bcl-

2, this may account for some of the drug resistance evident in B-CLL patients. This

present study was limited in the number of patient samples analysed, and further

investigations could monitor expression levels of Bcl-2 in conjunction with analysis

98

Chapter 5 Results 3

into apoptosome formation, possibly by monitoring activation of caspase-9, to

determine whether B-CLL cells respond to Fas signalling or stimulation with

cytotoxic drugs in a Type II manner.

Chapter Six General Discussion

Chapter Six - General Discussion and Suggestions for Future Work

This thesis has described an investigation into the significance of apoptosis in B cell

chronic lymphocytic leukaemia. The lymphoaccumulative nature of B-CLL implies

that dysregulation of the apoptotic process may be responsible for the development

and progression of the disease. Additionally, apoptosis is known to result from

treatment of malignant cells with chemotherapeutic drugs. The fact that drug

resistance is a major problem in CLL, indicates that there may be a problem in

apoptosis induction in B-CLL cells.

In order to investigate this relationship, preliminary work was performed in order to

assess the application of techniques for analysing apoptosis to specimens of freshly

isolated CLL cells. Use of flow cytometric techniques, supplemented with agarose gel

electrophoretic methods allowed quantification of the percentage of apoptotic cells in

any given sample of CLL cells, although the labelling techniques used altered as the

study progressed and new techniques became available.

Initial studies demonstrated a low in vivo level of apoptosis in B-CLL patients, and

confirmed the existence of ‘spontaneous apoptosis’ when freshly isolated B-CLL cells

were cultured in vitro. Variations in the level of spontaneous apoptosis between cases

indicated that B-CLL cells differed from patient to patient in their dependence on

external survival stimuli, which were not provided by the standard in vitro culture

environment. Preliminary findings in this study also showed that the sensitivity of B-

CLL cells to drug-induced apoptosis was closely related to the sensitivity of the cells

to spontaneous apoptosis. Taken together, these findings would appear to implicate

the degree of survival factor dependency of the malignant cells as an important factor

in determining the response of patients to chemotherapy, thus making this an

important area for future research. Candidates for survival stimuli in B-CLL include

anti-apoptotic members of the Bcl-2 family, type II cytokines (e.g. interleukin-4), and

members of the TNF/NGF receptor/ligand superfamily, in particular CD40 and its

ligand. The role of two of these factors (CD40 and interleukin-4) was investigated in

this thesis, where it was shown that both factors could inhibit some degree of

spontaneous or drug-induced apoptosis (see chapter 5). In relation to this, a

100


combination of CD40 stimulation and overexpression of Bcl-2 has been shown to

increase the clonogenic survival of chlorambucil-treated B-CLL cells, and as such

may contribute towards the acquisition of drug resistance (Walker et al, 1997), a

major problem in advanced B-CLL cases.

In this study, the effects of only two B cell survival factors have been investigated.

Other factors may be important in promoting survival of B cells. Enhanced survival

of B-CLL cells when cultured in direct contact with bone marrow stromal cells has

been reported, the effect mediated by p i and p2 integrins and linked to maintainence

of Bcl-2 levels (Lagneaux et al, 1998). The authors of this report postulate that this

contact derived survival stimulus could play a role in accumulation of B-CLL cells in

the bone marrow. These findings and the results contained within this thesis

demonstrate the extent to which B-CLL cells depend on extracellular stimuli for

enhanced survival and escape from apoptosis. As discussed above, this may also be a

crucial factor in determining the response of B-CLL cells to chemotherapy. Further

investigations in this area should incorporate a wider selection of B cell survival

factors and use a larger group of patient samples in order to better determine the

significance of the findings. The relative levels of the factors in the peripheral blood

and bone marrow of B-CLL patients, or the availability of such factors accessible

through contact with other cell types could also be investigated. In addition, samples

of B-CLL cells derived from bone marrow rather than peripheral blood may have an

altered dependence on growth factor stimulation for survival. One way in which this

may occur, the survival stimulus being derived from contact with bone marrow

stromal cells, is mentioned above (Lagneaux et al, 1998). Analysis of bone marrow

derived B-CLL cells could provide insights into the nature of more advanced or drug

resistant B-CLL.

