Induction of apoptosis and differentiation by fludarabine in ... - Nature

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 analog fludarabine (9-␤-arabinofuranosyl-2-fluoroadenine) and the macrocyclic lactone bryostatin 1 in the human monocytic leukemic cell line U937. Fludarabine exerted dose-dependent effects on U937 cell viability and growth which were associated with both induction of apoptosis, as well as cellular maturation. Incubation of cells with bryostatin 1 (10 nM; 24 h) after, but not before a 6-h exposure to 10 ␮M fludarabine resulted in a modest but significant increase in apoptosis, and was associated with greater than a 1 log reduction in clonogenicity. Subsequent exposure to bryostatin 1 also increased the percentage of fludarabine-treated cells displaying differentiation-related features (eg plastic adherence, CD11b positivity) compared to cells exposed to fludarabine alone. Bryostatin 1 did not increase the retention of the active fludarabine metabolite, Fara-ATP, nor did it increase 3H-F-ara-A incorporation into DNA. Despite its capacity to trigger cellular maturation, fludarabine exposure (either with or without bryostatin 1) failed to induce the cyclin-dependent kinase inhibitors (CDKIs) p21WAFI/CIP1 and p27KIP1. Nevertheless, dysregulation of p21 (resulting from stable transfection of cells with a p21WAFI/CIP1 antisense construct) reduced fludarabine-mediated differentiation, while inducing a corresponding increase in apoptosis. Enforced expression of Bcl-2 partially protected cells from fludarabinerelated apoptosis, an effect that was overcome, in part, by subsequent exposure of cells to bryostatin 1. Interestingly, Bcl-2overexpressing cells were as or in some cases, more susceptible to differentiation induction by fludarabine (± bryostatin 1) than their empty vector-containing counterparts. Collectively, these results indicate that the antiproliferative effects of fludarabine toward U937 leukemic cells involve both induction of apoptosis and cellular maturation, and that each of these processes may be enhanced by bryostatin 1. Keywords: fludarabine; bryostatin 1; U937 cells; apoptosis; differentiation; Bcl-2

Introduction The purine analog fludarabine phosphate (9-␤-arabinofuranosyl-2-fluoroadenine monophosphate; FAMP) is a deoxyadenosine analog which is a potent inhibitor of leukemic cell 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 subsequently phosphorylated intracellularly to form the active derivative, 9-␤-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). The cytotoxic actions of F-ara-ATP may stem from multiple actions, including inhibition of DNA polymerase ␣ and DNA primase, inhibition of ribonucleotide reductase, and nucleoside incorporation into DNA and/or RNA.2–4 In lymphoid cells, fludarabine induces apoptosis, an event that appears to be critically dependent upon DNA incorporation.5 Fludara-

Correspondence: S Grant, Division of Hematology/Oncology, Medical College of Virginia, Virginia Commonwealth University, PO Box 980230, Richmond, VA 23298, USA; Fax: 804 225 3788 Received 20 July 1998; accepted 14 January 1999

bine has shown significant activity in lymphoid malignancies such as chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin’s lymphoma, but has also been used in the treatment of acute myelogenous leukemia (AML), either as a single agent,6 or in combination with 1-␤-d-arabinofuranosylcytosine (ara-C).7 Bryostatin 1 is a macrocyclic lactone activator of protein kinase C (PKC), and is currently undergoing phase I and II evaluation.8 Bryostatin 1 variably induces maturation in human leukemic cell lines and primary AML blast specimens,9,10 although it is considerably weaker in this regard than the tumor-promoting phorbol diester, 12-phorbol-13-myristate acetate (PMA).11,12 The basis for the divergent spectrum of activity of bryostatin 1 and phorbols remains unknown, but may involve differential PKC isoform activation or translocation.13,14 Notably, bryostatin 1 is a potent down-regulator of PKC, a process recently linked to proteasomal degradation of the enzyme.15 Previous studies from this laboratory have demonstrated that bryostatin 1 increases the susceptibility of human leukemia cells (eg HL-60; U937) to apoptosis induced by ara-C in a dose- and sequence-dependent manner.16,17 In a human promyelocytic leukemic line (HL-60) insensitive to the differentiation-inducing actions of bryostatin 1, pretreatment with bryostatin 1 augmented ara-C-mediated apoptosis.17 In contrast, in the human monocytic leukemic cell line U937, exposure of ara-C-pretreated cells to bryostatin 1 significantly increased apoptosis, whereas exposure to bryostatin 1 before ara-C did not.18 Currently, it is unknown whether the ability 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 purpose of this study was to characterize the antiproliferative effects of the purine analog fludarabine in human myelomonocytic U937 leukemia cells in relation to induction of apoptosis and differentiation, and to determine what effect, if any, bryostatin 1 might have on these actions. Materials and methods

Cells U937 cells were derived from a patient with diffuse histiocytic leukemia as previously described.12 The cells were maintained in RPMI 1640 (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS), MEM vitamins, 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 antiapoptotic protein Bcl-2 were generated by electroporation of the parental line with a plasmid (pCEP4; Invitrogen, Carlsbad, CA, USA) containing Bcl-2 cDNA under the control of an

