Vol. 2, 1017-1030, June 1996 Clinical Cancer Research 1017
Inhibition of the Growth of Glioblastomas by CGP 41251, an
Inhibitor of Protein Kinase C, and by a Phorbol Ester
Tumor Promoter1
Martin Begemann,2 Sharafadeen A Kashimawo,
Yu-jeong A. Choi, Susan Kim,
Kim M. Christiansen, Gregg Duigou,
Marcel Mueller, Ira Schieren, Subrata Ghosh,
Doriano Fabbro, Nina M. Lampen,
Daniel F. Heitjan, Peter B. Schiff,
Jeffrey N. Bruce, and I. Bernard Weinstein
Columbia-Presbyterian Cancer Center [M. B., S. K., K. M. C., I. S.,
I. B. WI, Departments of Radiation Oncology [S. A. K., P. B. 5.1,
Neurological Surgery [Y. A. C., D. G., S. G., J. N. B.], Genetics and
Development [K. M. C., I. B. W.], and Division of Biostatistics in theSchool of Public Health [D. F. H.], Columbia-Presbyterian Medical
Center, New York, New York 10032; Department for Cancer andInfectious Diseases, Ciba-Geigy, CH-4002 Basel [M. M., D. F.],
Switzerland; and Department of Electron Microscopy, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021[N.M.L.1
ABSTRACT
Protein kinase C (PKC) plays a central role in signal
transduction pathways that mediate the action of certain
growth factors, tumor promoters, and cellular oncogenes.
To explore whether PKC might be an appropriate target forthe chemotherapy of human brain tumors, cell lines were
established from five glioblastomas, one mixed gliosarcoma
and glioblastoma, two astrocytomas, and one choroid plexuscarcinoma. The staurosponine derivative CGP 41251, aninhibitor of PKC, inhibited cell proliferation in all nine cell
lines with an IC50 in the range of 0.4 p�M. Drug withdrawaland clonogenicity assays showed that CGP 41251 induced an
irreversible growth arrest. Three cell lines were examined in
detail: two human glioblastoma cell lines, GB-i and GB-2,
and one gliosarcoma cell line, GS-i. All of these three cell
lines were highly aneuploid and displayed morphologies and
immunohistochemical markers characteristic of the glial lin-
eage. The compound 12-O-tetradecanoylphorbol-13-acetate
(TPA), a tumor promoter and activator of PKC, also inhib-ited the growth of these cell lines. CGP 41251 in combination
with TPA caused further growth inhibition. Cultures treated
with CGP 4i251 displayed an increase in the fraction of cellsin G2-M, a decrease of cells in S phase, and no consistent
effect on G0-G1. Immunohistochemical analyses demon-
strated that growth inhibition by CGP 41251 was associated
with the formation of giant nuclei with extensive fragmen-
tation and apoptotic bodies. These effects of CGP 41251were abrogated by withdrawal of serum from the medium
or by exposure of these cells to aphidicolin, actinomycin D,
cycloheximide, or TPA. In contrast to the effects seen with
the glioblastoma cell lines, nontransformed astrocyte lines
remained viable in the presence of 0.4 and 0.8 �iM CGP41251 and displayed only a slight increase in the fraction of
giant nuclei with fragmentation. The antitumor activity ofCGP 4i25i was demonstrated in vivo against xenografts of
the glioblastoma cell lines U87 MG and U373 MG. These
findings suggest that CGP 41251 might be a useful agent forthe treatment of glioblastomas.
INTRODUCTION
PKC3 is a family of phospholipid-dependent serine and
threonine kinases that has been shown to have the highest
activity in the central nervous system (1-3). About 13 different
isoforms of PKC have been described today, which differ in
their cofactor requirement and are differentially expressed dur-
ing development and in various tissues. Most of these isoforms
are activated by diacylglycerol, which is generated after activa-
tion of phospholipase C. Diacylglycerol binds to the regulatory
domain of various PKC isoforms. Phorbol esters, such as TPA,
also bind to the regulatory domain of these same isoforms and
are potent activators of PKC. Overexpression of PKC in R6 rat
fibroblasts using retroviral-mediated gene transfer demonstrates
that individual isoforms of PKC have different biological ef-
fects. Thus, high level expression of PKC�31 leads to growth
abnormalities, including increased frequency of focus formation
and growth in agar (4); overproduction of PKCa does not cause
any growth defects (5), whereas overexpression of PKC#{128}leads
to malignant transformation and tumorigenicity in athymic mice
(6).
Due to the central role of PKC in growth control, inhibition
of this enzyme might be a particularly promising approach for
cancer therapy (3). Recent studies indicate that the staurosponne
derivative CGP 41251 inhibits PKC in subcellular assays and
also inhibits tumor cell growth in cell culture and in athymic
mice (7-9). Because several isoforms of PKC are normally
expressed at high levels in the central nervous system and
Received 10/24/95; revised 2/12/96; accepted 3/1/96.
I This work was supported by National Cancer Institute Grants CA
02656 (to I. B. W.) and P20-CA60175-02 (to J. N. B.) and an award
from The Brain Tumor Society (to I. B. W.).
2 To whom requests for reprints should be addressed, at Columbia-
Presbyterian Cancer Center, Hammer 1509, 701 West 168th Street, New
York, NY 10032. Phone: (212) 305-6924: Fax: (212) 305-6889.
3 The abbreviations used are: PKC, protein kinase C; TPA, 12-0-
tetradecanoylphorbol-1 3-acetate; BrdUrd, bromodeoxyuridine; MAP2,
microtubulin-associated protein 2; DAPI, 4’,6-diaminidino-2-phenylin-
dole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide: GFAP, glial fibrillary acidic protein; NF16O, neurofilament 160.
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100
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0123456789
Day
-U-CGP41251 .4uM
-�--CGP41251 .8uM
-s’---CGP42700 .4uM
-D--CGP42700 .8uM
-D-TPA Ing/mi
-�‘--TPA lng/ml + CGP41251 .4uM-*--TPA Ing/mi + CGP42700 .4uM
.1
0 1 23456789
1018 Inhibition of the Growth of Glioblastomas by CGP 41251
Day
because glioblastomas are particularly refractory to treatment
with conventional chemotherapeutic agents, the present study
was undertaken to examine the effects of CGP 4 1 25 1 on a series
of human brain tumor-derived cell lines. This study demon-
strates that CGP 41251 is a potent growth inhibitor of glioblas-
toma cells and that this inhibition is, paradoxically, enhanced by
TPA. Growth curves with drug reversal as well as clonogenicity
studies suggest that CGP 4125 1 acts both through a cytocidal
and cytostatic mode. Flow cytometry studies indicate an accu-
mulation of cells in G,-M, and morphological studies indicate
that CGP 4 1 25 1 leads to the formation of giant nuclei and the
formation of apoptotic bodies. Furthermore, the antiproliferative
property of COP 4125 1 was confirmed in xenografts of human
glioblastoma cell lines U87 MG and U373 MG.