Other investigations performed during this study focused on components of the

apoptotic machinery. Proteases belonging to the caspase family are the machinery

enzymes involved in the degradation of an apoptotic cell, and are related to the ced-3

death gene of the nematode worm, C. elegans. They reside in the cell as inactive

zymogens which require cleavage at specific aspartate residues in order to attain their

active form. Caspase-3 and caspase-7 are two of the main ‘effector’ caspases,

101


responsible for initiating the terminal events of the apoptotic cascade such as DNA

degradation. Other caspases, including caspase-2, caspase-8 and caspase-9 have been

termed ‘initiator’ caspases due to their involvement in upstream events such as

Fas/CD95 receptor mediated apoptosis and initiation of apoptosis via mitochondria.

The important role that these proteases play is underlined by the finding that caspase-

3 null mice develop brain tumours. Also, it has recently been reported that in human

lymphoma the cellular location of caspase-3 can be an important prognostic indicator

(Donoghue et al, 1999). Work described in this thesis has demonstrated the

expression of caspase-2, caspase-3, caspase-7 and caspase-8 in B-CLL cells, all of

which, with the exception of caspase-2, are activated as a result of induction of

apoptosis by the chemotherapeutic drugs, chlorambucil and prednisolone. It has been

postulated that the inability of B-CLL cells to undergo apoptosis may have been due

to the absence of one or more caspases, however, this would not appear to be the case.

Caspase-8 pro-form was shown to be present in abundance, but was cleaved in only

small amounts in response to chlorambucil treatment. Since this caspase is primarily

involved in apoptosis mediated induced by the Fas/CD95 receptor, the ability of B-

CLL cells to undergo apoptosis in response to Fas stimulation was subsequently

investigated. This included an investigation into the expression of Fas inhibitory

proteins, and an examination of the ability of Fas-stimulated B-CLL cells to form a

‘death inducing signalling complex’ (DISC). In this study, a combination of CD40

and interleukin-4 was used to upregulate expression of the Fas receptor on B-CLL

cells. Despite this fact, it appears that the choice of interleukin-4 may not have been

prudent. A recent report has been published which describes a system for upregulating

Fas receptor using interleukins -2, -7 and -12 in combination with lipopolysaccharide

(LPS) (Williams et al, 1999). Fas receptor upregulated in this system was functional

in that the Fas bearing cells could be lysed by effector cells expressing Fas ligand.

The addition of interleukin-4 to the combination of stimulatory cytokines resulted in

delayed upregulation of Fas receptor, which was not functional in that apoptosis could

not be triggered by fas ligand. In the study presented in this thesis, although Fas

receptor was upregulated by CD40 and IL-4 stimulation, no increase in sensitivity to

Fas-induced apoptosis was evident, which may be due to the effects of interleukin-4.

However, in addition to the effects of interleukin-4, there may be a second factor

involved in determining Fas-mediated apoptosis sensitivity. The report described

102


above (Williams et al, 1999) used Fas ligand-expressing hybridoma cells as effectors,

whereas other reports, including this present study, used a monoclonal IgM anti-Fas

antibody to stimulate the Fas apoptosis pathway. It appears that this may be

significant. Analysis of the Fas sensitivity of cells carrying mutant Fas receptor

constructs and chimeric Fas/TNF constructs, demonstrated a difference in sensitivity

to fas ligand and anti-Fas antibody (Thilenius et al, 1997). The authors postulate that

the altered sensitivity observed in their system could be due to the different binding

characteristics of the antibody when compared with the ligand. It appears that the

large IgM molecule may cluster as many as ten Fas receptor molecules in a back-to-

back formation, rather than the cluster formed when Fas ligand binds to its receptor

(two hydrophobic faces together, ligand on the inside, receptor clustered on the

outside) (Hahne et al, 1995). Whether this could directly influence the downstream

signalling of Fas is not clear, but it could be one reason for the differences seen, and

as such should be considered in the planning and execution of any future research into

Fas-induced apoptosis.