Fludarabine and bryostatin 1 in human leukemia cells JA Vrana et al

CMV promotor and a hygromycin resistance marker as previously described.19 These cells, designated U937/Bcl-2, express approximately a seven-fold increase in Bcl-2 protein compared to their empty-vector counterparts (U937/pCEP4) and are maintained in 400 ␮g/ml hygromycin (GIBCO) for selection pressure. Levels of other Bcl-2 family members, 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 expressing the cyclin-dependent kinase inhibitor (CDKI) p21WAFI/CIPI in the antisense configuration.20 More recently, we have characterized the behavior of a U937 cell line that also exhibits p21WAFI/CIPI dysregulation.21 In response to the tumorpromoting phorbol PMA, antisense-expressing cells (U937/ASF4) are impaired in G1 arrest, cellular maturation, pRB dephosphorylation, and CDK2 inhibition compared to empty vector controls (U937/pREP4). These cells are maintained under selection pressure in medium containing 200 ␮g/ml hygromycin.

Drugs and chemicals Bryostatin 1 was provided by Dr Anthony Murgo, Cancer Treatment and Evaluation Program/Division of Cancer Treatment, National Cancer Institute (Bethesda, MD, USA). It was stored under light-protected conditions as a lyophilized powder at 4°C, and reconstituted in sterile dimethylsulfoxide (DMSO) prior to use. Material was subsequently diluted in RPMI medium to achieve the desired final concentration; in all cases, the final concentration of DMSO was ⬍0.1%, and found to exert negligible effects on the U937 cell responses. Fludarabine was provided by Dr Gary Williams, Berlex Laboratories (Richmond, CA, USA), stored under light-protected conditions at room temperature, and formulated in sterile water prior to use. 3H-fludarabine (10 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). F-araATP was kindly provided by Dr W Plunkett, MD Anderson Cancer Center (Houston, TX, USA). Deoxyadenosine triphosphate (dATP) was purchased from Sigma (St Louis, MO, USA).

Experimental format Logarithmically growing cells (approximately 4 × 105 cells/ml) were placed in 25 cm2T-flasks (Costar, Cambridge, MA, USA) and incubated in the presence of fludarabine (generally 10 ␮m) for 6 h. At the end of this period, cells were washed three times in drug and serum-free medium, and resuspended in fresh medium containing 10% FCS at 2 × 105 cells/ml in the presence or absence of bryostatin 1 (10 nm) for an additional 24 h. In some cases, cells were exposed to these agents in the opposite sequence, eg bryostatin 1 → fludarabine. At the end of the incubation period, cells were harvested and prepared for analysis as described below.

Assays of apoptosis Following appropriate drug exposures, cell morphology and apoptosis was measured by cytocentrifuge preparations stained with the Diff-Quik stain set (Dade Diagnostics, Aguada, PR, USA), as previously described.12 The percentage of cells exhibiting classic apoptotic features (eg cell shrinkage,

nuclear condensation, the formation of membrane-bound blebs, etc) was determined by examining 7–10 randomly selected fields and scoring at least 1000 cells/condition. In some cases, results were confirmed by monitoring DNA fragmentation following treatment of cell lysates with bisbenzamide staining as described previously.17

Differentiation studies Two separate methods were employed to monitor induction of leukemic cell maturation. First, after the designated incubation period (eg 24–72 h), the number of cells in suspension was determined using a hematocytometer. Subsequently, adherent cells were removed using a cell scraper, additional medium added, and the cell number assessed as above. The percentage of adherent cells, characteristic of a differentiated phenotype, was expressed relative to the total population (eg adherent cells + cells in suspension). Alternatively, cellular maturation was assessed by monitoring expression of the myelomonocytic surface marker CD11b by flow cytometry, as described previously.12 Briefly, treated cells were pelleted at 500 g and resuspended in cold phosphate-buffered saline (PBS) at 5 ×106 cells/ml. The cell suspension was mixed with fluorescein isothiocyanate-labeled antibody (CD11b or IgG2␣, Becton Dickinson, Mountain View, CA, USA) and placed on ice for 20 min. Cells were diluted in cold PBS and analyzed using a Becton Dickinson FACScan flow cytometer and Verity Winlist software. In some studies CD11b expression was measured separately in the adherent and suspension cell populations.

Clonogenic assays A previously described method was employed.17 Briefly, after drug treatment, cells were washed three times in drug-free medium, cell numbers normalized, and 500 cells/well plated in 12-well plates containing RPMI medium, 20% FCS, and 0.3% Bacto agar (Difco, Detroit, MI, USA). The plates were placed in a 37°C, 5% CO2, fully-humidified incubator for 12 days, after which colonies, consisting of groups of ⭓50 cells, were scored using an Olympus Model CK inverted microscope (Olympus, Lake Success, NY, USA).

