Vol. 3, 1 755-1762, October 1997 Clinical Cancer Research 1755
Impact of the Putative Differentiating Agents Sodium
Phenylbutyrate and Sodium Phenylacetate on
Proliferation, Differentiation, and Apoptosis
of Primary Neoplastic Myeloid Cells1
Steven D. Gore,2 Dvorit Samid, and Li-Jun Weng
The Johns Hopkins Oncology Center, Baltimore, Maryland 2 1287-8963 [5. D. G., L. J. W.], and The University of Virginia Health
Sciences Center, Charlottesville, Virginia 22903 [D. S.l
ABSTRACTSodium phenylacetate (PA) and sodium phenylbutyrate
(PB) are aromatic fatty acids that can effect differentiation
in a variety of cell lines at doses that may be clinically
attainable. We have studied the impact of these two agents
on lineage- and differentiation stage-specific antigen expres-
sion, proliferation, apoptosis, and clonogenic cell survival in
primary cultures of bone marrow samples from patients
with myeloid neoplasms at presentation and in remissionand from normal volunteers. PB inhibited the proliferation
of primary acute myeloid leukemia cells in suspension cul-
ture with an ID50 of 6.6 mr�i, similar to its ED50 in cell lines.At higher doses (�5 mM), PB also induced apoptosis. PB
inhibited clonogenic leukemia cell growth with a median
ID50 of less than 2 mM; however, colony-forming units-
granuiocyte/macrophage from patients with myelodysplasia
and normal volunteers were inhibited with a similar ID50. Incontrast to PB, its metabolite PA had no significant effect on
either acute myeloid leukemia proliferation or apoptosis.
Expression of the monocytic marker CD14 was increased in
monocytic and myelomonocytic leukemias in response to PB,
and to a lesser extent, PA. Surprisingly, both agents ap-peared to increase expression of the progenitor cell antigen
CD34, as well as the DR locus of the human leukocyte
antigen. These data indicate that PB, but not its metabolite
PA, has significant cytostatic and differentiating activity
against primary neoplastic myeloid cells at doses that may
be achievable clinically.
INTRODUCTION
Because of profound abnormalities of differentiation in
MDS3 and AML, these disorders have been targets for treatment
using agents that may effect differentiation, hopefully leading to
improved hematopoietic function. Although many cases of
AML can be cured using aggressive cytotoxic chemotherapy
and/or bone marrow transplantation, allogeneic bone marrow
transplantation represents the only known curative therapy for
patients with MDS ( 1-6). Unfortunately, the advanced median
age of patients with MDS and the lack of appropriate HLA-
matched donors for the majority of patients make this approach
available to few patients with this group of disorders. Aggres-
sive chemotherapy can induce remissions in approximately 50%
of patients with MDS; however, remissions appear to be short-
lived, with median remission durations of approximately I year
(7, 8). Although a variety of agents can effect cell cycle arrest
and terminal differentiation in leukemic cell lines and in primary
cultures of leukemic bone marrows, the clinical efficacy of most
agents has been limited by toxicity when doses approaching those
which cause differentiation in vitro are administered (9-I 1).
Recently, two aromatic fatty acids, PB and PA, have been
shown to have significant in vitro differentiating activity in
various models of hematological and epithelial malignancies
including HL-60 myeloid leukemia (granulocytic differentia-
tion; Ref. 12), KS62 leukemia cells (erythroid differentiation;
Ref. 12), high-grade glioma (13), prostate cancer ( 14), and
melanoma ( 1 5). Although millimolar concentrations of these
agents are required, similar to other differentiating agents such
as hexamethylene bisacetamide (16-18), both PB and PA have
been used clinically for the treatment of children with urea acid
cycle disorders (19-21) and the idiopathic hyperammonemia of
neutropenia associated with antileukemic treatment (22). In
those disorders, PA conjugates giutamine, forming phenylac-
etylglutamine, which is excreted in the urine. PB requires in vivo
metabolism to PA for its activity in these disorders. When used
for the treatment of urea acid cycle disorders, serum levels of PB
as high as 2.0 mM have been documented in the absence of
significant clinical toxicity (20, 23). The safety profile for these
Received 1/28/97; revised 5/30/97: accepted 6/19/97.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisen,ent in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by NIH Grant P30 CA06973. Presented inpart at the 1996 meeting of the American Association for Cancer
Research.