MATERIALS AND METHODS
Cell Lines and Cell Culture Conditions. All cell lines
were established in the Brain Tumor Laboratory of the Depart-
ment of Neurosurgery, Columbia-Presbyterian Medical Center.
In brief, fresh tumor specimens were placed in 15% FCS in the
operating room. In the tissue culture laboratory, the tumor cells
were mechanically dispersed with 18-gauge needles or enzy-
matically with trypsin, depending on the tumor consistency.
Dispersed cell explants were then placed in tissue culture dishes
containing DMEM with 20% FCS. The cell lines were also
maintained in DMEM supplemented with 20% FCS (Hybrimax:
Sigma Chemical Co., St. Louis, MO). TPA (LC laboratories,
Woburn, MA) and COP 41251 and COP 42700 (Ciba-Geigy,
Base!, Switzerland) were dissolved in DMSO. To obtain growth
curves, cells were plated at a density of l04/well in 6-well plates
(3-cm diameter) and grown in complete medium with or without
the indicated drugs (Fig. 1). Cell counts were determined on
alternate days in triplicate wells during the subsequent 8 days
with a Coulter Counter. Actinomycin D (Sigma) was dissolved
in DMSO and used at a final concentration of 2.5 pg/ml.
Cycloheximide (Sigma) was dissolved in ethanol and used at a
final concentration of 10 p.g/ml. Aphidicolin (Sigma) was dis-
solved in DMSO and used at a final concentration of I pg/ml.
In all cases, the final concentration of DMSO in the media was
less than 0. 1 %, which had a negligible effect on cell growth.
Protein Extraction and Immunoblotting. The various
cell lines were grown to subconfluency, washed three times with
PBS, and immediately lysed in a 1% SDS buffer [20 msi
Fig. I Growth curves of GB-I cells in the presence of the indicated
concentrations of TPA (A) and in the presence of the indicated concen-
trations of CGP 41251, CGP 42700, or these drugs plus TPA (B).DMSO, the solvent control for the studies with TPA; D20, the control
obtained in the absence of any drugs. In the statistical analyses, a model
with separate slopes of log cell counts for each TPA dose group was
estimated (A). A test for equality of all slopes was significant at P <
0.001. Slopes of the control group, the 0.01 group. and 0.1 group were
significantly positive, and slopes for the three remaining (high-dose)
groups were not significantly different from 0 at the 0.05 level. Bars,
SD. Data in B, TPA alone, CGP 41251 (at both doses) alone, and CGP
42700 (at 800 nM only) alone, all produced inhibition of proliferation (P< 0.001), although the effect of CGP 42700 was modest. The addition
of COP 41251 to TPA produced additional significant inhibition, as did
the addition of CGP 42700, although again the effect of COP 42700 was
modest. Bars, SD. For additional details, see “Materials and Methods.”
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Clinical Cancer Research 1019
Tris-HC1 (pH 7.5) and 2 mrvi EDTA] and boiled for 5 mm. The
protein content in these total extracts was determined according
to the method of Bradford (Bio-Rad). The equivalent of 50 �i.g
protein cell extracts was fractionated by electrophoresis in 7%
polyacrylamide gels, transferred to nitrocellulose, and exposed
to antibodies specific to various isoforms of protein kinase C as
indicated [PKCa, rabbit affinity purified IgO (OIBCO-BRL);
PKCs �, #{128},and �, rabbit polyclonal antisera; Refs. 9 and 10]. The
specificity of the antisera has been demonstrated previously (9).
Clonogenicity. Cells were plated in 100-mm dishes with
10 ml DMEM supplemented with 10% FCS (OIBCO) and
allowed to attach for about 24 h. The numbers of cells to be
plated were chosen such that 50 to 100 colonies would survive
after a specified treatment. COP 41251 or DMSO as control
were then added, and the cells were incubated for an additional
24, 48, 96, 120, and 144 h. Following these incubation periods,
the medium was removed by aspiration and replenished with
DMEM supplemented with 10% FCS without additives. The
cultures were then incubated for 14 days. Cells were fixed in
75% methanoll25% acetic acid and stained with crystal violet.
Collections of 50 or more cells were scored as colonies and
counted for each plate. Each data point was obtained in triplicate
assays. The survival fraction after drug treatment is given as a
percentage of the DMSO control. Control plating efficiencies
were 10% for OB-l, 14.5% for OB-2, 12% for GB-3, and 8%
for 05-1.
BrdUrd Incorporation Studies. BrdUrd labeling (Am-
ersham) was carried for 60 mm at 37#{176}Cin DMEM supple-
mented with 20% FCS. Subsequently, the cells were washed in
PBS and fixed in glacial acetic acid/ethanol for 30 mm. The
endogenous peroxidase activity was quenched by incubation
with methanol/3% peroxide for 30 mm at room temperature.
Subsequently, the dishes were treated with blocking solution
(horse serum, 2% BSA, 0.1% Triton X-100, and PBS) for 1 h.
Then anti-BrdUrd mouse monoclonal antibodies (Amersham)
were applied for 1 h, and the plates were washed in PBS. The
mouse monoclonal antibody was detected with the ABC system
from Vector Laboratories (Burlingame, CA). Staining was car-
ried out with diaminobenzidine using the diaminobenzidine
substrate kit from Vector Laboratories.
Flow Cytometry. Exponentially growing cultures were
trypsinized, collected, and washed twice with PBS. Cell pellets
were suspended in 1 ml PBS and fixed in 5 ml of 70% ethanol
and stored at 4#{176}C.On the day of the analysis, cells were
collected by centrifugation, and the pellets were resuspended in
0.2 mg/mi of propidium iodide containing 0.6% NP-40. RNAse
(1 mg/mI) was added, and the suspension was incubated in the
dark at room temperature for 30 mm. The cell suspension was
then filtered through a 60 jim Spectra mesh filter and analyzed
on a Coulter EPICS 753 flow cytometer for DNA content. The
percentage of cells in different phases of the cell cycle was
determined using a ModFit 5.2 computer program.
Immunohistochemistry. Cells were grown on LabTek
chamber slides that were precoated with poly-D-lysine (Collab-
orative Research) in DMEM (0.1 mg/ml for 1 h). After the
indicated exposure (see “Results”), the cells were fixed with
90% ethanol, 5% glacial acetic acid, and 5% H2O for 30 mm.
The cell preparation was blocked in 2% BSA and incubated
sequentially with various primary and secondary antibodies.
Antibodies against the following proteins were used: OFAP
(rabbit polyclonal antibodies from Sigma and mouse mono-
clonal antibody GAS from Boehninger-Manmheim); neuron-spe-
cific enolase (rabbit polyclonal antibodies; ICN); NF16O (mouse
momoclomal antibody; Accurate Scientific, NN16); neurofila-
ment 200 (rabbit polyclonal antiserum; Sigma); and MAP2
(mouse monoclonal antibody AP420; Sigma). The secondary
antibodies used were goat-antimouse-conjugated FITC (Sigma)
at a 1 :50 dilution and goat-antirabbit FITC-conjugated antibod-
ies (TAOO) at a 1:100 dilution. The nuclei were counterstained
with DAPI (Boehringer-Mannheim) at a final concentration of
1.5 p.g/ml in PBS for 30 mm at room temperature and visualized
with fluorescence microscopy. For quantitation of the nuclear
morphology under various culture conditions, the morphologies
of the nuclei were separated into six categories (oval, frag-
mented, multinucleated, mitotic, pyknotic, and multilobular).