Western analysis Whole cell pellets (5 × 106 cells/condition) were washed once in PBS, resuspended in 100 ␮l PBS, and lysed by the addition of 100 ␮l 2X loading buffer (1× = 30 mm Tris-base, pH 6.8, 2% SDS, 2.88 mm ␤-mercaptoethanol, 10% glycerol, 苲0.01% bromophenol blue). Lysates were sonicated, boiled for 10 min, centrifuged at 12 800 g for 5 min, and quantified using Coomassie protein assay reagent (Pierce, Rockford, IL, USA). Equal amounts of protein (25 ␮g) were separated by SDS-PAGE (5% stacker and 12% resolving gels) and electroblotted to Optitran nitrocellulose filters (Schleicher and Schleicher, Keene, NH, USA). The membrane was stained in 0.1% amido black and destained in 5% acetic acid to ensure transfer and equal loading. The blots were then blocked in PBSTween (PBS-T; 0.05%)/5% dry milk for 1 h at 22°C, and probed overnight at 4°C with primary antibody: p21CIP1/WAF1 or p27KIP1; both 1:500 (Transduction Laboratories, Lexington, KY, USA). Blots were washed 3 ×10 min in PBS-T and incu-

1047


1048

bated with a 1:2000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) in PBS-T for 1 h at 22°C. Blots were again washed 3 × 10 min in PBS-T and developed by enhanced chemiluminesence (Amersham, Arlington Heights, IL, USA). Western blots were reprobed with an anti-tubulin antibody; 1:2000 (Calbiochem, San Diego, CA, USA) to ensure equivalent loading and transfer.

cells are shown in Figure 1a. Following treatment of cells with 10 ␮m fludarabine for 6 h and a subsequent 24 h incubation in drug-free medium, a relatively small number of cells displayed apoptotic features (eg 苲l8%). Treatment of cells with bryostatin 1 alone for 24 h also weakly induced apoptosis in these cells. When cells were exposed to the sequence fludarabine → bryostatin 1, the percentage of apoptotic cells 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 Trioctylamine:1,1,2-Trichlorotrifluoroethane (Sigma-Aldrich). The aqueous phase was stored at −80°C until analysis. Immediately 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.

3

H-F-ara-A DNA incorporation

Cells were incubated with 10 ␮m 3H-fludarabine (final specific 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 6h 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 scintillography, 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 agents, Median Dose Analysis was employed23 using a commercially available software program (CalcuSyn; Biosoft, Ferguson, MO, USA). For these studies, the combination index (CI) was calculated for a two-drug combination involving a fixed concentration ratio. Using these methods, CI values ⬍1.0 indicate a synergistic interaction.

Results The sequence-dependent effects of sequential exposure of cells to fludarabine and bryostatin 1 on apoptosis in U937

Figure 1 (a) Cells were exposed to 10 nm bryostatin 1 (B) for 24 h either before or after a 6-h incubation with 10 ␮m fludarabine (F). The percentage of apoptotic cells was then determined as described in Materials and methods. Values represent the means for five separate experiments ± s.d. *Indicates significantly greater than values for the 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 ⭓50 cells, were scored on day 12. Values are expressed in relation 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 ⭐ 0.02. Median dose effect analysis of clonogenic growth for the sequence F → B; CI values ⬍1 correspond to synergistic interactions.


sequence fludarabine → bryostatin 1 (P ⭐ 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 ⬎1 log. The sequence bryostatin 1 → fludarabine also led to a significant reduction in colony formation (eg ⬎80%), although inhibitory effects were not as great as those observed for the opposite sequence (P ⭐ 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 ⬍1, 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 preincubation 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 correspondingly greater reduction in cellular proliferative potential, 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 adherence or CD11b expression, are shown in Figure 3a. In the case of the latter, separate determinations were conducted in the adherent and suspension cell populations. With both assays, a biphasic dose-response was observed, with maximal differentiation induction occurring at fludarabine concentrations of 0.5 to 1.0 ␮m, and declining at higher levels. For comparison, a fludarabine-induced apoptosis dose-response curve is shown in Figure 3b, demonstrating an increase in cell death at fludarabine concentrations greater or equal to those associated with maximal maturation responses (eg 1.0 ␮m). A discordance was noted between fludarabine-stimulated adherence and CD11 b positivity, in that the percentage of 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 0.5–2.0 ␮m. Unexpectedly, the extent of CD11b positivity was equivalent in the adherent and suspension cell populations, further indicating that these two maturation markers (cellular adherence and CD11b expression) are not induced in parallel in fludarabine-treated cells. Lastly, the effects of the combi-

1049

Figure 2 Following treatment of cells with 10 ␮m fludarabine for 6 h, cells were washed free of drug and resuspended in fresh medium ±10 nm bryostatin 1 for an additional 24 h. At the designated intervals, cells were removed, lysed, and intracellular levels of F-ara-ATP and 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. Medium (쏔), Bryostatin 1 (왕). (b) Effect of bryostatin 1 on intracellular ratios of F-ara-ATP and dATP 2 h (open bar) and 6 h (solid bar) after a 6-h incubation with fludarabine. Fludarabine followed by medium (F), fludarabine followed by bryostatin (F → B). (c) Alternatively, cells were exposed to 10 ␮m 3HF-ara-A (F) for 6 h followed (or preceded) by a 24-h incubation in medium (C) or 10 nm bryostatin 1 (B). The cells were then lysed, the DNA extracted, and incorporation of 3HF-ara-A into DNA determined as described in the text. In each case, values represent the means for three separate experiments ± s.d.