2 To whom requests for reprints should be addressed, at Johns Hopkins
Oncology Center, Oncology 2-109, 600 North Wolfe Street, Baltimore,
MD 21287-8963. Phone: (410) 955-8781: Fax: (410) 614-1005; E-mail:
3 The abbreviations used are: MDS, myelodysplastic syndromes: AML,
acute myeloid leukemia; HLA, human leukocyte antigen; PA, phenyl-
acetate; PB, phenylbutyrate: SF, steel factor; PE, phycoerythrin: FITC,
fluorescein isothiocyanate; FAB, French-American-British system of
leukemia classification: CFU-GM, granulocyte-monocyte colony form-
ing unit; CFU-L, leukemia colony forming unit: � dose of drug
which causes 50% of the maximum effect; � dose of drug causing
50% of maximal inhibition: HMBA, hexamethylene bisacetamide:
Ml-7, specific subtypes of AML according to the French-American-
British system of classification.
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1756 Phenylbutyrate in AML and Myelodysplasia
two agents makes them attractive drugs to be developed clini-
cally as cytostatic or differentiating agents in the treatment of
malignancy. The mechanisms by which PB and PA cause ter-
minal differentiation have not been determined. Both drugs have
been shown to inhibit DNA methylation, histone acetylation,
and protein isoprenylation (13, 24). More recently, these com-
pounds were found to stimulate the peroxisome proliferator-
activated receptor (PPARa), a member of the nuclear hormone
receptor superfamily known to control cell growth and differ-
entiation (25).
Although the ability of these agents to effect differentiation
in leukemic cell lines is encouraging, the correlation between
pharmacological effects in these tissue culture models and din-
ical leukemia samples is uncertain. We, therefore, sought to test
the activity of PB and PA as potential differentiation-inducing
or cytostatic agents in bone marrow cells from patients with
AML and MDS. The impact of PB and PA on lineage-associated
antigen expression, cell proliferation, apoptosis, and clonogenic
leukemic cell growth were studied to investigate the possible
utility of these drugs in the treatment of myeloid malignancies.
MATERIALS AND METHODS
Patients and Cells. Bone marrow samples were obtained
during routine clinical marrow aspirations from patients with
AML and MDS. All patients gave written informed consent for
the use of their bone marrow for research purposes as approved
by the Institutional Review Board under Department of Health
and Human Services guidelines. Mononuclear cells from hepa-
rinized samples of bone marrow aspirates were obtained by
density centrifugation (specific gravity, < 1 .077 g/dl; Ficoil-
Hypaque; Pharmacia, Piscataway, NJ). All bone marrow sam-
ples were studied on the day the marrow aspirate was obtained.
Cytocentrifuged preparations of the mononuclear cells were
stained with Wright’s stain, and differential blood counts were
performed manually. The myeloid leukemia cell line KGla was
maintained in RPMI 1640 (Sigma Chemical Co., St. Louis, MO)
and maintained at 37#{176}Cunder 5% CO2 as a control cell line for
proliferation assays. The growth factor-dependent myeloid leu-
kemia cell line TF-l was maintained under similar conditions
with the addition of interleukin 3 ( 10 ng/ml; a gift from Dr.
Lawrence Souza, Amgen Corp., Thousand Oaks, CA).
Suspension Culture. The impact of PB and PA (both
supplied by Elan Pharmaceuticals, Gainesville, GA) on leuke-
mid cells was studied in suspension culture. Samples were
plated in suspension culture medium as described previously
(26, 27) with 10% fetal bovine serum (HyClone Laboratories,
Inc. Logan, UT) in the presence of interleukin 3 and granulo-
cyte-macrophage/colony-stimulating factor (10 ng/mi of each
cytokine; granulocyte-macrophage/colony-stimulating factor
was also generously supplied by Dr. Lawrence Souza, Amgen),
and SF (SO ng/ml; a gift of Dr. Kristina Zsebo, Amgen). Cells
were plated at a concentration of 0.5 X 1 O6/mi in 25-cm2 flasks
(Nunclon z�; Nunc, Inc., Naperville, IL) for 3-7 days at 37#{176}C
under 5% CO2 before removing aliquots for determination of
cell proliferation, apoptosis, and expression of differentiation
stage-associated cell surface markers.