For each data point, four fields, each containing at least 150
nuclei, were evaluated.
Transmission Electron Microscopy. Cells were fixed
as a pellet in 2.5% glutaraldehyde in 1 ,4-piperazinediethanesul-
fonic acid buffer overnight, rinsed in 1 ,4-piperazinediethanesul-
fonic acid buffer, followed by postfixation in 2% 0504 for 1 h.
The samples were then rinsed in distilled H2O, and this was
followed by dehydration in a graded series of alcohol of 50, 75,
and 95% through absolute alcohol, followed by propylene ox-
ide, and then overnight in propylene oxide:PolyfBed 812 (1:1).
The samples were embedded in Poly/Bed 812 in a 60#{176}Coven.
Ultrathin sections were obtained with a Reichert Ultracut S
microtome. The thin sections were photographed using a JEOL
1200-EX electron microscope.
MTT Viability Assay. The MiT assay was performed as
described ( 1 1 ). Cells (2-5 X l0�) were seeded in each well
(0.38 cm2 area) of 96-well plates in DMEM plus 20% FCS. On
the following day, the medium was replenished with the drugs
at the indicated dosages. After various times (see “Results”), the
medium was removed, and 22 �il of MU solution (5 mg/mi in
PBS; Sigma) were added to 200 p.1 of 20% FCS in DMEM in
each well and incubated at 37#{176}Cfor 3 to 4 h. This medium was
then discarded, 100 p.1 of DMSO were added to each well, and
the plates were placed for 15 to 30 mm on a shaker. The color
change was recorded by measuring the absorbance at 570 mm on
an automated multiwell reader. Each data point represents the
mean of quintuplicate assays and was calculated as a fraction of
the control.
In Vivo Experiments. The U87 MG (HTB- 14) and U373
MO (HTB-17) human glioblastoma cell lines were obtained
from the American Tissue Culture Collection (12, 13). Both
tumor lines were maintained by serial passage by implanting
tumor fragments (approximately 25 mg) s.c. into the left flank of
female BALB/c nude mice (Bolmholtgard, Copenhagen, Den-
mark) with a 13-gauge Trokar needle under Forene (Abbott,
Cham, Switzerland) narcosis. Each group of mice contained six
animals. Drug treatment was started on day 5 after transplanta-
tion and continued until day 17. Compounds were prepared as
follows. For iv. applications, COP 41251 was dissolved in
DMSO, Tween 80, and NaC1. In brief, a stock solution contain-
ing 150 mg/ml COP 4125 1 was prepared by adding the appro-
priate amount of compound to DMSO containing 1% Tween 80
dissolved at room temperature. Prior to use, the clear stock
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1020 Inhibition of the Growth of Glioblastomas by CGP 41251
Table 1 Description of cell lines”
GB-l GB-2 GS-l
Age (yr)
Gender61
F71
M61
FDiagnosis
Protein expression
GFAP
Glioblastoma multiforme
+
Glioblastoma multiforme
+
Glioblastoma multiforme and gliosarcoma
+
NF16O - - -
MAP2 - -
Doubling time (h)Karyotype
Tumorigenicity in nude mice
58Aneuploid
+
46Aneuploid
+
62Aneuploid
-
a For additional detail, see “M aterials and Methods.”
solution was diluted 1 :20 (v/v) with sterile 0.9% NaC1, resulting
in a milky suspension (final concentration: 7.5 mg/mi COP
41251, 5% DMSO, 0.5% Tween 80, and 0.9% NaC1). For p.o.
applications, COP 4125 1 was prepared using Gelucire 44/14. In
brief, a waxy solid formulation of COP 41251 (18% w/w in
Gelucire 44/14; Oattefosse#{233}, France) was obtained from CIBA
Pharmaceutical. Prior to use, 16.7 mg of the active ingredient
corresponding to 83.3 mg of the waxy formulation were added
to 1 ml of sterile water, and the mixture was sonicated in an
ultrasonic bath for 10 mm, resulting in a milky suspension that
was given to the mice for 13 consecutive days once daily p.o.
(7). All placebo-treated animals received the matrix without
COP 41251 (gelucire/water) p.o. Tumor growth was followed
by measuring perpendicular tumor diameters. Tumor volumes were
calculated as described using the formula (‘rr X L X D2/6), where
L is the length and D is the diameter (14, 15). Mean tumor volume
(± SD of six animals) was expressed in cubic centimeters.
Statistical Methods. For experiments on cell prolifera-
tion, the cell counts were analyzed on the log scale. A linear
model was fit with a common intercept (number of cells at day
0) with a separate slope for each treatment group. The equality
of slopes was tested with the regression F test. For BrdUrd
incorporation studies, the data of the percentage of labeled
nuclei were analyzed on the logistic scale. The data were ana-
lyzed with an ANOVA model incorporating a main effect of cell
line, a slope for the drug dose, and an interaction of cell line and
dose. In the clonogenicity studies, the surviving fractions were
transformed to the log scale. The data were analyzed by a linear
model with a main effect of time, a slope for drug dose, and the
interaction of time and dose. We conducted separate analyses
for each cell line. In all analyses, we deleted the data from dose
group 0, because they were scaled to have mean SF = 1 . To
analyze the in vivo tumor growth data, the tumor volumes were
transformed to the log scale. We fit a mixed model with a
random animal effect and a separate slope for each treatment
group (placebo, p.o. administration, and i.v. administration). To
analyze the data on nuclear morphology, a separate two-way
table of treatment by morphology was formed for each of three
replicate assays. To eliminate cells with the smallest counts, our
analyses combined the multinucleated, multilobular, and py-
knotic categories. To compare the treatment groups, we exe-
cuted a Mantel-Haenszel test for general association, stratified
by replication. All analyses were carried out in SAS version
6.09 (SAS Institute, Cary, NC). We fit linear models in Proc
Table 2 Growth inhibition by CGP 41251 in glioblastomas,astrocytomas, and a choroid plexus carcinoma cell line
Patient Diagnosis
IC50 (p.M) for
CGP 41251”
GB-i Glioblastoma multiforme 0.2GB-2 Glioblastoma multiforme 0.4
GB-3 Glioblastoma multiforme IGB-4 Glioblastoma multiforme 0.6GB-S Glioblastoma multiforme 0.8GS-l Glioblastoma multiforme and gliosarcoma 0.2
AT-! Astrocytoma 0.1AT-2 Astrocytoma 0.4
CP-l Choroid plexus carcinoma 0.3
a Determined after a 6- to 8-day exposure of cultures to the drug, as
described in Fig. 1.