nation of bryostatin 1 and fludarabine on U937 cell maturation were assessed. When administered individually, bryostatin 1 (10 nm; 72 h) and fludarabine (10 ␮m; 6 h, washed, fresh medium; 72 h) induced expression of CD11b in 12.4 ±3.8% and 7.1 ±3.4% of cells respectively (Figure 3c). When cells were exposed to the sequence fludarabine → bryostatin


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 苲10%). However, the percentage of apoptotic cells was significantly greater in the suspension fraction (P ⬍ 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 ensuing 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 (쮿), adherent cells expressing CD11b (왔) or cells in suspension expressing CD11b (왖) 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 ⭐ 0.05; ** = P ⭐ 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 Fara-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 percentage of cells expressing CD11b increased to 26.3 ±6.3%. However, combination with bryostatin 1 did not lead to a further potentiation of maturation in cells exposed to lower concentrations of fludarabine (eg 0.5–1.0 ␮m; data not shown). These results suggest that sequential exposure of

Figure 4 Cells were incubated in the presence or absence of 1.0 ␮m F-ara-A for 72 h, after which the adherent and suspension cell populations were separately analyzed with respect to extent of apoptosis (open bars) and cloning efficiency (solid bars) as described in the text. Values represent the means for three separate experiments performed in triplicate ± s.d. *Indicates significantly greater than values obtained for adherent cells; P ⬍ 0.0001.


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 ⭓ 0.05; ** = Values significantly lower than those of empty-vector controls; P ⭐ 0.02. Values represent the means for three separate experiments ± s.d.

determine whether p21CIP1/WAF1 might nevertheless be involved in the apoptotic response to fludarabine, a U937 cell line stably expressing p21CIP1/WAF1 in the antisense configuration was employed (U937/p21ASF4). Following exposure to PMA, these cells display diminished p21CIP1/WAF1 induction, G1 arrest, pRb dephosphorylation, CDK2 inhibition, and acquisition of differentiation-related features compared to their empty vector counterparts (pREP4),21 in a manner analogous to that previously described in p21 antisense-expressing HL60 cells.20 Despite the fact that fludarabine failed to induce p21CIP1/WAF1, cells exhibiting p21CIP1/WAF1 dysregulation were significantly more sensitive to fludarabine-induced apoptosis compared to controls (Figure 5b; left panel). Conversely, dysregulation of p21CIP1/WAF1 significantly reduced the susceptibility of U937 to fludarabine-mediated differentiation induction (Figure 5b; right panel). The effects of Bcl-2 overexpression on the apoptotic response of cells to fludarabine were then examined (Figure 6). When cells were exposed to varying concentrations of 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 compared to their counterparts transfected with an empty vector (U937/pCEP4) (Figure 6a). When Bcl-2-overexpressing cells were sequentially exposed to fludarabine followed by bryostatin 1, the extent of apoptosis, while less than that observed in the pCEP4 line, was greater than that noted in control cells

treated with fludarabine alone (Figure 6b). These results are compatible with recent findings indicating that bryostatin 1 reverses, at least in part, resistance to ara-C-related apoptosis conferred by Bcl-2 overexpression in the HL-60 promyelocytic leukemia cell line.19 Lastly, effects on cellular maturation were compared in cells containing an empty vector and those overexpressing Bcl-2 (Figure 7). When fludarabine was administered alone, differentiation induction in Bcl-2 overexpressors was either equal to or, in most cases, greater than, that observed in their empty vector-containing counterparts (Figure 7a). Furthermore, when U937/Bcl-2 cells were sequentially exposed to fludarabine followed by bryostatin 1, the increase in CD11b positivity was at least as great or greater than that noted in the case of untransfected or empty vector controls (Figure 7b). These findings indicate that Bcl-2 overexpression does not protect cells from the maturation-related actions of fludarabine (or the fludarabine/bryostatin 1 combination), but may under some circumstances increase the susceptibility of cells to differentiation induction. Discussion The results described herein demonstrate that fludarabine induces both apoptosis and differentiation in U937 cells, and that each of these events is enhanced, at least to a degree, by the macrocyclic lactone bryostatin 1. Furthermore, sequential


1052

Figure 6 (a) Empty-vector control cells (U937/pCEP4) (쏔) and Bcl2-overexpressing cells (U937/Bcl-2) (쮿) were exposed to the designated concentration of fludarabine for 6 h, washed, and incubated in drug-free medium for 24 h prior to determination of the extent of apoptosis. * = Values significantly less than those obtained for U937/pCEP4; P ⭐0.05. (b) Alternatively, U937/pCEP4 (solid bars) and U937/Bcl-2 (open bars) cells were exposed to drug-free medium (C), 10 nm bryostatin 1 for 24 h (B), or the sequence 10 ␮m F-ara-A for 6 h → ± 10 nm bryostatin 1 for 24 h (F, F → B) which the percentage of apoptotic cells was determined. * = Greater than values obtained for U937/Bcl-2 cells; P ⭐ 0.05. In all cases, values represent the means for three to four separate experiments performed in triplicate ± s.d.

