Induction of Apoptosis and Differentiation By

Leukemia (1999) 13, 1046–1055  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Induction of apoptosis and differentiation by ﬂudarabine in human leukemia cells (U937): interactions with the macrocyclic lactone bryostatin 1 JA Vrana1, Z Wang1, AS Rao1, L Tang2, J-H Chen1, LB Kramer1 and S Grant1,2,3

Departments of 1Medicine 2Microbiology and Immunology, and 3Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA, USA

We have examined interactions between the purine nucleoside bine has shown significant activity in lymphoid malignancies ␤ analog fludarabine (9- -arabinofuranosyl-2-fluoroadenine) and such as chronic lymphocytic leukemia (CLL) and indolent the macrocyclic lactone bryostatin 1 in the human monocytic leukemic cell line U937. Fludarabine exerted dose-dependent non-Hodgkin’s lymphoma, but has also been used in the treat- effects on U937 cell viability and growth which were associated ment of acute myelogenous leukemia (AML), either as a single with both induction of apoptosis, as well as cellular maturation. agent,6 or in combination with 1-␤-d-arabinofuranosylcytos- Incubation of cells with bryostatin 1 (10 nM; 24 h) after, but not ine (ara-C).7 before a 6-h exposure to 10 ␮M fludarabine resulted in a modest Bryostatin 1 is a macrocyclic lactone activator of protein but significant increase in apoptosis, and was associated with kinase C (PKC), and is currently undergoing phase I and II greater than a 1 log reduction in clonogenicity. Subsequent 8 exposure to bryostatin 1 also increased the percentage of flu- evaluation. Bryostatin 1 variably induces maturation in darabine-treated cells displaying differentiation-related fea- human leukemic cell lines and primary AML blast speci- tures (eg plastic adherence, CD11b positivity) compared to mens,9,10 although it is considerably weaker in this regard than cells exposed to fludarabine alone. Bryostatin 1 did not the tumor-promoting phorbol diester, 12-phorbol-13-myristate increase the retention of the active fludarabine metabolite, F- acetate (PMA).11,12 The basis for the divergent spectrum of 3 ara-ATP, nor did it increase H-F-ara-A incorporation into DNA. activity of bryostatin 1 and phorbols remains unknown, but Despite its capacity to trigger cellular maturation, fludarabine may involve differential PKC isoform activation or translo- exposure (either with or without bryostatin 1) failed to induce 13,14 the cyclin-dependent kinase inhibitors (CDKIs) p21WAFI/CIP1 and cation. Notably, bryostatin 1 is a potent down-regulator p27KIP1. Nevertheless, dysregulation of p21 (resulting from of PKC, a process recently linked to proteasomal degradation stable transfection of cells with a p21WAFI/CIP1 antisense of the enzyme.15 construct) reduced fludarabine-mediated differentiation, while Previous studies from this laboratory have demonstrated inducing a corresponding increase in apoptosis. Enforced that bryostatin 1 increases the susceptibility of human leuke- expression of Bcl-2 partially protected cells from fludarabine- mia cells (eg HL-60; U937) to apoptosis induced by ara-C in related apoptosis, an effect that was overcome, in part, by sub- 16,17 sequent exposure of cells to bryostatin 1. Interestingly, Bcl-2- a dose- and sequence-dependent manner. In a human overexpressing cells were as or in some cases, more suscep- promyelocytic leukemic line (HL-60) insensitive to the differ- tible to differentiation induction by fludarabine (± bryostatin 1) entiation-inducing actions of bryostatin 1, pretreatment with than their empty vector-containing counterparts. Collectively, bryostatin 1 augmented ara-C-mediated apoptosis.17 In con- these results indicate that the antiproliferative effects of fluda- trast, in the human monocytic leukemic cell line U937, rabine toward U937 leukemic cells involve both induction of apoptosis and cellular maturation, and that each of these pro- exposure of ara-C-pretreated cells to bryostatin 1 significantly cesses may be enhanced by bryostatin 1. increased apoptosis, whereas exposure to bryostatin 1 before Keywords: fludarabine; bryostatin 1; U937 cells; apoptosis; differ- ara-C did not.18 Currently, it is unknown whether the ability entiation; Bcl-2 of bryostatin 1 to promote ara-C-induced apoptosis in human leukemia cells is restricted to this pyrimidine antimetabolite, or can be extended to include other nucleoside analogs. The Introduction purpose of this study was to characterize the antiproliferative effects of the purine analog fludarabine in human myelo- The purine analog fludarabine phosphate (9-␤-arabinofurano- monocytic U937 leukemia cells in relation to induction of syl-2-fluoroadenine monophosphate; FAMP) is a deoxyadeno- apoptosis and differentiation, and to determine what effect, if sine analog which is a potent inhibitor of leukemic cell any, bryostatin 1 might have on these actions. growth.1 FAMP is rapidly converted to the primary metabolite, 9-␤-arabinofuranosyl-2-fluoroadenine (F-ara-A) by ubiquitous phosphatases present in serum and plasma, and is sub- Materials and methods sequently phosphorylated intracellularly to form the active derivative, 9-␤-arabinofuranosyl-2-fluoroadenine triphosphate Cells (F-ara-ATP). The cytotoxic actions of F-ara-ATP may stem from multiple actions, including inhibition of DNA polymerase ␣ U937 cells were derived from a patient with diffuse histiocytic and DNA primase, inhibition of ribonucleotide reductase, and leukemia as previously described.12 The cells were main- nucleoside incorporation into DNA and/or RNA.2–4 In lymph- tained in RPMI 1640 (GIBCO, Grand Island, NY, USA) sup- oid cells, fludarabine induces apoptosis, an event that appears plemented with 10% fetal calf serum (FCS), MEM vitamins, to be critically dependent upon DNA incorporation.5 Fludara- sodium pyruvate, and penicillin and streptomycin (all ° GIBCO). Flasks were kept in a 37 C, 5% CO2, fully humidified incubator and cells were passaged twice weekly. Transfectant U937 cells stably overexpressing the anti- Correspondence: S Grant, Division of Hematology/Oncology, Medi- cal College of Virginia, Virginia Commonwealth University, PO Box apoptotic protein Bcl-2 were generated by electroporation of 980230, Richmond, VA 23298, USA; Fax: 804 225 3788 the parental line with a plasmid (pCEP4; Invitrogen, Carlsbad, Received 20 July 1998; accepted 14 January 1999 CA, USA) containing Bcl-2 cDNA under the control of an Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1047 CMV promotor and a hygromycin resistance marker as pre- nuclear condensation, the formation of membrane-bound viously described.19 These cells, designated U937/Bcl-2, blebs, etc) was determined by examining 7–10 randomly express approximately a seven-fold increase in Bcl-2 protein selected fields and scoring at least 1000 cells/condition. In compared to their empty-vector counterparts (U937/pCEP4) some cases, results were confirmed by monitoring DNA frag- and are maintained in 400 ␮g/ml hygromycin (GIBCO) for mentation following treatment of cell lysates with bisbenz- selection pressure. Levels of other Bcl-2 family members, amide staining as described previously.17 including Bcl-xL and Bax, have been found to be equivalent in the two lines. We have previously described a HL-60 cell line stably Differentiation studies expressing the cyclin-dependent kinase inhibitor (CDKI) p21WAFI/CIPI in the antisense configuration.20 More recently, we Two separate methods were employed to monitor induction have characterized the behavior of a U937 cell line that also of leukemic cell maturation. First, after the designated incu- exhibits p21WAFI/CIPI dysregulation.21 In response to the tumor- bation period (eg 24–72 h), the number of cells in suspension promoting phorbol PMA, antisense-expressing cells was determined using a hematocytometer. Subsequently,