Cell Proliferation and Apoptosis. Leukemic cell prolif-
eration was determined using a flow cytometric assay for the
nuclear antigen Ki67 (26-30). This antigen is expressed in all
cycling cells but is absent from cells in G0; we have shown
previously that Ki67 expression in marrow mononuclear cells
from patients with AML measured in this assay correlates well
with other markers of proliferation, including S-phase determi-
nation following incorporation of bromodeoxyuridine and thy-
midine incorporation (26). The percentage of Ki67-positive cells
following 3 days of exposure to drugs was compared to the
percentage of Ki67-positive cells in cultures grown in the ab-
sence of drug. Apoptosis was determined as the percentage of
cells with <2N DNA following staining with propidium iodide
(Sigma; Ref. 3 1 ). As with the Ki67 assay, the percentage of cells
with <2N DNA following exposure to the study drugs was
compared to the percentage of apoptotic cells in cultures grow-
ing in the absence of drug.
Expression of Differentiation Markers. Evidence of
differentiation of leukemic cells was sought through immuno-
phenotypic analysis following 7 days of suspension culture.
Cells were stained for the following antigen combinations as
described previously (32-34): PE-CD14/fluorescein-labeled
FITC-CD1S and CD34-PEIHLA-DR-FITC. CD14 and CD1S
denote monocytic and granulocytic differentiation, respectively;
loss of CD34 and HLA-DR expression occur early in differen-
tiation of myeloid progenitor cells (35). The percentage of cells
expressing a particular antigen was determined based on stain-
ing of comparable cells using identical concentrations (based on
protein content) of fluorochrome-labeled, isotype-matched irrel-
evant mouse monoclonal antibodies. The following antibodies
were purchased from Dako Corporation (Carpinteria, CA):
CD14 (Tuk4), CD1S (C3D-i), HLA-DR (CR3/43), and isotype-
matched controls. CD34-PE (HPCA-2) was purchased from
Becton Dickinson (Mountain View, CA).
Clonogenic Assays. Clonogenic assays were performed
as described previously (36, 37) with the exception of the
incorporation of SF (SO ng/ml) and PIXY321 (20 ng/ml; a gift
of Dr. Steven Gillis, Immunex, Seattle, WA) into the methyl
cellulose plates in addition to phytohemagglutinin-stimulated,
lymphocyte-conditioned media. When the impact of phenyibu-
tyrate on colony formation was studied, graded doses of the drug
were incorporated into methyl cellulose plates (PB and PA are
not metabolized in vitro; Ref. 38). Leukemic progenitors
(CFU-L) were scored on day S of culture; granulocyte-macro-
phage colonies (CFU-GM) were scored on day 14. As in pre-
vious studies, CFU-L were grown from bone marrow from
patients with AML in remission following plating of mononu-
clear cells depleted ofT cells (36, 37). In this setting, CFU-L are
recognized as compact colonies of uniformly sized cells that
arise early, are scored on day 5, and subsequently decline before
the appearance of CFU-GM. Such colonies do not grow from T
cell-depleted bone marrow from normal volunteers (36, 39). As
described previously, Wright’s stained cytospins of aspirated
colonies show blast cell morphology (36). Such colonies have
been demonstrated to have leukemia-specific aberrant surface
antigen expression (39); CFU-L grown from patients with acute
lymphoblastic leukemia in remission carry the clonal immuno-
globulin or T cell receptor gene rearrangement characteristic of
the original leukemia (36). CFU-L could be successfully cul-
tured from bone marrow from 50% of cases of AML in remis-
sion (data not shown).