administration of fludarabine followed by bryostatin 1 leads to a marked reduction in colony formation, which exceeds the extent to which cells display the classic morphologic features of apoptosis (and DNA fragmentation) at both early (24 h) and late (72 h) intervals. Previous studies have described a discordance between induction of apoptosis by agents such as aphidicolin and VP-16 and inhibition of colony formation,25,26 the basis for which may be multifactorial. For example, formation of colonies consisting of ⭓50 cells requires at least six cell divisions; consequently, an intervention that slows DNA synthesis could reduce the number of colonies in the absence of an increase in cell death. Alternatively, non-apoptotic cell death programs, including giant cell formation26 or mitotic forms of cell death27 could contribute to inhibition of clonogenicity. In this context, it has been postulated that early events associated with commitment to cell death and loss of self-renewal capacity exist, and that these can be dissociated from the classic signs of apoptotic morphology and accompanying biochemical features (eg caspase activation).28 By analogy, induction of leukemic cell dif-

Figure 7 (a) Logarithmically growing U937/pCEP4 (쏔) and U937/Bcl-2 (쮿) cells were continuously exposed to the designated concentration of F-ara-A for 72 h, after which the percentage of CD11b-expressing cells was determined by cytofluorometry. Values represent the means for three separate experiments performed in triplicate ± s.d. *, Significantly greater than values for U937/pCEP4 cells; P ⭐ 0.05. (b) U937/Bcl-2 cells were sequentially exposed to drug-free medium (C), 10 nm bryostatin 1 for 72 h (B), or the sequence 10 ␮m F-ara-A for 6 h, washed free of drug and incubated for and additional 72 h ± 10 nm bryostatin 1 (F, F → B). The percentage of CD11b+ cells was then determined as described above. Values represent the means for three separate experiments performed in triplicate ± s.d. Values for untransfected and empty vector-containing (pCEP4) U937 cells exposed to the sequence F → B are shown for comparison. *, Significantly less than values for U937/Bcl-2 cells; P ⭐ 0.05.

ferentiation can by itself limit leukemic cell self-renewal capacity, although the appearance of apoptosis in differentiating cells, generally as a late, presumably secondary event, is well documented.24 Lastly, a disproportionate susceptibility of clonogenic cells to apoptosis could also account for the observed discordance. The observation that administration of bryostatin 1 after, but not before, exposure of cells to fludarabine resulted in an increase in apoptosis is consistent with previous studies demonstrating the sequence-dependent potentiation of leukemic cell death following treatment with DNA-damaging and differentiation-inducing agents.29,30 It is also compatible with the finding that subsequent (but not prior) exposure to bryostatin 1 augments apoptosis in U937 cells treated with the pyrimidine analog ara-C.17 It is noteworthy that U937 cells are considerably more susceptible to apoptosis induced by ara-C (± bryostatin 1) than fludarabine, inasmuch as the extent of cell death induced by 1 ␮m ara-C exceeds that produced by a 10-fold higher concentration of fludarabine (unpublished observ-


ations). This phenomenon might reflect reduced conversion of fludarabine to its lethal triphosphate derivative in U937 cells, or alternatively, the relatively limited retention of F-ara-ATP following removal of drug in this cell line (Figure 2). In either case, subsequent treatment with bryostatin 1 failed to increase F-ara-ATP retention or incorporation of 3H-fludarabine into leukemic cell DNA, rendering it unlikely that these events account for the enhanced apoptosis observed with this drug combination. It appears likely that at least a component of the antiproliferative actions of fludarabine in U937 cells stems from its capacity to induce cellular maturation. Previous studies have shown that low concentrations of pyrimidine (eg ara-C) or purine (eg 6-thioguanine) analogs induce differentiation in leukemic cell lines, a phenomenon that may be related to antimetabolite-mediated inhibition of DNA synthesis.31,32 To the best of our knowledge, induction of leukemic cell maturation by fludarabine has not previously been described. While apoptosis accompanies induction of leukemic cell differentiation (eg by retinoic acid), generally as a relatively late event,24 a reciprocal relationship has been noted between early events associated with leukemic cell differentiation and apoptosis.33 In this regard, the modest capacity of fludarabine to induce apoptosis in these cells may be counterbalanced by an enhanced ability to trigger cellular maturation. Moreover, while bryostatin 1 has been reported to induce U937 cell maturation,34 we12 and others35 have found it to be considerably more limited in this capacity than tumor-promoting phorbols such as PMA. Nevertheless, sequential exposure of cells to fludarabine followed by bryostatin 1 did lead to a net increase in cellular differentiation, an event that very likely contributed to the antiproliferative effects of this drug combination. Since cellular maturation, like cell death, leads to a loss of leukemic cell self-renewal capacity, such a phenomenon could help explain the observed discordance between the increased but still relatively modest induction of apoptosis by fludarabine and bryostatin 1 and the more substantial reduction in clonogenic survival. In this context, it is worth noting that after exposure to lower concentrations of fludarabine (eg 0.3 or 1.0 ␮m), the reduction in cloning efficiency was comparable in the adherent and suspension cell fractions, despite enhanced apoptosis in the latter (Figure 4). It has previously been shown that following maturation induction (eg by PMA), leukemic cells initially become adherent, and after they undergo apoptosis, detach from the sides of the flask.36 Thus, induction of leukemic cell death and differentiation may both contribute, in a reciprocal manner, to the loss of clonogenic survival. The CDKI p21CIP1/WAF1 has been implicated in G1 arrest accompanying leukemic cell differentiation induced by PMA.37,38 In contrast, results from this laboratory20 and others37 indicate that DNA damage induced by antimetabolites such as ara-C does not trigger a p21 response, at least in p53-null human leukemia cells such as HL-60.39 Similarly, exposure of U937 cells to fludarabine failed to induce p21CIP1/WAF1, despite the ability of this agent to induce cellular maturation. We have also reported that bryostatin 1 is impaired in its capacity to induce p21CIP1/WAF1 in U937 cells when administered at low concentrations (eg 10 nm).12 Despite its association with enhanced differentiation, the combination of bryostatin 1 and fludarabine also failed to induce p21CIP1/WAF1 expression (Figure 5a), implicating alternative pathways in the maturation response to these agents. Nevertheless, the observation that p21 dysregulation (ie in p21 antisense-expressing cells; Figure 5b) attenuated fludarabine-