(U937/ASF4) are impaired in G1 arrest, cellular maturation, adherent cells were removed using a cell scraper, additional pRB dephosphorylation, and CDK2 inhibition compared to medium added, and the cell number assessed as above. The empty vector controls (U937/pREP4). These cells are main- percentage of adherent cells, characteristic of a differentiated tained under selection pressure in medium containing phenotype, was expressed relative to the total population (eg 200 ␮g/ml hygromycin. adherent cells + cells in suspension). Alternatively, cellular maturation was assessed by monitoring expression of the myelomonocytic surface marker CD11b Drugs and chemicals by flow cytometry, as described previously.12 Briefly, treated cells were pelleted at 500 g and resuspended in cold phos- Bryostatin 1 was provided by Dr Anthony Murgo, Cancer phate-buffered saline (PBS) at 5 ×106 cells/ml. The cell suspen- Treatment and Evaluation Program/Division of Cancer Treat- sion was mixed with fluorescein isothiocyanate-labeled anti- ment, National Cancer Institute (Bethesda, MD, USA). It was body (CD11b or IgG2␣, Becton Dickinson, Mountain View, stored under light-protected conditions as a lyophilized pow- CA, USA) and placed on ice for 20 min. Cells were diluted in der at 4°C, and reconstituted in sterile dimethylsulfoxide cold PBS and analyzed using a Becton Dickinson FACScan (DMSO) prior to use. Material was subsequently diluted in flow cytometer and Verity Winlist software. In some studies RPMI medium to achieve the desired final concentration; in CD11b expression was measured separately in the adherent all cases, the final concentration of DMSO was Ͻ0.1%, and and suspension cell populations. found to exert negligible effects on the U937 cell responses. Fludarabine was provided by Dr Gary Williams, Berlex Lab- oratories (Richmond, CA, USA), stored under light-protected Clonogenic assays conditions at room temperature, and formulated in sterile water prior to use. 3H-fludarabine (10 Ci/mmol) was pur- A previously described method was employed.17 Briefly, after chased from Moravek Biochemicals (Brea, CA, USA). F-ara- drug treatment, cells were washed three times in drug-free ATP was kindly provided by Dr W Plunkett, MD Anderson medium, cell numbers normalized, and 500 cells/well plated Cancer Center (Houston, TX, USA). Deoxyadenosine tri- in 12-well plates containing RPMI medium, 20% FCS, and phosphate (dATP) was purchased from Sigma (St Louis, MO, 0.3% Bacto agar (Difco, Detroit, MI, USA). The plates were ° USA). placed in a 37 C, 5% CO2, fully-humidified incubator for 12 days, after which colonies, consisting of groups of у50 cells, were scored using an Olympus Model CK inverted micro- Experimental format scope (Olympus, Lake Success, NY, USA).

Logarithmically growing cells (approximately 4 × 105 cells/ml) were placed in 25 cm2T-flasks (Costar, Cambridge, MA, USA) Western analysis and incubated in the presence of fludarabine (generally 10 ␮m) for 6 h. At the end of this period, cells were washed Whole cell pellets (5 × 106 cells/condition) were washed once three times in drug and serum-free medium, and resuspended in PBS, resuspended in 100 ␮l PBS, and lysed by the addition in fresh medium containing 10% FCS at 2 × 105 cells/ml in of 100 ␮l 2X loading buffer (1×=30 mm Tris-base, pH 6.8, the presence or absence of bryostatin 1 (10 nm) for an 2% SDS, 2.88 mm ␤-mercaptoethanol, 10% glycerol, ෂ0.01% additional 24 h. In some cases, cells were exposed to these bromophenol blue). Lysates were sonicated, boiled for agents in the opposite sequence, eg bryostatin 1 → fludarab- 10 min, centrifuged at 12 800 g for 5 min, and quantified ine. At the end of the incubation period, cells were harvested using Coomassie protein assay reagent (Pierce, Rockford, IL, and prepared for analysis as described below. USA). Equal amounts of protein (25 ␮g) were separated by SDS-PAGE (5% stacker and 12% resolving gels) and electro- blotted to Optitran nitrocellulose filters (Schleicher and Schle- Assays of apoptosis icher, Keene, NH, USA). The membrane was stained in 0.1% amido black and destained in 5% acetic acid to ensure trans- Following appropriate drug exposures, cell morphology and fer and equal loading. The blots were then blocked in PBS- apoptosis was measured by cytocentrifuge preparations Tween (PBS-T; 0.05%)/5% dry milk for 1 h at 22°C, and stained with the Diff-Quik stain set (Dade Diagnostics, Agu- probed overnight at 4°C with primary antibody: p21CIP1/WAF1 ada, PR, USA), as previously described.12 The percentage of or p27KIP1; both 1:500 (Transduction Laboratories, Lexington, cells exhibiting classic apoptotic features (eg cell shrinkage, KY, USA). Blots were washed 3 ×10 min in PBS-T and incu- Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1048 bated with a 1:2000 dilution of peroxidase-conjugated sec- cells are shown in Figure 1a. Following treatment of cells with ondary antibody (Kirkegaard and Perry Laboratories, Gaithers- 10 ␮m fludarabine for 6 h and a subsequent 24 h incubation burg, MD, USA) in PBS-T for 1 h at 22°C. Blots were again in drug-free medium, a relatively small number of cells dis- washed 3 × 10 min in PBS-T and developed by enhanced played apoptotic features (eg ෂl8%). Treatment of cells with chemiluminesence (Amersham, Arlington Heights, IL, USA). bryostatin 1 alone for 24 h also weakly induced apoptosis in Western blots were reprobed with an anti-tubulin antibody; these cells. When cells were exposed to the sequence fludar- 1:2000 (Calbiochem, San Diego, CA, USA) to ensure equival- abine → bryostatin 1, the percentage of apoptotic cells ent loading and transfer. approached 30%. When cells were exposed to bryostatin 1 before fludarabine, the extent of apoptosis also increased, albeit additively, but was less than that observed for the F-ara-ATP retention

