British journal oftfaematology. 1992. 82. 522-528

Effects of bryostatin 1 and rGM-CSF on the metabolism of 1 -P-D-arabinofuranosylcytosine in human leukaemic myeloblasts

S T E V E N G R A N T , ' ' w. D A V I D J A R V I S , ' A M Y J . TURNER. ' HUGH J . W A L L A C E ' A N D GEORGE R. PETTIT3

Division of Hcmatology and Oncology, Department of Medicine, and 'Department of Pharmacology, Medical College of Virginia, Richmond. Virginia, and 'Cancer Research Institute, Arizona State University, Tempe. Arizona

Received 16 March 1992; accepted for publication 23 Jutw 1992

Summary. The effects of the protein kinase C activator bryostatin 1 , either with or without recombinant granulo- cyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x lo-' M bryostatin 1 and 10-i 51 ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%: range 38-255%3, and no change in the remainder. In contrast. coincubation of cells with 1 .L 5 ng/ml rGM-CSF resulted in a significant increase in only one specimen. and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6,!9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincuba- tion of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA

incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3 / 8 additional specimens, and a decrease in one sample display- ing evidence of bryostatin I -induced macrophage differentia- tion. Incubation of cells with both rGM-CSF and bryostatin l for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of poten- tiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1- induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.

The nucleoside analogue of deoxycytidine. 1 -(I+-arabinofur- anosylcytosine (ara-C). is widely used in the treatment of acute myelogenous leukaernia (AML) in humans (Ellison et ni. 1968). Phosphnrylation proceeds by the pyrimidine salvage pathway enzyme, deoxycytidine kinase (dCK). and this process has been shown to represent the rate-limiting step in ara-C metabolism for ara-C concentrations greater than 1 0 ~ " M (White et al, 1987) . Ara-C is ultimately converted to its lethal triphosphate derivative. ara-CTP. which inhibits DNA polymerase 2 (Furth Sr Cohen. 1968) and is incorporated into elongating DNA strands. resulting in interference with DNA replication and eventually cell death

Correspondence: l)r Steven Grant. Division of Hematology/Oncology. Department of Medicine, MCI' Station Box 2 30. Medical College of Virginia. Richmond. Virginia 2 3298-02 30. L.S.A.

(Kufe ~t nl, 1980). Kecent reports have suggested a correla- tion between the formation of ara-CTP in leukaemic myelo- blasts and the response of patients with AML to ara-C containing regimens (Estey et a(. 1987). In addition to these biochemical considerations, ara-C is a prototypical S-phase specific agent. exerting its lethal effects only toward cells actively engaged in DNA synthesis (Young & Fischer. 1968). Since haeniatopoietic growth factors such as GM-CSF and 11,-3 are capable of inducing proliferative responses in leukaemic blasts (Miyauchi et al. 1987). it has been postu- lated that such factors might sensitize these cells to ara-C. For example, several groups. including ours, have demonstrated growth factor-mediated potentiation of ara-C cytotoxicity toward leukaemic myeloblasts, resulting from either S-phase recruitment and/or augmentation of ara-CTP formation (Cannistra et al. 1989; Lista et al, 1988; Karp et al , 1 Y Y O ;

522

Bryostatin 1 and ara-C in Leukaemic Myeloblasts 52 3

with acute myelogenous leukaemia. Only specimens consist- ing of at least 80% myeloblasts were included in these investigations. These studies have been approved by the Human Investigations Committee of the Medical College of Virginia. Samples were aspirated into sterile syringes con- taining 100 I.U. of preservative-free heparin, and diluted 1 : 5 in aMEM medium. Marrow samples were then passed through 1 6 gauge needles to disperse clumps, and the suspensions were gently layered over a cushion of Ficoll- Hypaque (sp.gr. 1.077-1.081; Sigma Chemicals, St. Louis, Mo.) contained in SO ml polystyrene centrifuge tubes. The tubes were centrifuged at 400 g at room temperature for 38 min, and the interface layer, containing blasts, aspirated with a sterile Pasteur pipette. The blasts were resuspended in EMEM medium containing 10% fetal calf serum (Hyclone, Logan, Utah), and cell densities determined utilizing a Model ZBI Coulter Counter. The cell concentrations were then adjusted accordingly, and the tubes placed in a 3 7°C 5% C 0 2 incubator until addition of agents as described below.

Drug exposure. 4 ml aliquots of the leukaemic blast cell suspension (cell density = 5 x lo6 cells/ml) were placed in 15 ml polystyrene centrifuge tubes are previously described for HL-60 cells (Grant et al, 1991a). To each tube was added

M ara-C (either labelled or unlabelled) and 12.5 x M bryostatin 1 or 1.2 S ng/ml rGM-CSF, alone or in combina- tion. Before removal for the biochemical determinations described below, the tubes were placed on their sides at a 45" angle in a 3 7OKhy C, 5% COz, fully humidified incubator for 4 h with periodic agitation. In some studies, cells were exposed to bryostatin 1 and/or rGM-CSF for 24 h followed by addition of ara-C; biochemical studies were performed after an additional 4 h of incubation.

