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 monitor- ing 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