After treatment, 20 × 106 cells/condition were washed in cold PBS, lysed in 0.6 n TCA, and extracted in 1:3.5 Trioctyl- amine:1,1,2-Trichlorotriﬂuoroethane (Sigma-Aldrich). The aqueous phase was stored at −80°C until analysis. Immedi- ately prior to column addition, the samples were thawed and extracted in succession with 0.5 m sodium periodate, 4 m methylamine, and 1 m rhamnose to convert the NTPs to their respective bases.22 Extracts were run on a Waters radial-pak 10 u SAX cartridge (Waters, Millipore, Bedford, MA, USA), monitored at 254 nm on a Beckman 160 detector (Beckman, Fullerton, CA, USA), and analyzed with a BioRad Model 700 Chromatography Workstation (Version 3.63 software; Biorad, Hercules, CA, USA). Samples were run at 3 ml/min for 22 min in 25% ammonium phosphate (0.75 m, pH 3.7)/75% ammonium phosphate (5 mm, pH 2.8), then ramped up to 100% of the 0.75 m ammonium phosphate over the next 40 min. Peaks were identiﬁed by relative retention time compared to authentic F-ara ATP and deoxyadenine triphosphate.

3H-F-ara-A DNA incorporation

Cells were incubated with 10 ␮m 3H-fludarabine (final spe- cific activity = 8.6 Ci/mmol) for 6 h, washed free of drug, and incubated in either drug-free medium or medium containing 10 nm bryostatin 1 for an additional 24 h. Alternatively, cells were incubated with 10 nm bryostatin 1 for 24 h prior to a 6- h exposure to 3H-fludarabine. At the end of the incubation period, the cells were lysed and the DNA extracted as previously described.2 Radioactivity was quantified by scintillo- graphy, and the amount of 3H-fludarabine incorporated into U937 cell DNA expressed as pmol fludarabine/␮g DNA.