Ara-CTPformation. The formation of ara-CTP in leukaemic blasts following a 4 h exposure to ara-C was determined by a previously published high-pressure liquid chromatographic method (Grant et al, 1991a). Briefly, after incubation with drugs, cells were washed twice with cold PBS and nucleotides extracted utilizing TCA and freon-octylamine according to the method of Khym (1975). The extract was then subjected to HPLC analysis employing Bio-Rad Model 800 Work- station, a Beckman Model 260 UV detector, a Waters Partisil SAX column, and a gradient separation method initially described by Plunkett et al (1987). UV absorbance was monitored at 280 nm, and peak areas corresponding to ara- CTP (retention time 28.6 min) were integrated automatically and compared to values obtained for known standards.

ara-C incorporation into DNA. After incubating cells with M [3H]ara-C for 4 h, the incorporation of ara-C into DNA

was determined by a previously described method (Bhalla et al, 1987). Following lysis of cells, DNA was isolated by proteinase and RNAase digestion, neutral phenol-chloro- form extraction, and ethanol precipitation. The DNA was quantitated spectrophotometrically, the radioactivity in designated aliquots determined utilizing a Beckman Model LS 8000 liquid scintillation counter, and ara-C DNA incorpora- tion expressed as pmol ara-C/pg DNA.

Statistical analysis. The significance between values obtained for control and treated cells was determined utilizing the Student's t test for paired observations.

Bhalla et al, 1988). Based in part upon these findings, clinical trials employing ara-C in conjunction with haematopoietic growth factors have been initiated in patients with refractory AML (Bethlehem et al, 1991).

Bryostatin 1 is a non-tumour promoting macrocyclic lactone activator of protein kinase C (PK-C) derived from the marine bryozoan, Bugula neritina (Pettit et aI, 1970, 1982), and has attracted considerable attention as a candidate antileukaemic agent in view of its ability to stimulate the in vitro growth of normal human multipotent progenitors (May et al, 1987). Concurrently, it has been shown to induce differentiation in some continuously cultured leukaemic cell lines (Stone et al, 1988) and some primary cultures of human leukaemic myeloblasts (Kraft et al, 1989). Bryostatin 1 has also been shown to inhibit the primary cloning efficiency (Jones et al, 1990) as well as the self renewal capacity (Grant et al, 1991 b) of leukaemic blasts in culture. Based upon these considerations, clinical phase I trials of bryostatin are currently in progress. In a recent communication it was reported that bryostatin 1 potentiated ara-CTP formation in the human promyelocytic leukaemia cell line HL-60, and that this effect was most pronounced in high-density cells exhibiting impaired ara-C phosphorylation (Grant et nl, 1991a). Subsequently, it was demonstrated that combina- tions of ara-C and bryostatin 1 (with or without rGM-CSF) were highly inhibitory toward the self-renewal capacity of leukaemic blast progenitors, whereas identical regimens spared a significant fraction of normal committed and primitive haematopoietic progenitors (Grant et al, 1992). Together, these observations raise the possibility that bryo- statin 1-mediated potentiation of ara-C phosphorylation might be responsible, at least in part, for the antileukaemic activity of this combination. The present studies were undertaken to determine what effect, if any, bryostatin 1 might have on ara-CTP formation in patient-derived leukae- mic myeloblasts exposed to a physiologic concentration of ara-C, and to compare this effect to that exerted by rGM-CSF. An additional goal was to determine whether coadminist- ration of bryostatin 1 and rGM-CSF might lead to a further potentiation of ara-C phosphorylation in these cells.

MATERIALS AND METHODS Reagents. Bryostatin 1 was isolated as previously described

(Pettit et al, 1970, 1982); immediately before use, it was dissolved in sterile DMSO, diluted in aMEM medium, and stored frozen at - 80OC. The final concentration of DMSO in all experiments was less than O.OS%, and has been shown in independent experiments not to affect leukaemic cell re- sponses to ara-C (Grant et al, 1991b). rGM-CSF was kindly provided by Dr Paul Trotta, Schering Plough Corporation, Bloomfield, N.J.; before use, it was diluted in sterile medium containing 1% BSA and stored frozen at -2OOC. Cytosine arabinoside hydrochloride was purchased from Sigma Chemicals, stored desiccated at - 20"C, and dissolved before application in sterile medium. [ 3H]ara-C ( 2 3 Ci/mmol) was purchased from Amersham Radiochemicals, Arlington, Ill., and determined to be > 99% pure by HPLC.