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Clinical Cancer Research 1757
Statistical Analysis. Changes in population means were
assessed using two-tailed Student’s t test for paired samples
(P < 0.05). To estimate the sensitivity of cells to the effects of
PB or PA, dose-response curves of a designated end point,
expressed as a percentage of control cell value, versus admin-
istered drug dose were plotted. Inhibition of Ki67 was plotted as
a linear curve; inhibition of CFU-L and CFU-GM were plotted
as log-linear curves as described previously (36, 37, 40). The
drug dose inducing 50% maximal drug effect was estimated
from the regression line of the dose-response curve derived
using least squares analysis of the raw or log-transformed data
(36, 37, 40).
RESULTS
Impact of PB and PA on AML Cell Proliferation andApoptosis. Because many agents that induce differentiation of
leukemia cells in vitro cause cell cycle arrest as a prominent
early effect (17, 41-44), we screened the impact of PB and PA
on the proliferation of primary AML cells. The AML patients
were phenotypically diverse (including FAB subtypes MO, 1, 2,
4, 4E, 5, and 7) and included seven patients with histories of
MDS. Seven patients had cytogenetics characteristic of high-
risk AML (45), and 1 1 expressed significant levels of the
progenitor cell antigen CD34, another feature of high-risk AML
(46). Bone marrow mononuclear cells from patients with AML
were placed in suspension culture in the presence of growth
factors and graded doses of PB or PA, as described in “Materials
and Methods.” Cultures were maintained for 3 days before
assessing the impact on proliferation, assayed as the percentage
of cells expressing Ki67 significantly compared to isotype-
matched controls. The mean results from I 6 patient samples
treated with PB and PA are displayed in Fig. 1A. PB reduced the
percentage of cells that expressed the proliferation-associated
antigen (Fig. 1A), as well as the total number of cells that were
Ki67 positive (data not shown). At 10 mtvi PB, proliferation was
profoundly inhibited, with Ki67-positive cells reduced to 14 ±
5% of control. In these primary cultures of AML cells, the
median ID50 of PB for inhibition of proliferation was approxi-
mately 6 mM. Growth arrest induced by PB was independent of
FAB classification; in fact, proliferation of all cases of AML
studied was inhibited by PB. In contrast to PB, PA had little effect
on AML cell proliferation, even at doses as high as 10 msi. Both
drugs were tested in concurrent samples from identical patients;
thus, this differential activity of the two aromatic fatty acids cannot
be attributed to biological heterogeneity of the samples treated.
Because declines in cell numbers in AML cultures exposed
to PB might be due to induction of cell death, we next tested the
effect of PB and PA on apoptosis. Aliquots of the day 3 cultures
were removed and assessed for the percentage of cells with <2
N DNA. Because primary AML cultures demonstrate a variable
degree of spontaneous apoptosis, results were normalized to the
percentage of apoptotic cells on day 3 of culture in the absence
of PB or PA (Fig. 1B). PB caused a dose-dependent increase in
apoptosis (132 ± 19% of control at S msi and 236 ± 36% of
control at 10 mrvi). Increased apoptosis was not seen at doses
below S mM. Similar to its lack of antiproliferative effect, PA
failed to induce apoptosis at doses as high as 10 ms�. In a subset
of patients, apoptosis was also assessed on day 7 of culture. No
significant increase in apoptosis was seen on day 7 after expo-
sure to PA (up to 10 mM). The changes in apoptosis induced by
PB were similar on day 7 and day 3 (data not shown).
Impact of PB and PA on Expression of Lineage- andDifferentiation Stage-specific Cell Surface Antigens.
Changes in the expression of differentiation-associated cell sur-
face antigens were studied after 7 days of treatment of suspen-
sion cultures. Fig. 2A displays a representative experiment using
PB; the sample studied was from a patient with acute my-
elomonocytic leukemia (FAB M4). PB induced a 7-fold increase
in expression of the monocytic marker CDI4. Significant in-
creases were seen at 2.5 and 5.0 m�i but not at 10 mrvi, a dose
that caused significant apoptosis. No change was seen in cx-
pression of the granulocytic marker CD1S. Unexpectedly, cx-
pression of the progenitor cell antigen CD34 also increased in
this patient, especially at higher doses of PB. The mean results
from 1 1 patient samples treated with PB are displayed in Fig.