Olm, mixed models in Proc Mixed, and executed Mantel-
Haenszel tests in Proc Freq software programs.
RESULTS
Characteristics of Cell Lines. Various properties of the
three major cell lines used in this study are summarized in
Tables 1 and 2. All three cell lines were positive for the
glial-specific protein OFAP and negative for the neural markers
MAP2 and NF16O. Cultures of GB-i and OB-2 cells were
heterogeneous by morphological criteria, displaying cells with
squamous, astrocytic, and polygonal morphologies. OB-l and
GB-2 cells were arranged in fascicular patterns and piled up. In
contrast, cultures of OS- 1 cells formed flat mosaic patterns of
polygonal cells. Occasionally, cells with astrocytic morpholo-
gies were found in 05-1 cultures. All three cell lines were
highly aneuploid and showed continuous chromosomal rear-
rangements during serial passages (data not shown). The cell
lines GB-I and GB-2 were tumorigenic when injected into
athymic mice (nu/nu). The histopathology of the grafted tumors
exhibited highly infiltrative tumor growth, nuclear polymor-
phism, pseudopalisading, and expression of OFAP. Two astro-
cyte lines were established from nontumorous tissue: A-l from
normal appearing brain tissue obtained from a patient with a
glioblastoma multiforme; and A-2 from brain tissue removed
from a patient with epilepsy. A-l was shown to be nontumori-
genic in athymic mice. A-l and A-2 both exhibited astrocytic
morphologies (data not shown).
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1 2 3456789101112
PKCa 84 -� � ___ - � -
PKC� 8463-
PKCc 112
84-
k� .i��Ii �
84- � _� �PKC�
63- �
Clinical Cancer Research 1021
Fig. 2 Immunoblot for PKCs
a, h, #{128},and �. GB-l cells werecultured for 2 days in the pres-
ence of DMEM supplemented
with 20% FCS alone (Lane 1)
or with 0.1% DMSO (Lane 2);
TPA at the following concen-trationsof0.Ol,0.l, 1, 10, and
100 ng/ml (Lanes 3-7): CGP41251 at 0.4 and 0.8 mM
(Lanes 8 and 9); CGP 42700 at
0.8 m�i (Lane /0); and CGP
41251 (0.8 mM) plus TPA at 1
or 10 ng/ml (Lanes 11 and 12).
In the PKC� immunoblot, thesolid arrow denotes the com-
plete PKC�, and the open ar-
row, the PKM form of PKC�.
For additional details, see “Ma-terials and Methods.”
Effects of TPA and CGP 4i25i on Cell Proliferation.
The cell line GB- I was first grown in the presence of increasing
concentrations of TPA (Fig. 1A). TPA markedly inhibited
growth with an IC�() of about 0. 1 ng/ml. Then the effect of the
PKC selective inhibitor COP 4125 1 on the growth of GB-i cells
was tested either alone or in combination with TPA. COP
42700, a close structural analogue of COP 4125 1 that does not
inhibit PKC (7, 9), and the solvent DMSO were used as controls.
As shown in Fig. 1B, proliferation ceased at 0.4 �.tM of the active
compound COP 4125 1, and cell killing was observed with 0.8
p.M of COP 41251 (P < 0.001). COP 42700 was not signifi-
cantly different from growth medium at 0.4 mM; there was a
modest but significant inhibition at 0.8 mivi. Thus, both TPA
(Fig. 1A) and COP 41251, as well as COP 42700 at a modest
level (Fig. I B), inhibit the proliferation of GB- 1 in a dose-
responsive manner. The inhibitory effect of 0.4 p.M COP 412�1
plus 1 ng/ml TPA was greater than the effect of either agent
alone (P < 0.001). The inhibitory effect of TPA plus COP
42700 was modest but significantly greater then that of TPA
alone (Fig. 1B; P < 0.001). This suggested that COP 41251
might be cytocidal when tested in combination with TPA.
Trypan blue exclusion studies, however, did not show increased
cell staining of COP 4 125 1 or COP 4 125 1 plus TPA-treated
cultures when compared to untreated cultures or to COP 42700
or DMSO-treated cultures (data not shown).
The growth-inhibitory effects of COP 4125 1 were analyzed
across a range of concentrations of COP 4125 1 in the GB-i and
eight additional brain tumor-derived cell lines, and the results
are summarized as IC5() (i.e., the concentration required to
produce about a 50% inhibition of growth) in Table 2. The five
glioblastoma-derived cell lines (GB-! to OB-S) displayed IC50s
between 0.2 to 1 p�M. This value was 200 n�i for the OS-i
gliosarcoma cell line; 0. 1 and 0.2 �j.M, for the two astrocytoma
cell line, AT- 1 and AT-2, respectively; and 0.3 �iM for the
choroid plexus carcinoma cell line CP-l (Table 2).
As with the OB-l cell line (Fig. 1B), when TPA was
combined with COP 4125 1, there was further inhibition of
growth of the OB-2 and 05-1 cell lines, and the compound COP
42700 caused a modest growth inhibition in OS- 1 and no
inhibition in OB-2. When the GB-i or 05-1 cells were exposed
to COP 4125 1 at 0.8 p�M for 4 days and the drug then removed,
the proliferation of the cells failed to resume. However, under
similar conditions, the growth inhibition obtained with the OB-2
cells was partially reversed when the drug was removed (data
not shown).
Effects of CGP 41251 and TPA on the Expression ofSpecific Isoforms of PKC. Fig. 2 displays an immunoblot
analysis of the expression profile of several isoforms of PKC in
GB-I cells and the effects of exposing these cells to various
drugs. PKCs a, �, #{128},and � were detected in total cell extracts but
not the PKC isoforms �3 and -y (data not shown). The molecular
weights of PKCs were as follows: Mr 86,000 (PKCa); Mr
7 1 ,000 (PKC�); a doublet at Mr 96,000 (PKC#{128});and Mr 78,000
(PKC�). We also detected an approximately Mr 58,000 species
cross-reacting with anti-PKC� antisera (Fig. 2, open arrow).
This moiety most likely represents the COOH-terminal catalytic
fragment of PKC� termed PKM (16, 17). The expression of
PKM has been demonstrated in rat brain (16, 17) and might play
a role in long-term potentiation (17). After exposing GB-I cells
for 48 h to TPA at 0.01, 0.1, and 1 ng/ml, there was no
significant effect on the expression levels of the four isoforms
(Fig. 2), although the 1 ng/ml dose caused marked growth
inhibition (Fig. 1A). Exposure to 10 and 100 ng/ml TPA caused
marked down-regulation of PKCs a and e. Exposure to COP
41251 at 0.4 or 0.8 J.LM, or the control compound COP 42700,
caused no appreciable loss of these four isoforms of PKC (Fig.
2, Lanes 8-JO). The combination ofCOP 41251 (0.8 jiM) with
TPA at 1 or 10 ng/ml (Fig. 2, Lanes ii and 12) showed
complete down-regulation of PKCs a and #{128}.Similar profiles of
these PKC isoforms were found with the OB-2 and OS-i cells
(data not shown).