Cells. Leukaemic myeloblasts were obtained with informed consent from the peripheral blood or bone marrow ofpatients

524 Steven Grant et nl

RESULTS

The effect of bryostatin 1 ( + rGM-CSF) on the 4 h intracellu- lar accumulation of ara-CTP by leukaemic myelohlasts obtained from 10 patients with AML is illustrated by the data presented in Table I. Several groups, including ours. pre- viously have found this exposure interval sufficient to permit close to steady-state intracellular ara-CTP levels to accumu- late (Rhalla et al. 1987). The individual samples were equally divided between hone marrow and peripheral blood sources, and were obtained from an equal number of patients who had either previously received ara-C or who were ara-C naive. The majority of specimens exhibited the MMZ or M 4 FAB classification. Although considerable heterogeneity was noted in the responses of individual specimens to both ara-C and the other agents. six of the 10 samples displayed a significant increase in ara-CTP formation in the presence of bryostatin 1. In the majority of cases the increase was approximately 100% or slightly less. although a 25Oo/, increase was noted in one specimen exhibiting very low levels of ara-CTP formation (no. 7 ) . No decreases in ara-CTP formation were observed in cells exposed to bryostatin 1. In contrast, cells simultaneously exposed to rGM-CSF exhibited an increase in ara-CTP accumulation in only one sample. no change in seven. and a decrease in two specimens. U‘hen cells were co-incubated with both bryostatin 1 and rGM-CSF. increases in ara-CTP formation were in general equivalent to those observed with bryostatin 1 alone. In only one sample (no. 1 ) did addition ofbryostatin 1 lead to a further {modest) increase in triphosphate formation compared to rCM-CSF alone.

Effects of bryostatin 1 (and rGM-CSF) on ara-C IINA incorporation in leukaemic myelohlasts were similar in most respects to those noted for ara-CTP formation. For example. exposure of cells to bryostatin 1 produced significant in- creases in ara-C incorporation in 6,’9 evaluable samples (mean increase 108%: range 60-1 6Z(%,i. and no decreases. It is noteworthy that increases were ohserved in two specimens (nos. 1 and 4) that did not exhibit augmentation of ara-C‘I’P formation in response to brvostatin I. Exposure of cells to rGM-CSF resulted in an increase in ara-C DNA incorporation in 4/9 evaluablc. samples. and a decrease in one. As noted above. co-exposure of cells to bryostatin 1 and rCM-CSF did not lead to further increases in ara-C DNA incorporation (compared to bryostatin 1 alone). with the single exception of sample 9. which displayed a modest increment. Interestingly. a combined exposure to rGM-CSF and bryostatin 1 resulted in a reduction in ara-C DNA incorporation compared to bryo- statin 1 alone in one specimen (no. 3 ) and a decrease compared to rGM-CSF alone in another (no. 6 ) .

An additional eight specimens were assayed for ara-CTP formation following a 2 4 h exposure to bryostatin 1 i r G M - CSF. As shown in Table 11. considerable heterogeneity was observed in the responses of individual patient samples to each of these agents. For example. exposure of cells to bryostatin 1 for 7-1 h resulted in a significant increase in ara- CTP accumulation in three samples (range 66-278%). no change in four samples. and a significant reduction in one (no. 8 ) . lncubation of cells with rGM-CSF resulted in

enhanced ara-CTP formation in three specimens and no decreases. Co-exposure of cells to bryostatin 1 and rGM-CSF produced effects equivalent to those observed with bryostatin 1 alone, including a decrease in ara-CTP formation in cells obtained from patient 8. Interestingly, cells from this patient exhibited a n increase in adherence, esterase positivity, and early morphologic changes consistent with macrophage maturation following exposure to bryostatin 1 (data not shown). This observaton raises the possibility that bryostatin 1 might selectively inhibit ara-C phosphorylation in those cells committed to a differentiation programme following exposure to this agent.

DISCUSSION

Several considerations and recent observations suggest a possible rationale for the combined use of ara-C and bryosta- tin 1 in leukaemic therapy. Both ara-C (Nara et a], 1986) and bryostatin 1 (Jones et nl. 1990) inhibit the clonogenic growth of AML cells. and both limit the secondary cloning efficiency (PE-2 capacity) of leukaemic blast progenitors (Nara et al, 1986: Grant et al. 1991b), a n in vitro characteristic which has been shown to correlate closely with clinical outcome (Curtis rt a]. 1984). Furthermore, our group recentlyreported that exposure of leukaemic blasts to ara-C and bryostatin 1 ( 5 rCM-CSF) eliminated leukaemic self renewal capacity in the large majority of primary leukaemic specimens assayed, while sparing a significant fraction of their normal committed (CFIJ-GM) and primitive (HPP-CFC) counterparts (Grant rt at. 1992 ). In studies employing the human promyelocytic leukaemic cell line HL-60 as a model, it was demonstrated that bryostatin 1 potentiated ara-C phosphorylation (and DNA incorporation). and that these effects were greatest in high-density cells displaying reduced ara-C metabolism (Grant r t nl. 1991a) . While the mechanism underlying this phenomenon could not he fully elucidated, it appeared likely that bryostatin 1 induced perturbations in ribo- and deoxyri- bonucleotide pools, and resultant allosteric regulation of dCK. contributed to the observed effects. The present studies extend these findings to primary cultures of human AMI, cells. and suggest that bryostatin 1 is capable of enhancing ara-C metabolism in at least some patient-derived samples.