2B. In this group of patients, a trend to increasing expression of
CDI4 was seen; this was due to four patients with monocytic or
myelomonocytic leukemias. In these four patients, 2.5 msi PB
increased CD14 expression to 229 ± 64% of control. Expres-
sion of the granulocytic marker CDIS was decreased signifi-
cantly in this cohort of patients. Expression of the progenitor
cell antigen CD34 was increased approximately 2-fold at 2.5
and S mM PB (this mean increase was statistically significant).
Concurrent with this increase in CD34 expression was an in-
crease in expression of HLA-DR, which is expressed on pro-
genitor cells and monocytes but not on maturing granulocytes.
The effect of PA (2.5-10 mM) on expression of these
antigens in six patients with AML was also studied. Although a
trend toward increased CD14 expression was seen at these
doses, no significant changes in mean values were seen (data not
shown). Lower doses of PA, tested in a total of I 6 patients, also had
no significant impact on antigen expression (data not shown).
Morphological evidence of increased differentiation was
not seen with either drug when compared with samples incu-
bated in growth factors alone after 7 days of culture (such
samples typically show increased size and granularity when
compared with the fresh leukemic samples).
To test whether PB regularly caused up-regulation of CD34
in hematopoietic cells which can express this progenitor cell
antigen, two CD34-expressing leukemic cell lines, KGla and
TF- I , were exposed to graded doses of PB in suspension for 3
and 7 days. Doses of PB ranged from 0.25 to S msi. CD34
expression was measured using flow cytometry. PB did not
significantly change the number of cells expressing CD34 nor
the mean fluorescence intensity of CD34 expression in either
cell line at any of the doses tested (data not shown).
Impact of PB on Clonogenic Cell Growth. Bone marrow
mononuclear cells from patients with AML represent a hetero-
geneous mixture of leukemic cells at a various stages of differ-
entiation. The Ki67 assay, which measures net cellular prolif-
eration, does not distinguish between proliferation of cells that
may be destined to terminally differentiate and cells that may
represent leukemic progenitor cells. We, therefore, tested the
impact of PB on the growth of clonogenic leukemic cells (CFU-
L). Graded doses of PB were incorporated directly into methyl
cellulose for constant exposure of cells to this agent as described
in “Materials and Methods.” CFU-L were scored on day S.
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A
0
C0
U.4-JCU)0
U)
0�
N-
0 2 4 6 8
Dose (mM)
----PA A --A--PA B
Fig. 1 Impact of PB and PA on proliferation andapoptosis of AML bone marrow cells. Bone mar-
row mononuclear cells from patients with AMLwere placed in suspension culture in the presence
10 of SF, PIXY321, and graded doses of PB or PA for3 days, as described in “Materials and Methods.”
Aliquots were removed, and the percentage of cellsexpressing the Ki67 nuclear antigen was deter-
mined using flow cytometry. The percentage ofapoptotic cells was determined as the percentage ofcells with <2 N DNA (P1). Results are reported as
the percentage of control cultures grown for 3 daysin the absence of drugs. In A, Ki67 results from 16patients treated with PB or PA. In 1 1 patients
(patient group A), the PA doses ranged from 0 to 2mM, and PB ranged from I to 10 mM; in 5 patients
(patient group B), doses of both drugs ranged from
0 to 10 mM. Data shown are means; bars, SE. B,impact of PB and PA on apoptosis; data are de-rived from the same experiments as in Fig. 1A.
-�---PB A
BC0
300
250
&Y�#{176}�:
0 2 4 6 8 10
1758 Phenylbutyrate in AML and Myelodysplasia
Dose, mM
Inhibition by PB of CFU-L growth was seen in bone
marrow samples from patients with newly diagnosed or relapsed
AML, AML in remission, and MDS. PB also inhibited the
growth of CFU-GM from bone marrow of patients with MDS
and normal volunteers. These data are summarized in Table 1.
In all patient populations tested, PB appeared to exert significant
suppression of CFU at doses significantly less than those re-
quired to inhibit net proliferation measured by the Ki67 assay.