mediated differentiation suggests that this CDK1 contributes in some yet-to-be determined way to drug-induced maturation. The latter finding is consistent with the recent observation that dysregulation of p21CIP1/WAF1 significantly reduces the differentiation response of U937 cells to PMA,21 raising the possibility that phorbol- and drug-induced maturation proceed, at least to a certain extent, along a common pathway. It is important to note that dysregulation of p21CIP1/WAF1 did increase the susceptibility of U937 cells to fludarabinemediated apoptosis. Recent studies have shown that interference with p21CIP1/WAF1-mediated G1 arrest in p53+ colon tumor cells potentiates apoptosis triggered by DNA-damaging agents, presumably by interfering with DNA repair mechanisms and/or by uncoupling S-phase and mitosis.40,41 A similar phenomenon may be operative in p53-null U937 leukemic cells exposed to fludarabine. Overexpression of Bcl-2 protects cells from the lethal actions of diverse chemotherapeutic agents,42 and the observation that U937/Bcl-2 cells were less susceptible to fludarabine-induced apoptosis than their empty vector-containing counterparts (Figure 6a) is consistent with these findings. Similarly, the partial restoration of fludarabine-mediated apoptosis by bryostatin 1 is similar to that which we have recently reported in the case of Bcl-2-overexpressing HL-60 cells exposed to ara-C,19 indicating that this phenomenon is not restricted to pyrimidine analogs. Furthermore, overexpression of Bcl-2 clearly did not antagonize maturation resulting from combined exposure of cells to fludarabine and bryostatin 1. In this regard, previous studies have demonstrated that enforced expression of Bcl-2 in HL-60 cells does not impair differentiation induction (eg by retinoic acid), although it does delay the onset of apoptosis.43,44 In the present studies, induction of differentiation by fludarabine (± bryostatin 1) was as least as great, (and in some cases, greater) in Bcl-2-overexpressing cells than in controls. This phenomenon might best be understood in the context of evidence demonstrating that apoptosis and differentiation represent alternative and, under some circumstances, mutually exclusive cellular fates.33 For example, U937 cells exhibiting dysregulation of PKC␦ undergo apoptosis rather than maturation in response to phorbol esters.45 More recently, differentiation or apoptosis induced by interferon-␥ in human neuroblastoma cells have been shown to represent mutually exclusive processes at the single cell level.46 Thus, dysregulation of early maturation events may sensitize cells to apoptosis; conversely, initiation of apoptosis and the accompanying cellular disruption may prevent cells from engaging a normal differentiation program. In light of these possibilities, it is tempting to speculate that Bcl2-mediated protection against fludarabine-mediated apoptosis renders cells more susceptible to the maturation-promoting actions of this agent. In summary, the results of the present studies demonstrate that fludarabine induces both apoptosis and differentiation in U937 cells, and that the latter process proceeds in the absence of detectable p21CIP1/WAF1 induction. Moreover, fludarabinemediated apoptosis and maturation are each enhanced by subsequent exposure of cells to bryostatin 1, and it appears likely that both processes contribute to the antiproliferative effects of this drug sequence. Lastly, bryostatin 1-mediated potentiation of fludarabine-induced cell death and differentiation also occurs in cells displaying enforced expression of Bcl-2. In view of the recent introduction of bryostatin 1 into clinical trials in humans,8 and preclinical evidence that it may enhance the cytotoxicity of established nucleoside analogs such as ara-C,16 chlorodeoxyadenosine,47 and fludarabine

1053


1054

(this study), further investigation of these drug combinations appears warranted.