BrdUrd Incorporation Studies. To further analyze the
antiproliferative properties of COP 41251 , the GB-!, OB-2, and
05-1 cell lines were selected for further detailed studies.
BrdUrd incorporation studies were performed to determine pos-
sible effects of COP 4125! on DNA synthesis. Fig. 3 summa-
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0
1022 Inhibition of the Growth of Glioblastomas by COP 41251
Ctrl
CGP41251 200 nM
CGP41251 400 nM
CGP41251 800 nM
10 20 30 40
Fig. 3 Effects of COP 4125 1 on BrdUrd incor-
poration. Cultures of OS-I, GB-i, and OB-2 cellswere treated for 4 days with 200, 400, or 800 nMCOP 4125 1 . The cultures were then incubated
with BrdUrd for 1 h, fixed, and prepared for
staining as described in “Materials and Methods.”
The figure indicates the BrdUrd labeling indices,
i.e., the percentage of nuclei positive for BrdUrd.
In the statistical analyses, the slope of the dose of
COP 41251 was significant (P < 0.001), as was
the main effect of the cell line. The statistical
interaction was not significant. Thus, the analyses
suggest a strong inhibitory effect of COP 41251,
which is present in all three of the cell lines
studied. Bars, SD.
% labeled nuclei
rizes BrdUrd labeling indices obtained after a 4-day treatment
with various concentrations of COP 41251. In untreated cul-
tures, the GB-2 cells exhibited the highest labeling index (about
33%), as compared to GB-l and OS-i cells (about 12%). The
addition of COP 41251 (200 to 800 nM) resulted in a dose-
dependent inhibition of BrdUrd incorporation in all three cell
lines. At 800 flM CGP 41251, the labeling index had decreased
by about 80% in all three cell lines when compared to the
untreated cultures. Time course studies with the GB-2 cell line
indicated that inhibition of BrdUrd incorporation began within
24 h after exposure to the drug and continued to decline there-
after (data not shown). Inspection of nuclei revealed that those
nuclei that were labeled were predominantly giant nuclei with a
fragmented morphology (see below), suggesting that these cells
were undergoing re-replication of DNA without entering mito-
sis. Exposure to TPA (100 ng/ml) for 2 days also led to a
decrease in BrdUrd incorporation with both the GB-2 and 05-1
cells, and this inhibitory effect was further augmented by the
addition of COP 41251 (data not shown).
Clonogenicity Assays. To further analyze the antiprolif-
erative effects of COP 41251, clonogenicity studies were per-
formed. GB-i, GB-2, GB-3, and OS-i cells were plated at low
density and incubated for various times with different doses of
COP 41251. Subsequently, the medium was replaced with regular
growth medium, and the cells were cultured in the absence of the
drugs for an additional 2 weeks, after which the surviving fractions
were determined (see “Materials and Methods”). As shown in Fig.
4, all of the four cell lines showed only partial inhibition when
exposed to COP 4125 1 for only 24 h. When exposed to the drug for
48 h or longer, marked inhibition and a clear dose-response rela-
tionship was seen in all of the cell lines, although OB-2 cells were
less responsive then the other three cell lines. In addition, at a given
dose of drug, an increased duration of treatment led to decreased
survival. For example, GB-l cells treated with 1 p.si COP 41251
for 24, 48, 96, and 144 h showed survival fractions of 24, 9, 0.7,
and 0.6%, respectively. The survival of the cells also declined in a
dose-response fashion. Thus, the survival fractions of GB-l cells
after a 96-h exposure to 100, 200, 400, 600, 800, and 1000 ruvi COP
41251 were 53, 43, 13, 1.7, 1.3, and 0.7%, respectively. Finally, the
survival fractions of the particular cell lines were compared at a
given dose for a given duration of exposure. At the longest expo-
sure (120 to 144 h), the LD� (i.e., the concentration required to
produce a 10% survival) for COP 41251 was 360, 740, 320, and
350 flM in GB-i, OB-2, OB-3, and OS-i cells, respectively. At the
maximum dose (1000 rust) and after the longest exposure (120 to
144 h), the surviving fractions were 0.i, 0.6, 3, and 6.2% for the
OB-3, OB-l, OS-l, and OB-2 cells, respectively. These results
demonstrate that OB-l and GB-3 cells are the most sensitive, OS-l
cells are intermediate, and OB-2 cells are least sensitive to inhibi-
tion by COP 4i251. In summary, the effects ofCOP 41251 on cell
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Clinical Cancer Research 1023
Fig. 4 Clonogenicity assays. Cultures of GB- I . GB-2, GB-3, and GS- 1 cells were plated at low density and then treated with various concentrationsof COP 4125 1 for different periods of times, as indicated, and then assayed for clonogenicity as described in “Materials and Methods.” The graphsindicate the surviving fractions as a function of dose and duration of exposure. There was a statistically significant interaction (P < 0.001 ) between
hours of exposure and the dose of COP 4 125 1 . This means that the slope of the SF dose curve depends on the hour of exposure. Moreover, in each
case, the slope of the SF-dose curve becomes more steep with increasing time of exposure, suggesting a time-dependent sensitivity.
survival depends on both the dosage and the duration of exposure
to the drug and the particular cell line.
Cell Cycle Analysis. To investigate possible effects on
cell cycle progression, exponentially dividing cultures of
GB-I and OB-2 cells were treated with the DMSO solvent
(control) or with COP 41251 (800 nM) for 4 days, and then
the cells were processed for flow cytometry (Table 3). With
both cell lines, the drug caused a 2- to 3-fold increase in the
fraction of the total cell population that was in O,-M and a
decrease in the fraction of cells in S phase. With the GB-i
cells, there was no significant change in the fraction of cells
in O�-O�, and in OB-2 cells, there was a slight decrease in
this fraction. The inactive compound, CGP 42700, had no
effect on cell cycle distribution in cultures of GB- 1 (Table 3)
and OB-2 cells (data not shown). Additional studies, not
shown here, indicated that exposure of GB-I and OB-2 cells
to TPA (100 ng/ml) also led to an increase of cells in O2-M
and a decrease in S phase: the combination of TPA plus COP
41251 further enhanced the accumulation of cells in O,-M.
Table 3 Cell cycle analysis
Exponentially growing cultures of GB-I and GB-2 cells were treated
for 4 days with the indicated drugs and then analyzed for DNA content by
flow cytometry. The data indicate the percentage of the total cell population
in the indicated phase of the cell cycle and are the mean values of duplicate
experiments. For additional details, see “Materials and Methods.”
G�-O
GB-I
S G2-M G�-G
GB-2
S G,-M-�-�
Control 59.2 30 10.8 75.3 15.0 9.7COP 41251 0.8 p.M 61.4 9.6 29 68.5 10.4 21.1
COP 42700 0.8 p.M 52 35.2 12.8 ND” ND ND
,‘ ND, not determined.