The heterogeneous effects that bryostatin 1 (and rGM-CSF) exerted on ara-CTP formation in leukaemic blasts obtained from different patients might have been anticipated from the results of previous studies employing these agents. For example. Kraft ct al (1989) reported that bryostatin 1 variably induced differentiation in primary cultures of human AML cells. as well as in different HL-60 sublines. Similarly. growth factors such as rGM-CSF have been shown to exert heterogeneous effects on the proliferation of AML cells in culture (Lendi eta! , 199 l) , as well as on leukaemic cell ara-CTP formation (Karp et ul, 1990). Furthermore. both bryostatin 1 and rGM-CSF are theoretically capable of altering ara-C metabolism by a variety of direct and indirect mechanisms, and these could potentially exert opposing effects on ara-C activation. For example, rGM-CSF might recruit blasts into S-phase, where the activity of dCK is known to he high (Colman el ul. 1975). In addition, rGM-CSF has

Tabl

e I.

Effe

ct of

bryo

stat

in 1

frG

M-C

SF o

n ar

a-C

TP

form

atio

n an

d ar

a-C

DN

A in

corp

orat

ion

in le

ukac

mic

mye

lobl

asts

.

Con

ditio

n

Pt

1 Pt

2

Pt 3

Pt

4

Pt 5

Pt

6

Pt 7

Pt

8

Pt 9

Pt

10

(BM

,AC

+M2)

(P

B,A

C-M

2)

(PB

,AC

+M4)

(B

M,A

C+M

2)

(BM

,AC

-M2)

(P

B,A

c+M

4)

(BM

,AC

+M2)

(PB

.AC

-M5)

(P

B.A

C-M

2)

(BM

,AC

+M2)

~

~~

~

4 hr

ara

-CTP

for

mat

ion

(pm

ol/l

Ob c

ells

) (1

) C

ontr

ol

8.7

f0.7

48

.3zk

5.3

12

.3f2

.1

19

.4fl

.X

39

.3f3

.6

20

.2h

2.4

0

.9f0

.2

(2)

Bry

osta

tin 1

8

.41

0.6

7

2,4

16

.9"

20.3

+3.

0d

24

.6f1

.9

54

.3f4

.2d

2

1.6

il.9

3

.2f0

.ba

(3)

rGM

-CSF

4.

6f0.

6'

53

.7f5

.2

6.9

k 0.

8'

14.3

f 2.

4 3

6.4

54

.2

20.8

+ 2.

9 2

.7f0

.4"

(4)

Bry

osta

tin l

+rG

M-C

SF

12

.7f2

.3"

79.6

zk6.

3a

25

.1f3

.3"

24

.6f3

.1

48

.6f3

.9

19

.8f8

.1

3.1

10

.5a

4 h

ara-

C D

NA

inco

rpor

atio

n (p

mol

ara

-C/p

g D

NA

) (1

) Con

trol

0.1

18 f0

.12

0.

1 jO

f0.0

13

0

.23

0f0

.03

1

0.01

7 f0

.00

2

04

31

f0.0

03

0.

025

f0.0

02

N

.D.

(2)

Bry

osta

tin 1

0.

247f

0.02

3a

0.32

9f0.

042"

0

.24

9f0

.02

0

0.0

46

f0.0

04

' 0,

049

f0.0

05"

0.0

29

h0

.00

3

N.D

. (3

) rG

M-C

SF

0.1 7

6f0.

018a

0.

123

f0.0

10

0.

107f

0.06

0'

0.01

5 f0

.00

1

0.0

39

f0.0

02

0.

043

10.0

03"

N.D

. (4

) B

ryos

tatin

1 +r

GM

-CSF

0.2

84f0

.026

a 0.

314f

0.04

6"

0,13

5 f0

.01

4b

0.0

1 8 f0

.00

2

0.04

2 f0

.00

4a

0.02

2 3

~0

.00

2 N

.D.

29

.61

2.6

7

2.4

f7.6

1

6.4

f3.2

6

6.4

~k

5.4

~

70

.45

5.2

3

3.4

f5.2

a 3

6.4

f3.9

7

1.3

f7.8

1

5.2

f2.6

53

.71t

4.2~

6

9.6

f 8.

2 34

.2 f4

.3a

0.39

2 f0

.04

5

0.06

8 f0

.00

7

0,0

08

fO.0

01

0.71

2 f 0.

082'

0,

077 f 0.

008

0.01

7 f 0.

001"

0

,60

8f0

.06

3a

0.0

97

f0.0

08

a 0.

009f

0.00

2 0,

841 f

0.0

81

" 0

.10

7f0

.01

0a

0.0

16

f0~

00

1"

Leuk

aem

ic m

yelo

blas

ts fr

om 1

0 pa

tient

s w

ere

incu

bate

d w

ith 1

0-'

M a

ra-C

(lab

elle

d an

d un

labe

lled)

for

4 h

in th

e pr

esen

ce of

bry

osta

tin 1

(12.

5 x

M)+

~GM

-CSF

(1.2

5 ng

/ml)

and

ara

-CT

P fo

rmat

ion

and

[3H

]-ar

a-C

DN

A i

ncor

pora

tion

dete

rmin

ed a

s de

scri

bed

in t

he t

ext.

Val

ues

repr

esen

t th

e m

eans

for

tri

plic

ate

dete

rmin

atio

ns f 1

SD. a

=sig

nifi

cant

ly g

reat

er t

han

cont

rol

(P<

0.05

);

-sig

nifi

cant

ly

less

tha

n co

ntro

l (P

<0.