The ID,0 for each population was variable, ranging from 0.2 mM
to as high as 8. 1 mrvi. However, the median ID50 was under 2 msi
for all patient populations tested. As with the Ki67 experiments,
CFU-L were significantly inhibited in patients with a variety of
FAB subtypes including M 1 , M2, M3, M4, M4Eo, and M6.
Samples studied from patients in remission included patients
with the following FAB subtypes: M 1 , M2, M4, M4Eo, and MS.
Again, no correlation was seen between FAB subtype and
response to PB. MDS samples were from patients with refrac-
tory anemia (1), refractory anemia with excess blasts (3), and
refractory anemia with excess blasts in transformation to acute
leukemia (1). No significant differences in PB sensitivity could
be detected between CFU-L and CFU-GM grown from patients
with MDS; the median ID,0 of PB for CFU-GM from normal
bone marrow was similar (1.7 mM).
DISCUSSION
The use of noncytotoxic agents to effect improved hema-
topoiesis in patients with bone marrow failure states has been a
focus of active laboratory and clinical investigation for at least
20 years. In MDS, the advanced age of most patients precludes
the widespread application of ailogeneic bone marrow trans-
plantation, the only present treatment with known curative po-
tential (1-6). A variety of agents that can induce differentiation
of leukemic cell lines have undergone clinical trials in MDS and
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A
Fig. 2 Impact of PB and PA on expressionof lineage- and differentiation stage-specificcell surface antigens. The cultures of AML
cells from Fig. 1 were harvested on day 7.
Cells were labeled using antibodies recog-nizing CD14, CD15, CD34, and HLA-DR.Data are reported as the percentage of day 7
control cultures. A, representative patientwith M4 AML. B, mean data from 11 patientsamples treated with PB (bars, SE). *, mean
value significantly different from 100% (P <
0.05).
800
700
600
500
400
300
200
100
0
400
300
200
100
0
CD14 CD15 CD34 HLA-DR
Differentiation Marker
B�2.5 �5 �1o
CD14 CD15 CD34 HLA-DR
Clinical Cancer Research 1759
05-
C0
U4-
0
CU)05-U)
0�
05-
C0
U4-
0
CU)05-U)
0�
LIII 0
Differentiation Marker
�2.5 �5
AML. Most of these agents have proven clinically toxic when
plasma levels approach the doses that have differentiating ad-
tivity in vitro. Examples include 1,25-dihydroxyvitamin D3 (hy-
percaicemia at doses well below those needed for differentia-
tion), HMBA (thrombocytopenia at miilimolar doses), low-dose
1-�3-D-arabinofuranosyicytosine (probably cytotoxic in those pa-
tients in whom responses have been achieved; Refs. 9-1 1, 47,
and 48). As with many agents studied previously, millimolar
doses of PB and PA are required to demonstrate differentiating
activity in hematopoietic as well as in epithelial tumor cell lines
(12-14, 49). However, when used for the treatment of metabolic
disorders in children and sickle cell anemia in adults, millimolar
plasma levels of PB have been documented in the absence of
toxicity (20, 23), suggesting that these agents may have promise
as potentially clinically useful differentiating agents for the
treatment of malignant disorders.
Table 1 Impact of PB administration on clonogenic cell growth
Bone marrow mononuclear cells or T cell-depleted mononuclearcells (AML in remission, MDS, and normal bone marrow) were platedin methylcellulose cultures as described in “Materials and Methods.”
Graded doses of PB were incorporated directly into the methylcelluloseplates. Each dose was studied in quadruplicate. CFU-L were scored onday 5; CFU-GM were scored on day 14. ID50 was estimated from theslope of the dose-response curve of the percentage of dlonogenic sur-
viva! (log-transformed) versus phenylbutyrate dose.
ID50 Rangen median (mM)
CFU-L (AML) 10 1.7 0.4-4.4CFU-L (AML in remission) 9 1 .3 0.4-8.1
CFU-L (MDS) 5 1 .0 0.2-2.0
CPU-GM (MDS) 4 2.1 1.4-3.9
CFU-GM (normal BM”) 5 1.7 0.1-4.5
a BM, bone marrow.