Formation of Giant Nuclei with Extensive Fragmenta-tion. To investigate possible changes at the level of cellular
morphology and differentiation, cultures of GB-i, OB-2, and
05-1 cells were incubated with COP 41 25 1, TPA, or a combi-
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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1024 Inhibition of the Growth of Olioblastomas by COP 4125!
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Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1025
Table 4 Nuclear morphology of OB-2 cells after exposure to COP 41251 and TPA
OB-2 cells were plated on LabTek chamber slides and incubated for 4 days with COP 41251 (0.8 p.M) or TPA (100 nglml) or the combinationof both. Then the cultures were fixed and processed for staining as described in “Materials and Methods.” Denoted are the means of triplicate assays
with standard variation in percent. In all three replications, there was significant association by the x2 test (P < 0.001) of treatment group with
morphology. A Mantei-Haenszel test for general association combining results across the three replicates is significant at P < 0.001 . The proportion
of fragmented cells in the COP 41251-treated group was significantly smaller than the fraction in the group treated with both COP 41251 and TPA
(P < 0.001 by the Mantei-Haenszei test).
Nuclear morphology
Treatment Oval Multi-nucleated Fragmented Mitotic Multi-lobular Pyknotic
Control 90.4 ± 8.4 0.8 ± 0.5 1.7 ± 0.3 3.9 ± 0.5 2.4 ± 0.9 0.8
COP4I251 28.7 ± 2.5 1.5 ± 0.2 65.2 ± 5.4 1.4 ± 1.1 2.1 ± 1.3 1.1
TPA 84.9 ± 3.3 3.4 ± 2.0 2.1 ± 1.5 4.6 ± 2.6 4.1 ± 1.2 0.9
COP41251 + TPA 27.4 ± 9.6 3.6 ± 0.3 54.4 ± 8.3 6.4 ± 4.4 2.2 ± 0.2 6 ± 5.7
nation of both drugs for various periods of times. Subsequently,
the cells were fixed and stained for the expression of various
neural- and glial-specific markers, and the nuclei were counter-
stained with DAPI. Fig. S shows a typical example of a culture
of OB-2 cells. Fig. 5, A and B, show that all of the cells
expressed the glial-specific marker OFAP. However, none of
the cells expressed the neural-specific markers NSE, NF16O, or
MAP2 before or after treatment with COP 4i25l, TPA, or the
two drugs combined (data not shown). Under control conditions
(Fig. 5, A and B), the nuclei were polymorphic and predomi-
nantly oval, with occasional mitoses. After exposure to COP
41251 at 0.8 p.M for 4 days (Fig. 5, C and D) the cells were
flatter and larger in size and acquired giant nuclei with extensive
fragmentation and apoptotic bodies (open arrow). The giant
nuclei in the COP 41251-treated cultures had the longest diam-
eter, which was 2.7-fold that of the untreated control cultures.
The cells that formed apoptotic bodies in this culture no longer
expressed OFAP (Fig. SC), nor did they express NF16O or
MAP2 when stained with the respective antibody (data not
shown). However, not all of the cells underwent these changes.
As shown in Fig. 5, C and D, some of the cells continued to
express OFAP and retained an oval nuclear morphology. In
contrast to the effects of COP 41251, exposure to TPA at 100ng/mi did not cause an increase in fragmented nuclei (Fig. SF).
Combined exposure to COP 4125 1 and TPA tended to resemble
the effects of COP 41251 alone (Fig. SH and data not shown).
To quantitate these observations, the morphologies of the nuclei
were evaluated on a large number of cells and assigned to the
following categories: oval, fragmented, multinucleated, mitotic,
pyknotic, and multilobular. As summarized in Table 4, when
compared to control untreated OB-2 cells. treatment with 0.8 p.M
CGP 41251 for 4 days caused the following changes. The
number of cells containing fragmented nuclei increased from 1.7
to 65%, and the number of mitotic nuclei decreased form 3.9 to
1.4%. On the other hand, following treatment with 100 ng/ml
TPA for 4 days, there was no increase in the number of frag-
mented nuclei (1.7% versus 2.1%), and there was a slight
increase in the number of mitotic cells, from 3.9 to 4.6%, and
multinucleated cells, from 0.8% to 3.4%. Treatment of OB-2
cells with 0.8 p.M COP 41251 plus 100 ng/ml TPA for 4 days
resulted in an increase in the number of fragmented nuclei from
1 .7 to 54% and an increase in the number of mitotic nuclei from
3.9 to 6.4%. Thus, TPA slightly inhibited the formation of
fragmented giant nuclei and protected the cells against the
inhibitory effects of COP 4 125 i with respect to mitosis.
The marked induction of nuclear fragmentation by COP
41251 and the inhibition of this process by TPA were also seen
with the GB-l and OS-i cells. Time course studies with the
GB-! cells showed that the induction of giant nuclei with
fragmentation and apoptotic bodies by 0.8 p.M COP 41251
occurred in about 50% of the cells within 24 to 48 h. Dose-
response studies with OB-2 cells showed that the concentration
of COP 41251 required to produce 50% of the maximum in-
duction of giant nuclei with fragmentation and apoptotic bodies
was about 0.4 p.M. Additional studies indicated that the induc-
tion of nuclear fragmentation by COP 41251 was markedly
inhibited when the cells were grown in 0.2% rather than 20%
FCS, or when the cells were treated with actinomycin D (2.5
p.g/ml), cycloheximide (10 p.g/ml), or aphidicolin (1 p.g/ml),
during the time of treatment with COP 41251 (data not shown).
The treated OB-2 cells were also examined by transmission
electron microscopy (Fig. 6). Various stages of apoptosis with
loss of cell-cell contact, nuclear condensation, and formation of
apoptotie bodies (Fig. 6B. cell b), as well as membrane bleb-
bing, vacuolization, and cellular fragmentation (Fig. 6B, cell a),
were observed. As commonly seen in cells undergoing apopto-
Fig. 5 Morphology of GB-2 cells after exposure to COP 4125! or TPA. The cells were cultured on LabTek chamber slides with COP 41251 or TPA
for 4 days and then fixed and stained with antibodies specific for OFAP (A, C, E, and G). The nuclei were stained with DAPI (B, D, F, and H). Thecontrol cultures (A and B) show OFAP-positive cells (A). Nuclei in the control culture (B) are predominantly oval and polymorphic with occasional
mitoses (arrow). The majority of cells treated with 0.8 mM COP 41251 for 4 days (C and D) form giant nuclei (*) with apoptotic bodies (open arrow).
Some of the COP 41251-treated cells are OFAP negative (C, *). Some OFAP-positive cells with oval nuclei are still present in the culture (C and
D, solid arrow). After treatment with TPA (100 ng/mi) for 4 days (E and F), most of the cells still display an oval nuclear morphology with occasionalmitoses (F, solid arrow). Cells treated with the combination of COP 41251 plus TPA (G and H) display oval and polymorphic nuclei, frequent mitoses(H, solid arrow), and multiple nuclei (H, open arrow). The combination led to a modest decrease of cells with giant nuclei and apoptotic bodies (H)
when compared to cells treated only with COP 41251 (D). H, bar, SO p.m.