05).

Abb

revi

atio

ns: B

M=b

one

mar

row

: PB

=per

iphe

ral

bloo

d: A

C+

=pri

or t

reat

men

t w

ith a

ra-C

: AC

- =

no p

rior

trea

tmen

t w

ith a

ra-C

: M2,

M4,

M5

=FA

B

clas

sifi

catio

n: N

.D. =

not

done

.

w b-

.

s 5 3 bl

D 3 a

Tabl

e 11

. Effe

ct o

f a

24 h

exp

osur

e to

bry

osta

tin 1

frG

M-C

SF o

n ar

a-C

TP

form

atio

n in

leuk

aem

ic m

yelo

blas

ts.

4 h

ara-

CT

P fo

rmat

ion

(pm

ol/1

06 c

ells

)

Pt 1

Pt

2

Pt 3

Pt

4

(PB

,AC

-M2)

(P

B.A

C +

M2)

(B

M,A

C + M

4)

(PB

A-

M2)

(1)

Con

trol

3

6.4

f3.4

6

.8f1

8

16.1

f

38

.6f2

.3

(2)

Bry

osta

tin 1

59

.21k

5.8~

1

8.9

12

' 4

2.8

k4

.4

78.5

k6.2

"

(4)

Bry

osta

tin 1

+rG

M-C

SF

62.4

f4.8

' 1

7.6

f3.2

4

3.9

f5.4

68

.3 f 5.

6d

(3)

rGM

-CSF

46

.3 f 3.

8"

9.6

f1.7

=

17

.3f2

.3

39.8

f4

.3

Pt 5

Pt

6

(BM

,AC

-M2)

(P

B,A

C+M

2)

33.2

f 3

.6

20.4

f 2.

1 1

8.9

f3.2

36

.4 %

4.3

32.4

& 3.

9 2

4.6

f3.2

3

3.8

f2.8

19

.8 f 2.

1

e ?

Pt 7

Pt

8

3

(BM

,AC

+M2)

(B

M,A

C+M

6)

r 0

*.

E * 5

14.1

212.

4 7

.3f1

.4

13.2

f 1.

8 3.

8% 1.

2b

19

.7f

1.8"

8

.9f2

.1

s-

15

.3f2

.9

3.6&

1.4b

z C

r

U

Leu

kaem

ic b

last

s fr

om e

ight

add

ition

al p

atie

nts

with

AM

L w

ere

incu

bate

d w

ith 1

2.5

x lo

-'

M b

ryos

tatin

1 f 1

.25

ng/m

l rC

M-C

SF f

or 2

4 h

and

ara-

CT

P fo

rmat

ion

E z n

dete

rmin

ed a

fter

an

addi

tiona

l 4 h

incu

batio

n w

ith 1

W5

M a

ra-C

. Val

ues

repr

esen

t the

mea

ns fo

r thr

ee re

plic

ate d

eter

min

atio

ns f 1

SD. "

=sig

nifi

cant

ly g

reat

er t

han

cont

rol:

h- -s

igni

fica

ntly

.

less

than

con

trol

(P

<0,

05).

Abb

revi

atio

ns a

re th

e sa

me

as th

ose

liste

d in

Tab

le I.

cg w1

h,

VI

526 Steven Grant et a1 been shown to increase (Bhalla ut al . 1988) and bryostatin 1 to decrease (Grant r t nl, 199 l a ) intracellular concentrations of deoxycytidine triphosphate. a metabolite which exerts a strong negative regulatory influence on dCK activity (Ives & Durham, 1970) . Moreover, induction of leukaemic cell maturation by either of these agents would also be expected to influence ara-C metabolism. Differentiated cells exhibit lower levels of dCK activity. and higher levels of cytidine deaminase activity, than their less differentiated counterparts (Chiba r t al. 1989). In this regard. it is noteworthy that the one sample that displayed a decrease in ara-CTP formation following a 24 h exposure to bryostatin I exhibited clear evidence of cellular maturation. This observation raises the possibility that bryostatin 1 might exert divergent effects on ara-C metabolism in different leukaemic cell populations. including reduced triphosphate formation in cells undergo- ing cellular maturation, and increased forma tion restricted to those cells unable to exhibit such a response.

The attempt to determine whether bryostatin 1 could augment the known capacity of rGM-CSF to potentiate ara-C phosphorylation in leukaemic blasts was prompted by evi- dence suggesting that activity of PK-C may regulate the responses of haematopoietic cells to growth factors. For example, Kanakura et nl ( 19 9 1 1 have shown that PI<-C activators such as TPA alter GM-CSF and IL-3 induced phosphorylation patterns in the M O T leukaemia cell line. and have postulated that these perturbations may be responsible for changes in cellular behaviour. It has also been demon- strated that coadministration of bryostatin 1 dramatically potentiates the response of committed myeloid progenitor cells to rGM-CSF (McCrady ( I t nl. 19 9 1 I and rIL- 3 (McCrady r t (11, 1990). However. in the current studies, a consistent pattern in the response of individual leukaemic specimens to bryostatin 1 or rGM-CSF was not detected, whether adminis- tered alone or together, as far as ara-C phosphorylation (or DNA incorporation) was concerned. For example. while some specimens exhibited an increase in ara-CTP formation in response to bryostatin 1 but not rGM-CSF (and rarely. vice versa). coadministration of these factors did not lead to a further augmentation of ara-C activation. These findings are consistent with results obtained in the HI.-60 cell line. in which rGM-CSF and rIL-3 were unable to mimic the effects of bryostatin 1 in potentiating ara-CTP formation (Grant c't r r l . 1991a). Together. these observations suggest that bryostatin 1 and rGM-CSF exert their effects on ara-C metabolism through non-interactive mechanisms.