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1760 Phenylbutyrate in AML and Myelodysplasia
Hematological malignancies provide excellent models in
which to study differentiating agents due to ready access to
primary patient material and well-documented sequential line-
age- and differentiation stage-specific phenotypic changes.
However, the hematological malignancies remain heterogene-
ous collections of diseases, and terminal differentiation of leu-
kemic blast cells to the neutrophil stage in vitro has been
difficult to document (with the exception of acute promyelocytic
leukemia). Cell cycle arrest has been a prominent early event
documented in a variety of model systems exposed to differen-
tiating agents and may represent an important surrogate end
point in the development of these drugs (17, 41-44). We have
studied the inhibition of proliferation in primary AML cultures
in response to PB and PA, in conjunction with changes in
lineage- and differentiation stage-specific cell surface antigens,
to explore whether the differentiating activities of PB and PA
noted in cell lines might be exploited clinically. This study is the
first of which we are aware to investigate the activity of these
agents in primary samples of malignant myeloid cells.
At doses of 2.5-S mM, PB inhibited cell proliferation of
primary AML cultures, whereas higher doses induced apoptosis.
In contrast to PB, PA had no significant impact on either
proliferation or apoptosis at doses as high as 10 m�i. This
finding suggests that the activities of PB are not due to its
metabolism to PA (which does not appear to occur in vitro; Ref.
38), and that the results are not due to a nonspecific effect of
high PB dosage on the tonicity of the culture medium. A similar
differential in activity between these two agents has been re-
ported recently in prostate cancer cell lines (14).
The Ki67 assay does not permit determination of the cell
cycle phase in which cells are arrested. Although initially de-
scribed as an antigen that expressed through all phases of the
cell cycle except G0, this was carefully studied in activated
lymphocytes (28, 29). We have demonstrated previously that
changes in the percentage of Ki67-positive cells in primary
AML cultures correlate well with changes in the percentage of
cells in S-phase measured using bromodeoxyuridine or thymi-
dine uptake assays (26). The low proliferative rate of primary
AML cultures makes more detailed cell cycle analysis difficult
in most patient samples.
Both PB and PA led to altered expression of differentia-
tion-associated antigens in primary leukemic cells. The increase
in CDI4 expression, particularly in leukemias with a monocytic
phenotype, suggests that these agents may be more effective in
promoting monocytic than granulocytic differentiation. This is
in contrast to the induced granulocytic differentiation of HL-60
cells (12). Although increased expression of HLA-DR might
represent further evidence of monocytic differentiation, the in-
crease in CD34 expression seen with both PB and PA is not part
of any normal differentiation program. This suggests that the
“differentiation” programs induced by PB and PA in malignant
myeloid cells may not be normally coordinated and integrated;
in normal adult bone marrow, no cell has been described that
coexpresses CD34 and CD14 (although CD14IHLA-DR coex-
pression is a normal monocytic phenotype; Refs. 35 and SO).
Up-regulation of CD34 expression does not appear to be a
regular feature of PB treatment, based on experiments in the
KGla and TF-i cell lines. Further experience with a wider
variety of leukemic samples may clarify whether up-regulation
of CD34 expression is a regular event when AML cells are
exposed to PB. Future correlation with other measures of mono-
cytic differentiation, such as CD1 lb expression and phagocytic
activity, may lend further support to the ability of PB to induce
terminal monocytic differentiation.
It is not possible from these experiments to determine
whether the induction of differentiation by PB led to subsequent
apoptosis of the leukemic cells or whether the two activities of
PB are separate. Flow cytometric techniques for apoptosis mon-
itoring available when these experiments were performed did
not allow for accurate simultaneous monitoring of surface anti-
gen expression and apoptosis. The recently developed annexin
V assay, which measures externalization of phosphatidyi serine,
an event that occurs relatively early in apoptosis, can be per-
formed on viable cells colabeled with surface antibodies (51-
53). This technique should allow direct determination of poten-
tial coupling between differentiation and apoptosis in PB
treatment of AML cells.
Although peak serum levels of 2 m�i PB have been re-
corded in patients treated with this agent for nonmalignant
disorders, it seems unlikely that millimolar concentrations of
any drug could be sustained chronically. Effective clinical use of
differentiating agents may require prolonged administration.