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Fig. 6 Transmission electron microscopy of OB-2 cells after exposure to 0.8 mM COP 4125 1 for 4 days. The control culture (A) shows glioblastoma
cells with typical muitilobed nuclei. Exposure to COP 41 25 1 for 4 days (B) leads to apoptosis. Cell b is in the process of apoptosis during the formation
of apoptotic bodies (arrow); cell a is disintegrating and shows extensive vacuolization. A and B, bar, 2 p.m.
1026 Inhibition of the Growth of Olioblastomas by COP 41251
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sis, organelles like mitochondria, Oolgi apparatus, and endo-
piasmatic reticula were often found to be intact.
Viability and Nuclear Morphology of Nontransformed
Astrocyte Cultures. To investigate the effects of COP 41251
on nontransformed astrocytes, primary human astrocyte lines
were established and examined for viability and nuclear mor-
phology after exposure to COP 41251. One nontransformed
astrocyte line (A-l) was obtained from a noninfiltrated brain
sample of a patient undergoing resection of a glioblastoma
multiforme, and the other (A-2) was derived from a brain
sample of a patient with epilepsy. The nuclear morphology of
these cell lines was evaluated after a 5-day exposure to various
concentrations of COP 41251 (Fig. 7). Only about 1% of the
A-l cells displayed apoptotic bodies and giant nuclei in the
absence or presence of 0.8 p.M COP 41251. The A-2 cells also
showed about 1% apoptotic cells in the control culture and 3, 4,
and 6% giant nuclei with fragmentation and apoptotic bodies
after a 5-day exposure to COP 41251 at 0.2, 0.4, and 0.8 p.M,
respectively. In contrast, OB-2 cells treated in parallel with
similar concentrations of COP 41 25 1 showed an increase from
5.2% under control conditions to 21, 34, and 85% after exposure
to 0.2, 0.4, or 0.8 p.M COP 41251, respectively. The selective
increase in nuclear fragmentation in OB-2 cells was confirmed
after treatment for only 24 h with COP 41251 (data not shown).
Furthermore, the viability of glioblastoma cells was compared to
that of the nontransformed cells after treatment with COP
41251, using the MU assay. Cultures of GB-!, OB-2, OS-i,
A-I , and A-2 cells were treated with COP 4125 1 at 0.8 p.M and
compared to DMSO-treated control cultures. There was a sig-
nificant decrease in viability among the tumors cell lines, and
30, 50, and 60% in the GB- 1 , GB-2, and OS- 1 cells, respec-
tively. In contrast, the two astrocyte lines A-i and A-2 showed
a viability of greater than 90%. Thus, normal astrocyte cell lines
are only minimally affected by exposure to COP 41251 in
contrast to glioblastoma cell lines.
Antitumor Activity. The antiproliferative effect of COP
4125 1 was further investigated in xenografts. Two previously
described human glioblastoma cell lines, U87 MO and U373
MO, were used for the tumorigenicity studies since they have
been shown to grow rapidly in nude mice in contrast to the
above-described GB-i and OB-2 lines. In this study, serially
passaged tumor fragments of U87 MO as well as U373 MO cells
were grafted s.c. into athymic mice, and the mice were observed
for tumor formation for the following 18 or 25 days, respec-
tively. The mice were treated once daily with COP 41251, with
either 75 mg/kg body weight p.o. or 10 mg/kg body weight iv.,
beginning on day 5 after transplantation and continuing to day
17. As shown in Fig. 8A, the growth ofthe transplanted U87 MO
tumor was inhibited by about 50% when the athymic mice were
treated with COP 41251, either when administered p.o. or iv.
Orowth of the U373 MO grafts was completely inhibited when
the animals were treated with COP 4125 1 (Fig. 8B). Further-
more, in the group of mice carrying the U373 MO tumor, the
drug was removed at day 1 7, and the animals were observed for
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
DMSO
CGP41251 200nM
CGP41251 400nM
CGP41251 800nM
Clinical Cancer Research 1027
Fig. 7 Nuclear morphology of nontransformed
astrocyte lines, A-l and A2, and the GB-2 glio-
blastoma-derived cell line after exposure to COP
4 125 1 . The cultures were seeded on LabTekchamber slides and maintained in the presence of
the indicated concentrations of COP 41251 for S
days. They were then fixed, and the DNA was
stained with DAPI and analyzed as described in
“Materials and Methods” and Table 4. Bars, SD.
the subsequent 9 days. There was negligible regrowth of the
tumor during the latter period of time. Thus, COP 41 25 1 is also
a potent inhibitor of the tumorigenicity of human glioblastoma
cells in athymic mice.
DISCUSSION
Olioblastoma multiforme, also designated grade IV astro-
cytoma, is a common form of primary human brain tumors (I 8).
It is associated with a poor prognosis, even with the most
advanced current types of multiniodal therapy (19). Previous
studies have demonstrated the expression of several isoforms of
PKC in astrocytomas of various grades (20-24) and provided
evidence for a role of PKC in regulating the proliferation of both
astrocytes and astrocytoma cells in vitro (25, 26). This study
demonstrates that both a potent activator of PKC, TPA, as well
as a potent inhibitor of PKC, COP 41251, inhibit the in vitro
proliferation of human glioblastoma cells and certain other types
of human brain tumor-derived cell lines (summarized in Table
2). Furthermore, the inhibition obtained with TPA plus COP
4125 1 is greater than that obtained when each agent is used
separately, suggesting that they inhibit growth by different
mechanisms. Consistent with this conclusion is the finding that
0 10 20 30 40 50 60 70 80 90 100
% of nuclei displaying fragmentation
cultures treated with COP 41251 form giant nuclei, whereas
cultures treated with TPA contain multinucleated cells.
At the present time, it is not apparent why both TPA, an
activator of PKCs, and the PKC inhibitor COP 4125 1 cause
growth inhibition of the glioma cell lines (Fig. I ). This is
apparently not simply because TPA causes down-regulation of
PKCs, since TPA-mediated growth inhibition was seen at doses
of TPA (0. 1 to 1 ng/ml; Fig. 1A) that did not produce detectable
down-regulation of PKCs a, �, #{128},or � (Fig. 2). Alternatively,
stimulation of the activity of one or more specific isoforms of
PKC in these cells by TPA might lead to growth inhibition.