Because of the relatively small sample population assayed. it cannot be ascertained whether cells obtained from patients who have received ara-C previously exhibit a different response to bryostatin 1 compared to cells from ara-C-naive patients. In this regard, it may be relevant that cells displaying resistance to ara-C may exhibit alterations in the activity of enzymes involved in de r~ovo pyrmidine biosynthe- sis ie.g. CTP synthetase: Brockman eta!. 1975 1, which could. in turn, influence responsiveness to bryostatin 1. Prospective analysis of a larger number of cell samples obtained from both treated and untreated patients will be necessary to resolve this issue definitively.

In view of previous evidence that the combination of

bryostatin 1 and ara-C preferentially inhibits the growth of leukaemic sersus normal haematopoietic progenitor cells in vitro (Grant et nl, 1992). it would clearly be of interest to compare the effect of bryostatin 1 on ara-C metabolism in leukaemic myeloblasts as well as in their normal counter- parts. Unfortunately. it is difficult to make direct comparisons since normal bone marrow aspirates represent a heteroge- neous cell population, of which true progenitors comprise a very small minority (e.g. 0.1%). The development of more sensitive techniques capable of measuring ara-C metabolites in progenitor cell-enriched populations of normal human bone marrow cells will therefore be required to document differential effects on ara-C metabolism in the two cell types.

In summary, the present findings demonstrate that bryo- statin 1 exerts heterogeneous effects on the metabolism of ara-C in patient-derived human leukaemic myeloblasts, and that these effects appear to be distinct from those induced by rGM-CSF. Moreover. it is apparent that this agent can potentiate ara-CTP formation (and ara-C DNA incorporation) in a subset of leukaemic specimens, including some that do not exhibit such a n increase in response to rGM-CSF. In this respect, the response of such specimens to bryostatin 1 is similar to that previously observed in high-density HL-60 cells. Identifying the mechanism(s) responsible for bryostatin 1 -mediated actions in individual leukaemic samples will necessitate a broad range of simultaneously performed studies, including assessment of both dCK and cytidine deaminase activity, cell cycle and deoxyribonucleotide pool perturbations, induction of leukaemic cell differentiation, as well as examination of different schedules of drug administra- tion. Whatever the mechanism, the present findings raise the possibility that bryostatin 1 induced potentiation of ara-C metabolism may contribute to synergistic antileukaemic interactions between these two agents, a t least in some leukaemic cells. It may be relevant to these speculations that Kufe and coworkers have recently reported that a variety of agents. including ara-C and bryostatin 1. induce up-regula- tion of immediate early response genes (e.g. c-jun, c-fos) in leukaemic cells through a PK-C dependent mechanism (Sherman et nI. 1990; Kharbanda et al, 1990). Furthermore, these events have been temporally related to the loss of leukaemic cell clonogenic potential and the appearance of endonucleosomal DNA cleavage characteristic of apoptosis or programmed cell death (Gunji et al, 1991). It is conceiv- able, therefore. that bryostatin 1 might potentiate the lethal effects of ara-C toward leukaemic cells by enhancing ara-C induced DNA damage. Preliminary results from our labora- tory suggest that this phenomenon does indeed occur in HL-(70 cells (unpublished observations), and parallel studies employing primary cultures of AML cells are currently being implemented.

ACKNOWLEDGMENTS

This work was supported by American Cancer Society Award CH-523. Cancer Center Support Core Grant CA-16059, and Cancer Biology Training Grant CA-09564-09, the Virginia Commonwealth Grants-in-Aid Program, the Fannie E. Rippel

Bryostatin 1 and ara-C in Leukaemic Myeloblasts 52 7 Grant, S.. Pettit, G.R., Howe. C. & McCrady. C. (1991b) Effect of the

PK-C activating agent bryostatin 1 on the clonogenic response of leukemic blast progenitors to recombinant granulocyte-macro- phage colony stimulating factor. Leukemia, 5, 392-398.

Grant, S., Traylor. R.. Bhalla, K.. McCrady. C. & Pettit, G.R. (1992) Effect ofa combined exposure to cytosine arabinoside. bryostatin 1 and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells. Leukemia, 5, 432-439.

Gunji, H.. Kharbander, S. & Kufe, D. (1 991) Induction of internucleo- soma1 DNA fragmentation of human myeloid leukemia cells by 1 - b-D-arahinofuranosykytosine. Cancer Research. 51, 741-743.

Ives. D.H. & Durham. J.P. (1970) Deoxycytidine kinase. 111. Kinetics and allosteric regulation of the calf thymus enzyme. Iournal of Biological Chemistry, 245, 2 2 8 5-2 2 94.

