impact of the putative differentiating agents sodium ... · we have studied the impact of these two...

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:

[email protected].

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).



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.



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


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



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


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.




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.




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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