Thus, it has been observed that overexpression of specific iso-
forms of PKC either stimulate or inhibit growth in the presence
of TPA, depending on the cell type (4, 27). Furthermore, over-
expression of PKCS in CHO cells results in growth inhibition
(28), and when these derivatives were treated with TPA, the
cells arrested in O,-M and there was an accumulation of cells in
telophase (28). It is of interest that although TPA is a tumor
promoter in mouse skin, TPA inhibits the in vitro growth of not
only human glioblastoma cells, as indicated in the present study,
but also the growth of a variety of human melanoma (29), breast
(30), and colon (27) cancer cell lines. In the latter cases, the
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
I -.-- Placebo treated controls� -ern- 75 mg/kg p.o. once dailyL-�--� 10 mg/kg i.v. once daily
- I I I I
5 8 11 14 18
days after tumor transplantation
-.- Placebo treated controls-0- 75 mg/kg p.o. once daily-k- 10 mg/kg iv. once daily
1028 Inhibition of the Growth of Olioblastomas by COP 41251
4 M. Begemann, unpublished results.
A 2.5’
2.0’
C’)
E’’C)
a)N(I)
0
E� 1.0’
B
0.5�
0.0
1.00
0.75
C’)
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.� 0.50
0
E
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5 10 15 20
days after tumor transplantation
Fig. 8 Antitumor activity of COP 41251. Antitumor activity was
tested using xenografts of the human glioblastoma multiforme cells U87
MG (A) and U373 MO (B), as described in “Materials and Methods.”
The compound was administered from day 5 to day 17 following s.c.transplantation of the tumor cells, as indicated. In both in vivo tumorgrowth experiments, admmistration of COP 41251 significantly inhib-
ited tumor growth (P < 0.001). In both cases, the placebo and treated
curves were significantly different, and the two modes of administration
of COP 41251 were not significantly different (A, P = 0.9; B, P - 0.6).Bars, SD.
precise mechanism of inhibition is also not known. Other acti-
vators of PKC that lack tumor-promoting activity, for example
the bryostatins (30-33), might therefore, be useful agents in
cancer therapy.
The present studies establish the fact that the staurosporine
derivative COP 4125 1 is a potent inhibitor of the in vitro growth
of nine early-passage cell lines derived from the following
human tumors: five glioblastoma multiforme, one mixed glio-
blastoma and gliosarcoma, two astrocytomas, and one choroid
plexus carcinoma. Using growth-curve studies, the IC�0 was in
the range of 0.4 p.M, although the OB-3 gliobiastoma cell line
was relatively resistant, since inhibition of growth required 0.8
to 1 p.M COP 4125 1 (Table 2). However, in clonogenicity
studies, this line was highly sensitive (Fig. 4). More detailed
studies suggest that CGP 4 125 1 inhibited the proliferation of
these cell lines through both cytostatic and cytocidal mecha-
nisms. However, although COP 41251 has a high selectivity for
inhibition of PKC (7), our studies do not exclude the possibility
that its growth-inhibitory effects in the above-described cell
types is due, at least in part, to inhibition of other protein
kinases. Of particular interest might be inhibitory effects on the
functions of the platelet-derived growth factor and epidermal
growth factor receptors (8), since these receptors have been
shown to be frequently activated in glioblastomas through mu-
tations and amplifications of the corresponding genes (34-37).
We hypothesize that COP 4125 1 inhibits the growth of
glioblastoma cells, at least in part, by arresting cells at the O2-M
transition point, and to a variable extent during � . This
hypothesis is consistent with the flow cytometry data (Table 3),
the inhibition of BrdUrd labeling (Fig. 3), the markedly reduced
number of mitotic figures, and the increased number of giant
nuclei with fragmentation and apoptotic bodies seen in the
treated cultures (Fig. 5). Studies in yeast have shown that PKC
is required for the 02-M transition (38). Furthermore, yeast cells
deficient in the cdc2/cyclin B complex are unable to transit from
the 02 to the M phase and form giant nuclei (39). In recent
studies, we have found that glioblastoma cells treated with COP
41251 exhibit a decrease in cdc2/cyclin B histone Hi kinase
activity.4 Other inhibitors of PKC have been found to arrest cells
in 02 or result in inhibition of cdc2 kinase activity (40-45).
High concentrations of staurosporine lead to the arrest of trans-
formed cells in 02 but arrest nontransformed cells in both G�
and 0, (� 45). However, further studies are required to deter-
mine the precise effects of COP 41251 on cell cycle progression
and cyclin-CDK activity.
A striking finding in the present study is that COP 412�1
induces nuclear fragmentation and other morphological changes
26 characteristic of apoptosis (Fig. 5). A common feature of apop-tosis is internucleosomal DNA fragmentation, which was inves-
tigated by preparation of high molecular weight DNA from
treated cultures as well as through various in situ procedures.
However, DNA fragmentation was not observed after treatment
with COP 41251 in these gliobiastoma cells (data not shown).
Nevertheless, apoptotic cell death has been shown to occur
without DNA fragmentation (46-48). The absence of DNA
Research. on March 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1029
fragmentation might reflect the absence of endonucleases in
these particular cell types or, alternatively, different pathways
leading to cell death. The COP 41251-induced nuclear fragmen-
tation with apoptotic bodies was partially inhibited by TPA (Fig.
5), which is consistent with the ability of certain phorbol esters
to inhibit apoptosis in thymocytes (49). Serum deprivation,
actinomycin D, and cycloheximide markedly inhibited COP
4125 1-induced nuclear fragmentation, suggesting that de nos’o
RNA and protein synthesis and cell cycle progression are re-
quired for COP 41 25 1 to induce apoptosis, which presumably
occurs in 02. Another staurosporine derivative, K252a, has been
shown to uncouple cell cycle control in rat diploid fibroblasts,
leading to tetraploidization, which was inhibited also by serum
deprivation, cycloheximide, and actinomycin D (43). Other in-
vestigators (50) have recently reported that tamoxifen, which
also inhibits PKC, also induces apoptosis in human malignant
gliomas, but they did not examine cell cycle-related effects.
Nuclear fragmentation and micronucleation, as well as 02 ar-
rest, were described in leukemic lymphocytes after exposure to
staurosporine, the parental compound, from which COP 41251
was derived (44). Immunohistochemical studies (data not
shown) provide evidence that OB-l, OB-2, and OS-! cell lines
used in the present study express a mutant p53 protein. This
suggests that COP 4125 1 induces apoptosis by a p53-independ-
ent pathway and, therefore, may be useful against a variety of
human tumors. Furthermore, cultures of normal astrocytes were
relatively resistant to the toxic effects of COP 41251, including
the induction of nuclear fragmentation (Fig. 7).
Finally, COP 4125 1 when given either by the oral route or
i.v. markedly inhibited the tumorigenicity of two previously
characterized human glioblastoma cell lines, U87 MO and U373
MO, following s.c. transplantation into athymic mice. This
finding extends previous studies indicating that COP 41251
inhibits the growth of other types of human cancer cells in
athymic mice (7). Thus, COP 41251 might be a useful agent
when administered either alone or in combination with other
agents for the therapy of human brain tumors.
ACKNOWLEDGMENTS
We are deeply grateful to Philippe Rousselot and Christine Neyt fortechnical advice, as well as valuable comments on the manuscript. We
thank Jingmei Xu for assistance with the statistical analyses.
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(Washington DC), 233: 305-312, 1986.
2. Nishizuka, Y. The molecular heterogeneity of protein kinase C and
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1996;2:1017-1030. Clin Cancer Res M Begemann, S A Kashimawo, Y A Choi, et al. promoter.inhibitor of protein kinase C, and by a phorbol ester tumor Inhibition of the growth of glioblastomas by CGP 41251, an
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