18

Acknowledgements This work was supported by CA 63753 and CA77141 from the NCI, by a Translational Research Award from the Leukemia Society of America (LSA 6407–97), and by Berlex Laboratories, Inc.

19

20

References 1 Avramis VI, Plunkett W. Metabolism and therapeutic efficacy of 9-␤-d-arabinofuranosyl-2-fluoroadenine against murine leukemia P388. Cancer Res 1982; 42: 2587–2591. 2 Huang P, Chubb S, Plunkett W. Termination of DNA synthesis by 9-␤-d-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity. J Biol Chem 1990; 265: 16617–16625. 3 Spriggs D, Robbins G, Mitchell T, Kufe D. Incorporation of 9-␤d-arabinofuranosyl-2-fluoroadenine into HL-60 cellular RNA and DNA. Biochem Pharmacol 1986; 35: 247–252. 4 Tseng WC, Derse D, Cheng YC, Brockman RW, Bennett LL Jr. In vitro biological activity of 9-␤-d-arabinofuranosyl-2-fluoroadenine and the biochemical actions of its triphosphate on DNA polymerases and ribonucleotide reductase from HeLa cells. Mol Pharmacol 1982; 21: 474–477. 5 Huang P, Plunkett W. Fludarabine-and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event. Cancer Chemother Pharmacol 1995; 36: 181–188. 6 Spriggs DR, Stopa E, Mayer RJ, Schoene W, Kufe DW. Fludarabine phosphate (NSC 312878) infusions for the treatment of acute leukemias: phase I and neuropathological study. Cancer Res 1986; 46: 5953–5958. 7 Gandhi V, Estey E, Keating MJ, Plunkett W. Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia during therapy. J Clin Oncol 1993; 11: 116– 124. 8 Grant S, Roberts J, Poplin E, Tombes MB, Kyle B, Welch D, Carr M, Bear HD. Phase Ib trial of bryostatin 1 in patients with refractory malignancies. Clin Cancer Res 1998; 4: 611–618. 9 Kraft AS, Williams F, Pettit GR, Lilly MB. Varied differentiation responses of human leukemias to bryostatin 1. Cancer Res 1992; 49: 1287–1293. 10 Stone RM, Sariban E, Pettit GR, Kufe DW. Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells. Blood 1998; 72: 208–213. 11 Carey JO, Posekany KJ, deVente JE, Pettit GR, Ways DK. Phorbol ester-stimulated phosphorylation of PU.1: association with leukemic cell growth inhibition. Blood 1996; 87: 4316–4324. 12 Vrana JA, Saunders AM, Chellappan SP, Grant S. Divergent effects of bryostatin 1 and phorbol myristate acetate on cell cycle arrest and maturation in human myelomonocytic leukemia cells (U937). Differentiation 1998; 63: 33–42. 13 Szallasi Z, Smith CB, Pettit GR, Blumberg PM. Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts. J Biol Chem 1994; 269: 2118–2124. 14 Hocevar BA, Fields AP. Selective translocation of ␤II-protein kinase C to the nucleus of human promyelocytic leukemia (HL-60) cells. J Biol Chem 1991; 266: 28–33. 15 Lee HW, Smith L, Pettit GR, Vinitsky A, Smith JB. Ubiquitinization of protein kinase C-␣ and degradation by the proteosome. J Biol Chem 1996; 271: 20973–20976. 16 Grant S, Jarvis WD, Swerdlow PS, Turner AJ, Traylor RS, Wallace HJ, Lin PS, Pettit GR, Gewirtz D. Potentiation of the activity of 1-␤-d-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in Hl-60 cells: association with enhanced fragmentation of mature DNA. Cancer Res 1992; 52: 6270–6278. 17 Jarvis WD, Povirk LF, Turner AJ, Traylor RS, Gewirtz DA, Pettit GR, Grant S. Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-␤-d-arabinofuranosylcytosine-