Statistical analysis

The significance of the difference between experimental conditions was determined utilizing the Student’s t-test for unpaired observations. To assess the interaction between 23 agents, Median Dose Analysis was employed using a com- Figure 1 (a) Cells were exposed to 10 nm bryostatin 1 (B) for 24 h mercially available software program (CalcuSyn; Biosoft, Fer- either before or after a 6-h incubation with 10 ␮m fludarabine (F). The guson, MO, USA). For these studies, the combination index percentage of apoptotic cells was then determined as described in (CI) was calculated for a two-drug combination involving a Materials and methods. Values represent the means for five separate fixed concentration ratio. Using these methods, CI values experiments ± s.d. *Indicates significantly greater than values for the → р Ͻ1.0 indicate a synergistic interaction. sequence B F; P 0.05. (b) Following exposure to fludarabine and bryostatin 1 as above, cells were washed free of drugs and plated in soft agar as described in the text. Colonies, consisting of groups of у50 cells, were scored on day 12. Values are expressed in relation Results to control colony formation, and represent the means for six to seven experiments performed in triplicate ± s.d. *Indicates significantly less than values for the sequence B → F; P р 0.02. Median dose effect The sequence-dependent effects of sequential exposure of analysis of clonogenic growth for the sequence F → B; CI values Ͻ1 cells to fludarabine and bryostatin 1 on apoptosis in U937 correspond to synergistic interactions. Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1049 sequence fludarabine → bryostatin 1 (P р 0.05). In each case, parallel effects on DNA fragmentation were observed (data not shown). Reductions in clonogenicity following exposure of cells to fludarabine or bryostatin 1 administered individually or sequentially are illustrated in Figure 1 b. In each case, inhibition of colony formation was considerably greater than the extent of apoptosis observed at the end of the drug incubation interval (Figure 1a). When cells were exposed to the sequence fludarabine → bryostatin 1, the reduction in colony formation was Ͼ1 log. The sequence bryostatin 1 → fludarabine also led to a significant reduction in colony formation (eg Ͼ80%), although inhibitory effects were not as great as those observed for the opposite sequence (P р 0.05). Median dose effect analysis employing varying concentrations of fludarabine (0.2–20 ␮m) and bryostatin 1 (0.2–20 nm) at a fixed ratio (1000:1) in the sequence fludarabine → bryostatin 1 resulted in combination index values Ͻ1, indicating a synergistic interaction (Figure 1c). To determine whether enhancement of fludarabine-related apoptosis by bryostatin 1 might stem from altered drug metabolism, retention of the active fludarabine metabolite F-ara-ATP was monitored over time in fludarabine-treated cells subsequently incubated in drug-free or bryostatin 1-containing medium (Figure 2). It can be seen that degradation of the triphosphate derivative was relatively rapid with either treatment, in that levels became virtually undetectable by 12 h of incubation. Furthermore, exposure of cells to bryostatin 1 did not result in a discernable difference in the rate of F-ara-ATP degradation (Figure 2a). Ratios of F-ara-ATP to its physiologic counterpart, dATP at 2 h and 6 h after a 6-h incubation with fludarabine were also unaltered by bryostatin 1 treatment (Figure 2b). Finally, incorporation of 3H-fludarabine into U937 cell DNA was not significantly altered by either a 24-h prein- cubation with or subsequent exposure to 10 nm bryostatin 1 (Figure 2c). Thus, enhanced fludarabine metabolism could not be invoked to explain the modulatory effects of bryostatin 1 on fludarabine actions. Because of the discordance between the extent of apoptosis induced by exposure to fludarabine/bryostatin 1 and the cor- respondingly greater reduction in cellular proliferative poten- tial, an attempt was made to determine whether additional mechanisms (eg cellular maturation) might be involved in this phenomenon. Fludarabine dose-response curves for induction of U937 cell differentiation, reflected by the extent of cellular Figure 2 Following treatment of cells with 10 ␮m fludarabine for adherence or CD11b expression, are shown in Figure 3a. In 6 h, cells were washed free of drug and resuspended in fresh medium the case of the latter, separate determinations were conducted ±10 nm bryostatin 1 for an additional 24 h. At the designated intervals, in the adherent and suspension cell populations. With both cells were removed, lysed, and intracellular levels of F-ara-ATP and assays, a biphasic dose-response was observed, with maximal dATP determined by HPLC as described in the text. (a) Effect of bryostatin 1 on intracellular levels of F-ara-ATP as a function of time. differentiation induction occurring at fludarabine concen- ̅ ␮m Medium ( ), Bryostatin 1 ( ). (b) Effect of bryostatin 1 on intracellular trations of 0.5 to 1.0 , and declining at higher levels. For ratios of F-ara-ATP and dATP 2 h (open bar) and 6 h (solid bar) after comparison, a fludarabine-induced apoptosis dose-response a 6-h incubation with fludarabine. Fludarabine followed by medium curve is shown in Figure 3b, demonstrating an increase in cell (F), fludarabine followed by bryostatin (F → B). (c) Alternatively, cells death at fludarabine concentrations greater or equal to those were exposed to 10 ␮m 3HF-ara-A (F) for 6 h followed (or preceded) associated with maximal maturation responses (eg 1.0 ␮m). A by a 24-h incubation in medium (C) or 10 nm bryostatin 1 (B). The 3 discordance was noted between fludarabine-stimulated cells were then lysed, the DNA extracted, and incorporation of H- F-ara-A into DNA determined as described in the text. In each case, adherence and CD11 b positivity, in that the percentage of values represent the means for three separate experiments ± s.d. adherent cells displayed increases over basal levels at drug concentration of 0.01–1.0 ␮m, whereas increased CD11b expression occurred primarily at fludarabine concentrations of nation of bryostatin 1 and fludarabine on U937 cell matu- 0.5–2.0 ␮m. Unexpectedly, the extent of CD11b positivity was ration were assessed. When administered individually, bryo- equivalent in the adherent and suspension cell populations, statin 1 (10 nm; 72 h) and fludarabine (10 ␮m; 6 h, washed, further indicating that these two maturation markers (cellular fresh medium; 72 h) induced expression of CD11b in 12.4 adherence and CD11b expression) are not induced in parallel ±3.8% and 7.1 ±3.4% of cells respectively (Figure 3c). When in fludarabine-treated cells. Lastly, the effects of the combi- cells were exposed to the sequence fludarabine → bryostatin Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1050 U937 cells to fludarabine and bryostatin 1 can lead to an increased maturation response, although this effect is only apparent at higher concentrations of fludarabine (10 ␮m). In parallel studies, utilizing the 10 ␮m concentration of fludarabine, the percentage of apoptotic cells at the 72-h interval was 8.9 ± 0.9% vs 14.2 ± 3.8% for fludarabine and the fludarabine → bryostatin 1 sequence, respectively (data not shown), consistent with results of previous studies demonstrating the late appearance of apoptosis in leukemic cells undergoing differentiation.24 The effects of a 72 h exposure to a lower concentration of fludarabine (1.0 ␮m) on induction of apoptosis and inhibition of cloning efficiency were then separately examined in the suspension and adherent cell populations (Figure 4). Contrary to expectations, the reduction in cloning efficiency (relative to controls) following fludarabine treatment was equivalent in both groups (eg ෂ10%). However, the percentage of apoptotic cells was significantly greater in the suspension fraction (P Ͻ 0.0001). Similar results were observed following treatment with a lower dose of fludarabine (0.3 ␮m) (data not shown). These findings suggest that the loss of self-renewal in fludarabine-treated cells displaying certain differentiation-related features (eg plastic adherence) is equivalent to that which results from enhanced apoptosis (eg in the suspension cell fraction). Induction of leukemic cell maturation (eg by the tumor-promoting phorbol PMA) is associated with increased expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1.20 To determine whether a similar mechanism is operative in U937 cells undergoing differentiation in response to bryostatin 1 and fludarabine, Western blot analysis was employed (Figure 5a). As we have recently reported,12 10 nm bryostatin 1 failed to induce p21CIP1/WAF1 at any point during the 72-h exposure interval. Furthermore, neither fludarabine alone, nor the combination of fludarabine and bryostatin 1 led to increased p21CIP1/WAF1 expression over this time period. In contrast, exposure of cells to 10 nm PMA resulted in clear induction of p21CIP1/WAF1 at 24 h, which increased modestly over the ensu- ing 48 h. In separate studies, the combination of fludarabine and bryostatin 1 was not associated with increased expression of the CDK1 p27KIP1 (data not shown). These findings suggest that differentiation induction by fludarabine (± bryostatin 1) can proceed in the absence of p21CIP1/WAF1 induction. To

Figure 3 (a) Logarithmically growing cells were exposed to the designated concentration of F-ara-A for 72 h after which the percentage of cells displaying plastic adherence (࡯), adherent cells expressing CD11b (̄) or cells in suspension expressing CD11b (̆) was determined as described in Materials and methods. Open symbols correspond to cells incubated in drug-free medium. (b) Alternatively, the percentage of cells exhibiting apoptotic features following a 72-h incubation with various concentrations of F-ara-A was determined as described above. *Indicates signiﬁcantly greater than control values; P р 0.05; ** = P р 0.01. (c) Cells were exposed to either drug-free medium (C) or 10 10 nm bryostatin 1 (B) for 72 h, or to 10 ␮m F- ara-A for 6 h followed by a 72-h incubation in medium (F) or 10 nm bryostatin 1 (F → B). The percentage of CD1 1b + cells was determined by ﬂow cytometry. In each case, values represent the means for three separate experiments ± s.d.

1 (F-ara-A; 10 ␮m; 6 h, washed, BRY: 10 nm; 72 h), the per- Figure 4 Cells were incubated in the presence or absence of ␮ centage of cells expressing CD11b increased to 26.3 ±6.3%. 1.0 m F-ara-A for 72 h, after which the adherent and suspension cell However, combination with bryostatin 1 did not lead to a populations were separately analyzed with respect to extent of apoptosis (open bars) and cloning efficiency (solid bars) as described further potentiation of maturation in cells exposed to lower in the text. Values represent the means for three separate experiments concentrations of fludarabine (eg 0.5–1.0 ␮m; data not performed in triplicate ± s.d. *Indicates significantly greater than shown). These results suggest that sequential exposure of values obtained for adherent cells; P Ͻ 0.0001. Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1051