From the analysis performed in this study on the apical events of Fas-induced

apoptosis in B-CLL cells, it appears that there is no evidence for overexpression of

the caspase-8 inhibitory protein, c-FLIP, although it is plausible that other, yet to be

identified proteins, may exert their effects at this point. One possibility is the

existence of a SODD-like ‘silencer’ protein, as is found complexed with the TNF

receptor (Jiang et al, 1999). In relation to the ability of B-CLL cells to form a ‘death

inducing signalling complex’, necessary for apoptosis induced via the Fas receptor, it

appears that, whilst the cells contain the death domain containing adapter molecule

FADD, along with significant amounts of caspase-8, a DISC is not readily assembled

upon stimulation of the cells with antibodies against the Fas receptor. It may be the

case therefore, that apoptosis in B-CLL cells could be mediated mainly via the

mitochondria. Since the mitochondrial pathway of apoptosis can be inhibited by anti-

apoptotic members of the Bcl-2 family, the fact that Bcl-2 is known to be

overexpressed in many instances of B-CLL could be significant. This may also have

implications in the development of drug resistance in B-CLL, since many

chemotherapeutic agents have been shown to induce apoptosis via the mitochondrial

pathway. Further research could be performed in order to characterise the pathway of

organelle involvement and caspase activation induced during apoptosis of B-CLL

103


cells. Should further analysis be undertaken, it should include monitoring of

expression levels of the Bcl-2 family in conjunction with analysis of loss of

mitochondrial membrane potential, release of cytochrome c, and activation of the

apoptosome caspase, caspase-9.

Overall, this study has confirmed that chronic lymphocytic leukaemia is a disease

linked closely with the process of apoptosis. Arguably the most significant findings

described here are the discovery that B-CLL cells possess the necessary caspases and

adapter molecules to execute the apoptotic process, and that the aberration which

contributes to the longevity of B-CLL cells most probably lies in the control or

upstream signalling events of apoptosis. The importance that members of the Bcl-2

family play in this disease has again been highlighted, and this, in conjunction with

the findings related to B-CLL cell dependency on extracellular survival factors, may

hold the key to understanding the nature of the malignant B-CLL cell.

104

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118

Appendix

Appendix

Patient 121

Pm -treatm ent, fM shly isolated cells

Ml*33*.

M2

J 1 10* FL1 -Height

T natm en t day 7, fieshly isolated cells Post-treatment, freshly isolated cells

Ml92.3 *A

in®

M2

‘ 7.5%

1 '0 * 1 0 1 1 0 * 1 0 1 1 0 *

FL1-Height

CO

M2

UJ

FL1 -Height

2Pie-treatm ent, Control 24 h

Ml77.6%h- M2

in ® i n • V n t V r

22.4%

10* 1 0 ’ 10* 1 0 J 10 * FL1-Height

Treatm ent day 7, Control 24 h

Ml

M2

1 0 ' 10FL1-Height

M159.9%

i- M240.1%

10* 1 0 1 10* 1 0 J 10* FL1 -Heigiift

Patient 8 A

Pre-treatment, freshly isolated cells

S w l| 93%* 1 . M2| I '----- T l %

O JW hy-s- .... i .iwm ■. ....10* 10’ 102 1 0 J 10*

FL1-Height

Day 7, freshly isolated cells M1

V)

CD>UJ

. 93.7% M2

5.9%

1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height

Post-treatment, freshly isolated cells M1

93.2%

1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height

Pre-treatment, Control 24 h M1co 1

v>■ECD>UJ

63.6%M2

34.6%

10* 10 ’ 102 10* 10*

FL1-Height

Day 7, Control 24 h M1

■ECD>

59.5%i--- M2

40.3%

1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height

Post-treatment, Control 24 h M1

«■ECD>UJ

51.2%I--- M2

48.6%

1 0 * 1 0 ’ 102 1 0 J 10* FL1-Height

Figure A l. Flow cytometry histograms for in vivo analysis, chapter 3, section 3.6. Cells were taken from patients 121 and 8A prior to, during and post treatment. The cells were analysed using the Annexin V assay at 0 h and 24 h of in vitro culture. The histograms shown are plots of Annexin V-FITC (X-axis) against number of cells (Y- axis). The two peaks are live cells (Annexin V negative) and apoptotic cells (Annexin V positive). Markers were set on the pre-treatment, 0 h sample for each case, and remained in place for all further analysis.

119

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