Exposure of ML-! cells to lower doses of PB (0.5 mM) induces
cell cycle arrest several days after the effects of higher doses are
seen.4 The increased sensitivity of clonogenic precursors to PB
suggest that this agent may have important activity at doses that
might be feasible to administer chronically. However, the lack
of a clear therapeutic differential between leukemic and myelo-
dysplastic progenitor cells and normal CFU-GM raise concerns
that doses that might effectively suppress and/or differentiate
malignant clones may have toxic effects on normal progenitors.
HMBA had similarly overlapping dose-response curves be-
tween leukemic and normal progenitor cells and was found to
cause thrombocytopenia at targeted doses (10, 18). However,
PB has been successfully administered to patients with urea acid
cycle disorders and sickle cell anemia for extended periods of
time, and no hematological toxicity has been reported (54, 55).
Significant hematological toxicity was not reported in a Phase I
trial of PA in patients with malignancy (56).
It is important to specifically define the desired clinical
activity of “differentiating” agents. HMBA has been docu-
mented to increase the percentage of neutrophils with a clonal
cytogenetic abnormality in a patient with MDS, suggesting that
this agent effected more normal, but still clonal, hematopoiesis
( I 1 ). In contrast, when all-trans retinoic acid is used to induce
remission in acute promyelocytic leukemia, the malignant cells
terminally differentiate, apparently to cional extinction (57).
4 5. D. Gore and L-J. Weng. unpublished data. Bone marrow mononu-clear cells or T cell-depleted mononuclear cells (AML in remission,
MDS, and normal bone marrow) were plated in methylcellulose culturesas described in “Materials and Methods.” Graded doses of phenylbu-
tyrate were incorporated directly into the methylcellulose plates. Each
dose was studied in quadruplicate. CFU-L were scored on day 5;
CFU-GM were scored on day 14. ID3() was estimated from the slope of
the dose-response curve of the percentage of clonogenic survival (log-transformed) versus phenylbutyrate dose.
Research. on March 26, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1761
Remission is then characterized by normal cytogenetics, sug-
gesting that the terminal differentiation of the leukemic cells
induced by retinoic acid is followed by a relative growth ad-
vantage of normal progenitors. Thus, even if the therapeutic
window for PB between leukemic or myelodysplastic progeni-
tors and normal CFU-GM is narrow, it is possible that this agent
and related compounds could act similarly to retinoic acid in
acute promyelocytic leukemia: effecting terminal differentiation
and inhibiting proliferation of the myeiodysplastic or leukemic
clone, and enabling outgrowth of residual normal hematopoietic
progenitors. In fact, both all-trans and 1 3-cis-retinoic acids
cause marked in vitro inhibition of normal bone marrow
CFU-GM and erythroid burst-forming unit at doses that are
achieved in the therapy of acute promyelocytic leukemia (58, 59).
Finally, it is possible that combinations of differentiating
agents may be more effective than single agents. The dose-
response curve for butyric acid-induced differentiation of HL-60
cells is markedly shifted to the left when cells are treated
simultaneously with retinoic acid (60). Retinoic acid has also
been shown to augment the granulocytic differentiation of
HL-6O cells induced by submaximal doses of phenylacetate
(12). It is possible that such combinations may increase the
efficacy of PB at lower doses. Ultimate elucidation of the
genetic abnormalities underlying the aberrant differentiation in
myelodysplasia will enable more focused monitoring of the
effects of differentiating agents and will hopefully lead to mo-
lecularly targeted treatments. Until such targeted therapies can
be developed, PB appears to have significant activity against
neoplastic myeloid cells, and because of its attractive clinical
toxicity profile, PB represents an excellent candidate for clinical
trials in this group of disorders.
ACKNOWLEDGMENTS
We acknowledge the technical support of Margit Lucsay and the
expert assistance of Lisa Minick in manuscript preparation.
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1997;3:1755-1762. Clin Cancer Res S D Gore, D Samid and L J Weng cells.differentiation, and apoptosis of primary neoplastic myeloidphenylbutyrate and sodium phenylacetate on proliferation, Impact of the putative differentiating agents sodium
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