Figure 5 Cells were exposed to 10 ␮m F-ara-A for 6 h, washed, and incubated in either fresh medium (F-ara-A) or medium containing 10 nm bryostatin 1 (F-ara-A→BRY) for an additional 72 h. Control, incubation in drug-free media; and BRY, cell treated with 10 nm bryostatin 1. (a) Aliquots of the cell suspension were lysed at 24-h intervals, proteins (10 ␮g/condition) separated by PAGE gel electrophoresis, and probed with anti-p21 monoclonal antibody as described in the text. As a control, protein extracted from cells exposed to 10 nm PMA for 24–72 h was also subjected to Western blot analysis. The results of a representative study are shown; two additional experiments yielded equivalent findings. The blot was reprobed with an anti-tubulin antibody to control for gel loading and transfer. (b) Alternatively, empty-vector control cells (U937/pREP4) and p21 antisense-expressing cells (U937/p21AS/F4) were exposed to 10 ␮m F-ara-A for 24 h, after which the percentage of apoptotic cells was determined (left panel), or incubated with 1 ␮m F-ara-A for 72 h, after which the percentage of CD11b+ cells was monitored by flow cytometry (right panel). * = Values significantly greater than those obtained in empty-vector controls; P у 0.05; ** = Values significantly lower than those of empty-vector controls; P р 0.02. Values represent the means for three separate experiments ± s.d. determine whether p21CIP1/WAF1 might nevertheless be treated with fludarabine alone (Figure 6b). These results are involved in the apoptotic response to fludarabine, a U937 cell compatible with recent findings indicating that bryostatin 1 line stably expressing p21CIP1/WAF1 in the antisense configur- reverses, at least in part, resistance to ara-C-related apoptosis ation was employed (U937/p21ASF4). Following exposure to conferred by Bcl-2 overexpression in the HL-60 promyelocytic PMA, these cells display diminished p21CIP1/WAF1 induction, leukemia cell line.19 Lastly, effects on cellular maturation were

G1 arrest, pRb dephosphorylation, CDK2 inhibition, and compared in cells containing an empty vector and those over- acquisition of differentiation-related features compared to expressing Bcl-2 (Figure 7). When fludarabine was adminis- their empty vector counterparts (pREP4),21 in a manner anal- tered alone, differentiation induction in Bcl-2 overexpressors ogous to that previously described in p21 antisense-expressing was either equal to or, in most cases, greater than, that HL60 cells.20 Despite the fact that fludarabine failed to induce observed in their empty vector-containing counterparts (Figure p21CIP1/WAF1, cells exhibiting p21CIP1/WAF1 dysregulation were 7a). Furthermore, when U937/Bcl-2 cells were sequentially significantly more sensitive to fludarabine-induced apoptosis exposed to fludarabine followed by bryostatin 1, the increase compared to controls (Figure 5b; left panel). Conversely, dys- in CD11b positivity was at least as great or greater than that regulation of p21CIP1/WAF1 significantly reduced the suscepti- noted in the case of untransfected or empty vector controls bility of U937 to fludarabine-mediated differentiation induc- (Figure 7b). These findings indicate that Bcl-2 overexpression tion (Figure 5b; right panel). does not protect cells from the maturation-related actions of The effects of Bcl-2 overexpression on the apoptotic fludarabine (or the fludarabine/bryostatin 1 combination), but response of cells to fludarabine were then examined (Figure may under some circumstances increase the susceptibility of 6). When cells were exposed to varying concentrations of cells to differentiation induction. fludarabine for 6 h followed by a 24-h incubation in drug-free medium, a significantly smaller percentage of Bcl-2-overexpressing cells (U937/Bcl-2) exhibited apoptotic features com- Discussion pared to their counterparts transfected with an empty vector (U937/pCEP4) (Figure 6a). When Bcl-2-overexpressing cells The results described herein demonstrate that fludarabine were sequentially exposed to fludarabine followed by bryosta- induces both apoptosis and differentiation in U937 cells, and tin 1, the extent of apoptosis, while less than that observed in that each of these events is enhanced, at least to a degree, by the pCEP4 line, was greater than that noted in control cells the macrocyclic lactone bryostatin 1. Furthermore, sequential Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1052