21

22 23 24 25 26

27

28

29 30

31 32

33

34

35

36

induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem Pharmacol 1994; 47: 839–852. 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 1: relationship to differentiation and ara-C-induced apoptosis. Exp Cell Res 1996; 228: 65–75. Wang S, Vrana JA, Bartimole TM, Freemerman AJ, Jarvis WD, Kramer LB, Krystal G, Dent P, Grant S. Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-␤-d-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2. Mol Pharmacol 1997; 52: 1000–1009. Freemerman AJ, Vrana JA, Tombes RM, Jiang H, Chelleppan SP, Fisher PB, Grant S. Effects of antisense p21 (WAF1/CIPI/MDA6) expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (HL-60). Leukemia 1997; 11: 504–513. Wang Z, Su ZZ, Fisher PB, Van Tuyle G, Grant S. Evidence of a functional role for the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 in the reciprocal regulation of PKC activatorinduced apoptosis and differentiation in human myelomonocytic leukemia cells. Exp Cell Res 1998; 244: 105–116. Khym JX. An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin Chem 1975; 21: 1245–1252. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27–55. Martin SJ, Bradley JG, Cotter TG. HL-60 cells induced to differentiate toward neutrophils subsequently die via apoptosis. Clin Exp Immunol 1990; 79: 448–453. Yin DX, Schimke RT. BCL-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells. Cancer Res 1995; 55: 4922–4928. Lock RB, Stribinskiene L. Dual modes of cell death induced by etoposide in human epithelial tumor cells allow Bcl-2 to inhibit apoptosis without affecting clonogenic survival. Cancer Res 1996; 56: 4006–4012. Radford IR. Mouse lymphoma cells that undergo interphase death show markedly increased sensitivity to radiation-induced DNA double-strand breakage as compared with cells that undergo mitotic death. Int J Rad Biol 1991; 59: 353–369. Amarante-Mendes GP, Finucane DM, Martin SJ, Cotter TG, Salvesan GS, Green DR. Anti-apoptotic oncogenes prevent caspasedependent and independent commitment for cell death. Cell Death Diff 1998; 5: 298–306. Bhatia U, Traganos F, Darzynkiewicz Z. Induction of cell differentiation potentiates apoptosis triggered by prior exposure to DNA damaging drugs. Cell Growth Diff 1995; 6: 937–944. Waxman S, Huang Y, Scher BM, Scher M. Enhancement of differentiation and cytotoxicity of leukemia cells by combinations of fluorinated pyrimidines and differentiation inducers: development of DNA double-strand breaks. Biomed Pharmacother 1992; 46: 183–192. Griffin J, Munroe D, Major P, Kufe D. Induction of differentiation of human myeloid leukemia cells by inhibitors of DNA synthesis. Exp Hematol 1982; 10: 774–781. Ishiguro K, Schwartz EL, Sartorelli AC. Characterization of the metabolic forms of 6-thioguanine responsible for cytotoxicity and induction of differentiation of HL-60 of acute promyelocytic leukemia cells. J Cell Physiol 1984; 121: 383–390. Selvakumaran M, Reed JC, Liebermann D, Hoffman B. Progression of the myeloid differentiation program is dominant to transforming growth factor-␤1-induced apoptosis in M1 myeloid leukemic cells. Blood 1994; 84: 1036–1042. Asiedu C, Biggs J, Lilly M, Kraft AS. Inhibition of leukemic cell growth by the protein kinase C activator bryostatin 1 correlates with the dephosphorylation of cyclin-dependent kinase 2. Cancer Res 1995; 55: 3716–3720. Ng SB, Guy GR. Two protein kinase C activators, bryostatin 1 and phorbol-12-myristate-13 acetate, have different effects on haemopoietic cell proliferation and differentiation. Cell Signal 1992; 4: 405–416. Solary E, Bertrand R, Pommier Y. Apoptosis of human leukemic


37 38

39 40 41 42

HL-60 cells induced to differentiate by phorbol ester treatment. Leukemia 1994; 8: 792–797. Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, elHouseine ME. Induction of p21 (WAF1/CIP1) during differentiation. Oncogene 1994; 9: 3389–3396. Jiang H, Lin J, Su ZZ, Collart FR, Huberman E, Fisher PB. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21WAF1/CIP1 expression in the absence of p53. Oncogene 1994; 9: 3397–3406. Wolf D, Rotter V. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc Natl Acad Sci USA 1985; 82: 790–794. Waldman T, Lengauer C, Kinzler KW, Vogelstein B. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 1996; 381: 713–716. Waldman T, Zhang Y, Dillehay L, Yu J, Kinzler K, Vogelstein B, Williams J. Cell-cycle arrest versus cell death in cancer therapy. Nature Med 1997; 3: 1034–1036. Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapyinduced apoptosis in a human leukemia cell line. Blood 1993; 81: 151–157.

43 Park JR, Robertson K, Hickstein DD, Tsai S, Hockenbery DM, Collins SJ. Dysregulated bcl-2 expression inhibits apoptosis but not differentiation of retinoic acid-induced HL-60 granulocytes. Blood 1994; 84: 440–445. 44 Naumovksi L, Cleary ML. Bc12 inhibits apoptosis associated with terminal differentiation of myeloid leukemia cells. Blood 1994; 83: 2261–2267. 45 deVente J, Kiley S, Garris T, Bryant W, Hooker J, Posekany K, Parker P, Cook P, Fletcher D, Ways DK. Phorbol ester treatment of U937 cells with altered protein kinase C content and distribution induces cell death rather than differentiation. Cell Growth Diff 1995; 6: 371–382. 46 Montaldo PG, Chiesa V, Bado M, Raffaghello L, Rozzo C, Ponzoni M. Induction of differentiation and apoptosis by interferon-␥ in human neuroblastoma cells in vitro as a dual and alternative early biologic response. Cell Death Diff 1998; 4: 150–158. 47 Mohammad RM, Katato K, Almatchy VP, Wall N, Liu KZ, Schultz CP, Mantsch HH, Varterasian M, al-Katib AM. Sequential treatment of human chronic lymphocytic leukemia with bryostatin 1 followed by 2-chlorodeoxyadenosine: preclinical studies. Clin Cancer Res 1998; 4: 445–453.

1055