Jones, R.J., Sharkis. S.J., Miller, C.B., Rowinsky. E.K.. Burke, P.J. & May. W.S. (1990) Bryostatin 1. a unique biologic response modifier: Anti-leukemic activity in vitro. Blood, 75, 131 9-1 323.

Kanakura, Y.. Druker. B., Wood, K., Mamon, H., OKuda, K.. Roberts, T. & Griffen. J.D. (1 991) Granulocyte-macrophage colony-stimu- lating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene raf-1 in a human factor- dependent myeloid cell line. Blood, 77, 243-248.

Karp, J.E., Burke, P.J. & Donehower. R.C. (1990) Effects of rGM-CSF on intracellular ara-C pharmacology in vitro: comparability with drug-induced humoral stimulatory activity. Leukemia. 8, 5 53- 556.

Kharbanda, S.M.. Sherman. M.L. & Kufe. D.W. (1990) Transcrip- tional regulation of c-jun expression by arabinofuranosylcytosine in human myeloid leukemia cells. Journal of Clinical Investigation,

Khym, J.X. (1975) An analytical system for the rapid separation of tissue nucleotides at low pressure on conventional ion- exchangers. Clinical Chemistry. 21, 1245-1 252.

Kraft, A., William, F., Pettit, G. & Lilly. M.B. (1989) Varied differentiation responses of human leukemias to bryostatin 1 . Cancer Research. 49, 1287-1293.

Kufe, D.W.. Major. P.P.. Eagan. E.M. & Beardsley. E.P. (1980) Correlation of cytotoxicity with incorporation of ara-C into DNA. Journal of Biological Chemistry, 255, 8997-9000.

Lendi, R.M.. Gulati, S.C., Strife, A.. Lambek, C., Perex. R. & Clarkson. B.D. (1991) Proliferative response of human myeloid leukemia cells and normal marrow enriched progenitor cells to human recombinant growth factors IL-3, GM-CSF, and G-CSF alone and in combination. Leukemia. 5, 386-391.

Lista. P., Brizzi. M., Avanzi, G., Veglra. F., Resegotti. L. & Pegoraro. L. (1 988) Induction of proliferation of acute myeloblastic leukemia (AML) cells wtih hematopoietic growth factors. Leukemia Research. 12,441-447.

May, W.S.. Sharkis, S.J.. Esa. A.H.. Gebbia, V.. Kraft. A S . . Pettit. G.R. & Sensenbrenner. L.L. (1 987) Antineoplastic bryostatins are multipotential stimulators of human hematopoietic progenitor cells. Proceedings of the National Academy of Sciences of the United States ol America, 8483-848 7.

McCrady, C., Lei, F., Pettit, G. & Grant. S. (1990) Modulation of the response of highly purified human hematopoietic progenitor cells (MY-10+) to hematopoietic growth factors by bryostatin 1. Blood. 76, (Suppl. 1). 412.

McCrady. C., Staniswalis, J., Pettit, G.R., Howe. C.W.S. & Grant, S. (1991) Etrect of pharmacologic manipulation of protein kinase C by phorbol dibutyrate and bryostatin 1 on the clonogenic response of human granulocyte-macrophage progenitors to recombinant GM-CSF. British Journal of Haematology, 77, 5-1 5.

Miyauchi. J., Kelleher, C.A., Yang. Y., Wong, G., Clark, S.. Minden.

86. 1517-1523.

Foundation, the Robert B. Dalton Endowment Fund, the Bone Marrow Transplantation Core Research Laboratory of the Medical College of Virginia, and Outstanding Investigator Grant CA-44344-02A1 from the National Cancer Institute.

Portions of this work have been presented in preliminary form a t the Sls t Annual Meeting of the American Association for Cancer Research, Washington, D.C., 1990.

REFERENCES

Bethlehem, P., Valent. P.. Andreeff. M.. Tafuri, A., Haimi. J., Gorischek. C., Muhm, M.. Sillaber, C.. Haas, O., Vieder. L., Maurer, D., Schulz. C.. Speiser. K.. Kier. P.. Hinterberger. W. & Lechner. K. (1 99 1 ) Recombinant human granulocyte/macrophage colony- stimulating factor in combination with standard induction chemo- therapy in de novo acute myeloid leukemia. Blood. 77, 700-71 1 .

Bhalla, K., Birkhofer, M.. Arlin. Z., Grant, S. & Graham, G. (1988) Mect of rCM-CSF on the metabolism of cytosine arabinoside in normal and leukemic human bone marrow cells. Leukemia. 12,

Bhalla, K., MacLaughlin. W., Cole, J., Arlin, Z.. Baker, M.. Graham, G. & Grant, S. (1 987) Deoxycytidine preferentially protects normal versus leukemic myeloid progenitor cells from cytosine arabino- side-mediated cytotoxicity. Blood, 70, 568-571.