Figure 6 (a) Empty-vector control cells (U937/pCEP4) () and Bcl- Figure 7 (a) Logarithmically growing U937/pCEP4 ( ) and ࡯ 2-overexpressing cells (U937/Bcl-2) (࡯) were exposed to the desig- U937/Bcl-2 ( ) cells were continuously exposed to the designated nated concentration of fludarabine for 6 h, washed, and incubated concentration of F-ara-A for 72 h, after which the percentage of in drug-free medium for 24 h prior to determination of the extent of CD11b-expressing cells was determined by cytofluorometry. Values apoptosis. * = Values significantly less than those obtained for represent the means for three separate experiments performed in tripli- ± U937/pCEP4; P р0.05. (b) Alternatively, U937/pCEP4 (solid bars) and cate s.d. *, Significantly greater than values for U937/pCEP4 cells; р U937/Bcl-2 (open bars) cells were exposed to drug-free medium (C), P 0.05. (b) U937/Bcl-2 cells were sequentially exposed to drug-free ␮ 10 nm bryostatin 1 for 24 h (B), or the sequence 10 ␮m F-ara-A for medium (C), 10 nm bryostatin 1 for 72 h (B), or the sequence 10 m 6h→ ± 10 nm bryostatin 1 for 24 h (F, F → B) which the percentage F-ara-A for 6 h, washed free of drug and incubated for and additional ± → + of apoptotic cells was determined. * = Greater than values obtained 72 h 10 nm bryostatin 1 (F, F B). The percentage of CD11b cells for U937/Bcl-2 cells; P р 0.05. In all cases, values represent the was then determined as described above. Values represent the means ± means for three to four separate experiments performed in triplicate for three separate experiments performed in triplicate s.d. Values ± s.d. for untransfected and empty vector-containing (pCEP4) U937 cells exposed to the sequence F → B are shown for comparison. *, Signifi- cantly less than values for U937/Bcl-2 cells; P р 0.05. administration of fludarabine followed by bryostatin 1 leads to a marked reduction in colony formation, which exceeds ferentiation can by itself limit leukemic cell self-renewal the extent to which cells display the classic morphologic fea- capacity, although the appearance of apoptosis in differen- tures of apoptosis (and DNA fragmentation) at both early tiating cells, generally as a late, presumably secondary event, (24 h) and late (72 h) intervals. Previous studies have is well documented.24 Lastly, a disproportionate susceptibility described a discordance between induction of apoptosis by of clonogenic cells to apoptosis could also account for the agents such as aphidicolin and VP-16 and inhibition of colony observed discordance. formation,25,26 the basis for which may be multifactorial. For The observation that administration of bryostatin 1 after, but example, formation of colonies consisting of у50 cells not before, exposure of cells to fludarabine resulted in an requires at least six cell divisions; consequently, an inter- increase in apoptosis is consistent with previous studies dem- vention that slows DNA synthesis could reduce the number onstrating the sequence-dependent potentiation of leukemic of colonies in the absence of an increase in cell death. Alter- cell death following treatment with DNA-damaging and differ- natively, non-apoptotic cell death programs, including giant entiation-inducing agents.29,30 It is also compatible with the cell formation26 or mitotic forms of cell death27 could contrib- finding that subsequent (but not prior) exposure to bryostatin 1 ute to inhibition of clonogenicity. In this context, it has been augments apoptosis in U937 cells treated with the pyrimidine postulated that early events associated with commitment to analog ara-C.17 It is noteworthy that U937 cells are consider- cell death and loss of self-renewal capacity exist, and that ably more susceptible to apoptosis induced by ara-C (± bryo- these can be dissociated from the classic signs of apoptotic statin 1) than fludarabine, inasmuch as the extent of cell death morphology and accompanying biochemical features (eg cas- induced by 1 ␮m ara-C exceeds that produced by a 10-fold pase activation).28 By analogy, induction of leukemic cell dif- higher concentration of fludarabine (unpublished observ- Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1053 ations). This phenomenon might reflect reduced conversion of mediated differentiation suggests that this CDK1 contributes fludarabine to its lethal triphosphate derivative in U937 cells, in some yet-to-be determined way to drug-induced matu- or alternatively, the relatively limited retention of F-ara-ATP ration. The latter finding is consistent with the recent obser- following removal of drug in this cell line (Figure 2). In either vation that dysregulation of p21CIP1/WAF1 significantly reduces case, subsequent treatment with bryostatin 1 failed to increase the differentiation response of U937 cells to PMA,21 raising F-ara-ATP retention or incorporation of 3H-fludarabine into the possibility that phorbol- and drug-induced maturation pro- leukemic cell DNA, rendering it unlikely that these events ceed, at least to a certain extent, along a common pathway. account for the enhanced apoptosis observed with this drug It is important to note that dysregulation of p21CIP1/WAF1 did combination. increase the susceptibility of U937 cells to fludarabine- It appears likely that at least a component of the antiprolifer- mediated apoptosis. Recent studies have shown that inter- CIP1/WAF1 + ative actions of fludarabine in U937 cells stems from its ference with p21 -mediated G1 arrest in p53 colon capacity to induce cellular maturation. Previous studies have tumor cells potentiates apoptosis triggered by DNA-damaging shown that low concentrations of pyrimidine (eg ara-C) or agents, presumably by interfering with DNA repair mech- purine (eg 6-thioguanine) analogs induce differentiation in anisms and/or by uncoupling S-phase and mitosis.40,41 A simi- leukemic cell lines, a phenomenon that may be related to anti- lar phenomenon may be operative in p53-null U937 leukemic metabolite-mediated inhibition of DNA synthesis.31,32 To the cells exposed to fludarabine. best of our knowledge, induction of leukemic cell maturation Overexpression of Bcl-2 protects cells from the lethal by fludarabine has not previously been described. While actions of diverse chemotherapeutic agents,42 and the obser- apoptosis accompanies induction of leukemic cell differen- vation that U937/Bcl-2 cells were less susceptible to fludarab- tiation (eg by retinoic acid), generally as a relatively late ine-induced apoptosis than their empty vector-containing event,24 a reciprocal relationship has been noted between counterparts (Figure 6a) is consistent with these findings. Simi- early events associated with leukemic cell differentiation and larly, the partial restoration of fludarabine-mediated apoptosis apoptosis.33 In this regard, the modest capacity of fludarabine by bryostatin 1 is similar to that which we have recently to induce apoptosis in these cells may be counterbalanced by reported in the case of Bcl-2-overexpressing HL-60 cells an enhanced ability to trigger cellular maturation. Moreover, exposed to ara-C,19 indicating that this phenomenon is not while bryostatin 1 has been reported to induce U937 cell restricted to pyrimidine analogs. Furthermore, overexpression maturation,34 we12 and others35 have found it to be consider- of Bcl-2 