Brockman. R.W.. Shaddix, S.C., Williams, N.. Nelson. J.A.. Rose, L.M. & Schabel, F.M., Jr (1975) The mechanism of action of 3- deazauridine in tumor cells sensitive and resistant to arabinofura- nosylcytosine. Annals of the New York Academy of Sciences, 255,

Cannistra, S.A.. Groshek, P. & Griffin, 1.D. (1989) Granulocyte macrophage colony-stimulating factor enhances the cytotoxic effects of cytosine arabinoside in acute myeloblastic leukemia and in the myeloid blast crisis phase of chronic myelogenous leukemia. Leukemia, 3 , 328-334.

Chiba. F.P.. Tihan, T., Eher. R.. Koller, U., Wallner, C.. Gobl. R. & Linkesch. W. (1989) Effect of cell growth and cell differentiation on 1-/l-Darabinofuranosylcytosine metabolism in myeloid cells. Bri- tish lournal ofHaematology. 71, 451-455.

Colman. C.N.. Stoller. R.G., Drake, J.C. & Chabner, B. (1975) Deoxycytidine kinase: properties of the enzyme from human leukemic granulocytes. Blood. 46, 791-803.

Curtis, J.E., Messner, H.A.. Hassellock. R.. Elhakem, T.M. & McCul- loch, E.A. (1984) Contributions of host and disease-related attributes to the outcome of patients with acute myelogenous leukemia (AML). Blood, 2, 253-259.

Ellison, R.R., Holland. J.F., Weill. M., Jacquillat, C., Boiron, M.. Bernard, J., Savitsky, A.. Rosner, E., Sussof, B., Silvers, R.T., Karmas. A,, Cuttner. 7.. Spurr, C.. Hayes. D.M., Blom, J., Leone, L.A.. Haurani, F., Kyle, R., Hutchinson, J., Forcier. R.J. & Moon, J.H. (1968) Arabinofuranosylcytosine: a useful agent in the treatment of acute leukemia in adults. Blood, 32, 507-533.

Estey, E., Plunkett. W., Dixon. D., Keating. M., McCredie, K. & Freirich. E. (1987) Variables predicting response to high-dose cytosine arabinoside therapy in patients with refractory acute leukemia. Leukemia, 8, 580-583.

Furth. I.J. & Cohen. S.S. (1968) Inhibition of mammalian DNA polymerase by the 5’-triphosphate of 1 -p-D-arabinofuranosylcyto- sine and the 5’-triphosphate of 9-/f-~-arabinofuranosyladenine. Cancer Research, 28, 2061-2067.

Grant, S.. McCrady, C., Boise, L.. Westin. E. & Pettit, R. (1991a) EfSects of bryostatin 1 on the metabolism and cytotoxicity of 1-p-11- arabinofuranocyslcytosine in human leukemia cells. Biochemical Pharmacology, 42, 853-867.

8 10-81 3 .

501-521.

528 Stevrri Grant et (11

M.. Minken. M.M. McCulloch. E.,4. I 19871 The efforts of three recombinant growth factors. TI.-3. GM-CSF and G-CSF on the blast cells of acute myelohlastic leukemia maintained in short term suspension culture. Blood. 7 0 , 657-663.

Nara. N., Curtis. J . E . . Senn. J.S.. Pritchler. D.L. 8: hlcCulloch. E.A. (1 986) The sensitivity to cytosine arabinoside of the blast progenitors of acute myeloblastic leukemia. B h d . 67. 762-i69.

Pettit. G.R., Day. J.F.. Hartwell. J.L. 8 Wood. H.B. (1970) Antineo- plastic components of marine animals. ,Vanturf. 277. 962-96 3 .

Pettit. G.R., Herald, C.L. Douhek. D.L.. Herald. I1.L.. Arnold, E. 8 Clardy. j. ( 1 982 I Isolation and structure of hryostatin 1 . joiirnnl of the American Chemical SocietLy. 104. 6186-6487.

Plunkett. W.. Liliemark. 1.0.. Adams. TAI.. Norzak. R.. Estey. E.. Kantarjian. H. & Keating. U.1. (19871 Saturation of 1- / -1 ) - arabinofuranosvlcvtosine <’-triphosphate accumulation in leukemia cells during high dose 1 -/l-i)-arabiriofuranosylcytosine therapy. Cunccr Rcwurch. 47, 3 0 0 5 - 3 0 1 1 ,

Sherman. M.L., Stone. R.M.. Datta. R.. Bernstein, S.H. & Kufe. D.W. ( 19901 Transcriptional and post-transcriptional regulation of c- j i i n expression during monocytic differentiation of human myeloid leukemic cells. Journal of Biological Chumistry. 265, 3320-3323 .

Stone. R.M.. Sarihan, E.. Pettit, G.R. & Kufe, D.W. (1988) Bryostdtin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells. Blood. 72. 208-2 1 3 .

LVhite. J.C.. Rathmell. J.P. & Capizzi. R.L. (1987) Membrane transport intluences the rate of accumulation of cytosine arabino- side in human leukemia cells. Journal of Clinical Investigation, 79, 380- 38 7 .

Young. K.S.K. 8 Fischer. G.A. (1968) The action ofarabinofurano- sylcytosine on synchronously growing populations of mammalian cells. Rioc~!ietnii.ul and Bioph!gsicaI Research Communications, 32, 23-29.