clearly did not antagonize maturation resulting from ably more limited in this capacity than tumor-promoting phor- combined exposure of cells to fludarabine and bryostatin 1. In bols such as PMA. Nevertheless, sequential exposure of cells this regard, previous studies have demonstrated that enforced to fludarabine followed by bryostatin 1 did lead to a net expression of Bcl-2 in HL-60 cells does not impair differen- increase in cellular differentiation, an event that very likely tiation induction (eg by retinoic acid), although it does delay contributed to the antiproliferative effects of this drug combi- the onset of apoptosis.43,44 In the present studies, induction of nation. Since cellular maturation, like cell death, leads to a differentiation by fludarabine (± bryostatin 1) was as least as loss of leukemic cell self-renewal capacity, such a phenom- great, (and in some cases, greater) in Bcl-2-overexpressing enon could help explain the observed discordance between cells than in controls. This phenomenon might best be under- the increased but still relatively modest induction of apoptosis stood in the context of evidence demonstrating that apoptosis by fludarabine and bryostatin 1 and the more substantial and differentiation represent alternative and, under some cir- reduction in clonogenic survival. In this context, it is worth cumstances, mutually exclusive cellular fates.33 For example, noting that after exposure to lower concentrations of fludara- U937 cells exhibiting dysregulation of PKC␦ undergo bine (eg 0.3 or 1.0 ␮m), the reduction in cloning efficiency apoptosis rather than maturation in response to phorbol was comparable in the adherent and suspension cell fractions, esters.45 More recently, differentiation or apoptosis induced despite enhanced apoptosis in the latter (Figure 4). It has pre- by interferon-␥ in human neuroblastoma cells have been viously been shown that following maturation induction (eg shown to represent mutually exclusive processes at the single by PMA), leukemic cells initially become adherent, and after cell level.46 Thus, dysregulation of early maturation events they undergo apoptosis, detach from the sides of the flask.36 may sensitize cells to apoptosis; conversely, initiation of Thus, induction of leukemic cell death and differentiation apoptosis and the accompanying cellular disruption may pre- may both contribute, in a reciprocal manner, to the loss of vent cells from engaging a normal differentiation program. In clonogenic survival. light of these possibilities, it is tempting to speculate that Bcl- CIP1/WAF1 The CDKI p21 has been implicated in G1 arrest 2-mediated protection against fludarabine-mediated apoptosis accompanying leukemic cell differentiation induced by renders cells more susceptible to the maturation-promoting PMA.37,38 In contrast, results from this laboratory20 and actions of this agent. others37 indicate that DNA damage induced by antimetab- In summary, the results of the present studies demonstrate olites such as ara-C does not trigger a p21 response, at least that fludarabine induces both apoptosis and differentiation in in p53-null human leukemia cells such as HL-60.39 Similarly, U937 cells, and that the latter process proceeds in the absence exposure of U937 cells to fludarabine failed to induce of detectable p21CIP1/WAF1 induction. Moreover, fludarabine- p21CIP1/WAF1, despite the ability of this agent to induce cellular mediated apoptosis and maturation are each enhanced by maturation. We have also reported that bryostatin 1 is subsequent exposure of cells to bryostatin 1, and it appears impaired in its capacity to induce p21CIP1/WAF1 in U937 cells likely that both processes contribute to the antiproliferative when administered at low concentrations (eg 10 nm).12 effects of this drug sequence. Lastly, bryostatin 1-mediated Despite its association with enhanced differentiation, the com- potentiation of fludarabine-induced cell death and differen- bination of bryostatin 1 and fludarabine also failed to induce tiation also occurs in cells displaying enforced expression of p21CIP1/WAF1 expression (Figure 5a), implicating alternative Bcl-2. In view of the recent introduction of bryostatin 1 into pathways in the maturation response to these agents. Never- clinical trials in humans,8 and preclinical evidence that it may theless, the observation that p21 dysregulation (ie in p21 anti- enhance the cytotoxicity of established nucleoside analogs sense-expressing cells; Figure 5b) attenuated fludarabine- such as ara-C,16 chlorodeoxyadenosine,47 and fludarabine Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1054 (this study), further investigation of these drug combinations induced apoptosis in HL-60 human promyelocytic leukemia cells. appears warranted. Biochem Pharmacol 1994; 47: 839–852. 18 Grant S, Turner AJ, Freemerman AJ, Wang Z, Kramer L, Jarvis WD. Modulation of protein kinase C activity and calcium-sensitive isoform expression in human myeloid leukemia cells by bryostatin Acknowledgements 1: relationship to differentiation and ara-C-induced apoptosis. Exp Cell Res 1996; 228: 65–75. This work was supported by CA 63753 and CA77141 from 19 Wang S, Vrana JA, Bartimole TM, Freemerman AJ, Jarvis WD, the NCI, by a Translational Research Award from the Leuke- Kramer LB, Krystal G, Dent P, Grant S. Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-␤-d-arabino- mia Society of America (LSA 6407–97), and by Berlex Labora- furanosylcytosine-induced apoptosis in human leukemia cells that tories, Inc. overexpress Bcl-2. Mol Pharmacol 1997; 52: 1000–1009. 20 Freemerman AJ, Vrana JA, Tombes RM, Jiang H, Chelleppan SP, Fisher PB, Grant S. Effects of antisense p21 (WAF1/CIPI/MDA6) References expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (HL-60). Leukemia 1997; 11: 504–513. 1 Avramis VI, Plunkett W. Metabolism and therapeutic efficacy of 21 Wang Z, Su ZZ, Fisher PB, Van Tuyle G, Grant S. Evidence of a 9-␤-d-arabinofuranosyl-2-fluoroadenine against murine leukemia functional role for the cyclin-dependent kinase inhibitor P388. Cancer Res 1982; 42: 2587–2591. WAF1/CIP1/MDA6 2 Huang P, Chubb S, Plunkett W. Termination of DNA synthesis by p21 in the reciprocal regulation of PKC activator- 9-␤-d-arabinofuranosyl-2-fluoroadenine. A mechanism for cyto- induced apoptosis and differentiation in human myelomonocytic toxicity. J Biol Chem 1990; 265: 16617–16625. leukemia cells. Exp Cell Res 1998; 244: 105–116. 3 Spriggs D, Robbins G, Mitchell T, Kufe D. Incorporation of 9-␤- 22 Khym JX. An analytical system for rapid separation of tissue d-arabinofuranosyl-2-fluoroadenine into HL-60 cellular RNA and nucleotides at low pressures on conventional anion exchangers. DNA. Biochem Pharmacol 1986; 35: 247–252. Clin Chem 1975; 21: 1245–1252. 4 Tseng WC, Derse D, Cheng YC, Brockman RW, Bennett LL Jr. In 23 Chou TC, Talalay P. Quantitative analysis of dose-effect relation- vitro biological activity of 9-␤-d-arabinofuranosyl-2-fluoroadenine ships: the combined effects of multiple drugs or enzyme inhibitors. and the biochemical actions of its triphosphate on DNA poly- Adv Enzyme Regul 1984; 22: 27–55. merases and ribonucleotide reductase from HeLa cells. Mol Phar- 24 Martin SJ, Bradley JG, Cotter TG. HL-60 cells induced to differen- macol 1982; 21: 474–477. tiate toward neutrophils subsequently die via apoptosis. Clin Exp 5 Huang P, Plunkett W. Fludarabine-and gemcitabine-induced Immunol 1990; 79: 448–453. apoptosis: incorporation of analogs into DNA is a critical event. 25 Yin DX, Schimke RT. BCL-2 expression delays drug-induced Cancer Chemother Pharmacol 1995; 36: 181–188. apoptosis but does not increase clonogenic survival after drug 6 Spriggs DR, Stopa E, Mayer RJ, Schoene W, Kufe DW. Fludarabine treatment in HeLa cells. Cancer Res 1995; 55: 4922–4928. phosphate (NSC 312878) infusions for the treatment of acute leu- 26 Lock RB, Stribinskiene L. Dual modes of cell death induced by kemias: phase I and neuropathological study. Cancer Res 1986; etoposide in human epithelial tumor cells allow Bcl-2 to inhibit 46: 5953–5958. apoptosis without affecting clonogenic survival. Cancer Res 1996; 7 Gandhi V, Estey E, Keating MJ, Plunkett W. Fludarabine 56: 4006–4012. potentiates metabolism of cytarabine in patients with acute mye- 27 Radford IR. Mouse lymphoma cells that undergo interphase death logenous leukemia during therapy. J Clin Oncol 1993; 11: 116– show markedly increased sensitivity to radiation-induced DNA 124. double-strand breakage as compared with cells that undergo 8 Grant S, Roberts J, Poplin E, Tombes MB, Kyle B, Welch D, Carr mitotic death. Int J Rad Biol 1991; 59: 353–369. M, Bear HD. Phase Ib trial of bryostatin 1 in patients with refrac- 28 Amarante-Mendes GP, Finucane DM, Martin SJ, Cotter TG, Sal- tory malignancies. Clin Cancer Res 1998; 4: 611–618. vesan GS, Green DR. Anti-apoptotic oncogenes prevent caspase- 9 Kraft AS, Williams F, Pettit GR, Lilly MB. Varied differentiation dependent and independent commitment for cell death. Cell responses of human leukemias to bryostatin 1. Cancer Res 1992; Death Diff 1998; 5: 298–306. 49: 1287–1293. 29 Bhatia U, Traganos F, Darzynkiewicz Z. Induction of cell differen- 10 Stone RM, Sariban E, Pettit GR, Kufe DW. Bryostatin 1 activates tiation potentiates apoptosis triggered by prior exposure to DNA protein kinase C and induces monocytic differentiation of HL-60 damaging drugs. Cell Growth Diff 1995; 6: 937–944. cells. Blood 1998; 72: 208–213. 30 Waxman S, Huang Y, Scher BM, Scher M. Enhancement of differ- 11 Carey JO, Posekany KJ, deVente JE, Pettit GR, Ways DK. Phorbol entiation and cytotoxicity of leukemia cells by combinations of ester-stimulated phosphorylation of PU.1: association with leu- fluorinated pyrimidines and differentiation inducers: development kemic cell growth inhibition. Blood 1996; 87: 4316–4324. of DNA double-strand breaks. Biomed Pharmacother 1992; 46: 12 Vrana JA, Saunders AM, Chellappan SP, Grant S. Divergent effects 183–192. of bryostatin 1 and phorbol myristate acetate on cell cycle arrest 31 Griffin J, Munroe D, Major P, Kufe D. Induction of differentiation and maturation in human myelomonocytic leukemia cells (U937). of human myeloid leukemia cells by inhibitors of DNA synthesis. Differentiation 1998; 63: 33–42. Exp Hematol 1982; 10: 774–781. 13 Szallasi Z, Smith CB, Pettit GR, Blumberg PM. Differential regu- 32 Ishiguro K, Schwartz EL, Sartorelli AC. Characterization of the lation of protein kinase C isozymes by bryostatin 1 and phorbol metabolic forms of 6-thioguanine responsible for cytotoxicity and 12-myristate 13-acetate in NIH 3T3 fibroblasts. J Biol Chem 1994; induction of differentiation of HL-60 of acute promyelocytic leu- 269: 2118–2124. ␤ kemia cells. J Cell Physiol 1984; 121: 383–390. 14 Hocevar BA, Fields AP. Selective translocation of II-protein kinase C to the nucleus of human promyelocytic leukemia (HL-60) 33 Selvakumaran M, Reed JC, Liebermann D, Hoffman B. Progression of the myeloid differentiation program is dominant to transforming cells. J Biol Chem 1991; 266: 28–33. ␤ 15 Lee HW, Smith L, Pettit GR, Vinitsky A, Smith JB. Ubiquitinization growth factor- 1-induced apoptosis in M1 myeloid leukemic of protein kinase C-␣ and degradation by the proteosome. J Biol cells. Blood 1994; 84: 1036–1042. Chem 1996; 271: 20973–20976. 34 Asiedu C, Biggs J, Lilly M, Kraft AS. Inhibition of leukemic cell 16 Grant S, Jarvis WD, Swerdlow PS, Turner AJ, Traylor RS, Wallace growth by the protein kinase C activator bryostatin 1 correlates HJ, Lin PS, Pettit GR, Gewirtz D. Potentiation of the activity of with the dephosphorylation of cyclin-dependent kinase 2. Cancer 1-␤-d-arabinofuranosylcytosine by the protein kinase C activator Res 1995; 55: 3716–3720. bryostatin 1 in Hl-60 cells: association with enhanced fragmen- 35 Ng SB, Guy GR. Two protein kinase C activators, bryostatin 1 and tation of mature DNA. Cancer Res 1992; 52: 6270–6278. phorbol-12-myristate-13 acetate, have different effects on haemo- 17 Jarvis WD, Povirk LF, Turner AJ, Traylor RS, Gewirtz DA, Pettit poietic cell proliferation and differentiation. Cell Signal 1992; 4: GR, Grant S. Effects of bryostatin 1 and other pharmacological 405–416. activators of protein kinase C on 1-␤-d-arabinofuranosylcytosine- 36 Solary E, Bertrand R, Pommier Y. Apoptosis of human leukemic Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al 1055 HL-60 cells induced to differentiate by phorbol ester treatment. 43 Park JR, Robertson K, Hickstein DD, Tsai S, Hockenbery DM, Col- Leukemia 1994; 8: 792–797. lins SJ. Dysregulated bcl-2 expression inhibits apoptosis but not 37 Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, el- differentiation of retinoic acid-induced HL-60 granulocytes. Blood Houseine ME. Induction of p21 (WAF1/CIP1) during differen- 1994; 84: 440–445. tiation. Oncogene 1994; 9: 3389–3396. 44 Naumovksi L, Cleary ML. Bc12 inhibits apoptosis associated with 38 Jiang H, Lin J, Su ZZ, Collart FR, Huberman E, Fisher PB. Induction terminal differentiation of myeloid leukemia cells. Blood 1994; of differentiation in human promyelocytic HL-60 leukemia cells 83: 2261–2267. activates p21WAF1/CIP1 expression in the absence of p53. Oncogene 45 deVente J, Kiley S, Garris T, Bryant W, Hooker J, Posekany K, 1994; 9: 3397–3406. Parker P, Cook P, Fletcher D, Ways DK. Phorbol ester treatment of 39 Wolf D, Rotter V. Major deletions in the gene encoding the p53 U937 cells with altered protein kinase C content and distribution tumor antigen cause lack of p53 expression in HL-60 cells. Proc induces cell death rather than differentiation. Cell Growth Diff Natl Acad Sci USA 1985; 82: 790–794. 1995; 6: 371–382. 40 Waldman T, Lengauer C, Kinzler KW, Vogelstein B. Uncoupling 46 Montaldo PG, Chiesa V, Bado M, Raffaghello L, Rozzo C, Ponzoni of S phase and mitosis induced by anticancer agents in cells lack- M. Induction of differentiation and apoptosis by interferon-␥ in ing p21. Nature 1996; 381: 713–716. human neuroblastoma cells in vitro as a dual and alternative early 41 Waldman T, Zhang Y, Dillehay L, Yu J, Kinzler K, Vogelstein B, biologic response. Cell Death Diff 1998; 4: 150–158. Williams J. Cell-cycle arrest versus cell death in cancer therapy. 47 Mohammad RM, Katato K, Almatchy VP, Wall N, Liu KZ, Schultz Nature Med 1997; 3: 1034–1036. CP, Mantsch HH, Varterasian M, al-Katib AM. Sequential treat- 42 Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy- ment of human chronic lymphocytic leukemia with bryostatin 1 induced apoptosis in a human leukemia cell line. Blood 1993; followed by 2-chlorodeoxyadenosine: preclinical studies. Clin 81: 151–157. Cancer Res 1998; 4: 445–453.