μM ara-C Research Articles

Reduced cellular transport and activation of fluoropyrimidine nucleosides and resistance in human lymphocytic cell lines selected for arabinosylcytosine resistance

Arabinofuranosylcytosine (araC) resistant H9-araC0.05 and H9-araC0.5 sublines were obtained following in vitro exposure of H9 cells to 0.05 and 0.5 μM araC, respectively. These cell lines were 83.3- and 266.7-fold, 21- and 80-fold, and 2.4- and 4.0-fold more resistant to 5-fluorouridine (FUR), 5-fluoro-2′-deoxyuridine (FdUR), and 5-fluorouracil (FU), respectively. Compared with H9 cells, the cellular accumulation of FUR was 2.2 and 0.2%, FdUR 15.6 and 0.9%, and FU 56.9 and 66.5% in H9-araC0.05 and H9-araC0.5 cells, respectively. An araC resistant HL60 cell line (promyelocytic cell line) was 5.0- and 1.7-fold resistant to FUR and FdUR, respectively, but displayed no resistance to FU. The lower FUR and FdUR nucleotide levels in the resistant cells were a result of reduced cellular transport and uridine kinase (UR kinase) and thymidine kinase (TK) activities. Compared with the parental cell line, the p-nitrobenzyl thioinosine (an inhibitor of nucleoside transport) binding sites also were lower in the araC resistant cells. There was no difference in the expression of multidrug-resistant protein and thymidylate synthase mRNA in the parental and the resistant cell lines. Data presented here suggest that araC exposure of H9 cells, in addition to araC resistance, induced/selected cells that were resistant to FUR and FdUR. These cells had altered cellular drug transport and lower TK and UR kinase activities. Further studies to understand molecular mechanisms of this phenomenon are warranted.

Optimizing antimetabolite-based chemotherapy for the treatment of childhood acute lymphoblastic leukaemia.

Optimizing antimetabolite-based chemotherapy for the treatment of childhood acute lymphoblastic leukaemia.

Inhibition of protein kinase C activator-mediated induction of p21 CIP1 and p27 KIP1 by deoxycytidine analogs in human leukemia cells : Relationship to apoptosis and differentiation

Events accompanying sequential exposure of U937 leukemic cells to the deoxycytidine (dCyd) analogs 1-[β- d-arabinofuranosyl]cytosine (ara-C) or 2′,2′-difluorodeoxycytidine (gemcitabine; dFdC) followed by two protein kinase C (PKC) activators [bryostatin 1 (BRY) or phorbol 12′-myristate 13′-acetate (PMA)] exhibiting disparate differentiation-inducing abilities were characterized. A 24-hr exposure to 10 nM BRY or PMA after a 6-hr incubation with 1 μM ara-C or 100 nM dFdC resulted in equivalent increases in apoptosis, caspase-3 activation, and polyADP-ribose polymerase degradation, as well as identical DNA cleavage patterns. BRY and PMA did not modify retention of the lethal ara-C metabolite ara-CTP or alter ara-CTP/dCTP ratios. Unexpectedly, pretreatment of cells with ara-C or dFdC opposed BRY- and PMA-related induction of the cyclin-dependent kinase inhibitors (CDKIs) p21 CIP1 and/or p27 KIP1. These effects were not mimicked by the DNA polymerase inhibitor aphidicolin or by VP-16, a potent inducer of apoptosis. Inhibition of PKC activator-induced CDKI expression by ara-C and dFdC did not lead to redistribution of proliferating cell nuclear antigen but was accompanied by sub-additive or antagonistic effects on leukemic cell differentiation. Sequential exposure of cells to ara-C followed by BRY or PMA led to substantial reductions in clonogenicity that could not be attributed solely to apoptosis. Finally, pretreatment of cells with ara-C attenuated PMA- and BRY-mediated activation of mitogen-activated protein kinase, an enzyme implicated in CDKI induction. Collectively, these findings suggest that pretreatment of leukemic cells with certain dCyd analogs interferes with CDKI induction by the PKC activators PMA and BRY, and that this action may contribute to modulation of apoptosis and differentiation in cells exposed sequentially to these agents.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promoters of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promotors of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM of cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase--mediated biotinylated--deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Comparison of the induction of apoptosis in human leukemic cell lines by 2′,2′-difluorodeoxycytidine (gemcitabine) and cytosine arabinoside

The induction of DNA fragmentation by cytosine arabinoside (araC) and 2′,2′-difluorodeoxycytidine (dFdC, gemcitabine) was compared in human leukemic cell lines. For both araC and dFdC this process was time- and concentration-dependent and resulted in loss of clonogenic survival of HL-60 myeloid leukemic cells. There was a marked difference in the potency between these two analogs in inducing apoptosis. A 6 h exposure to 5 μM araC was required to produce DNA laddering in HL-60 cells, whereas dFdC at a concentration 100-fold less (0.05 μM) was sufficient to produce similar results. Pre-incubation of HL-60 cells with staurosporine, a non-specific protein kinase C inhibitor, increased the level of apoptosis induced by a 3 h exposure to araC or dFdC, suggesting the possible involvement of this family of enzymes in this process. Also, dFdC was able to increase the expression of both c- jun and c- fos in Molt-3 leukemic cells with a concentration known to induce apoptosis in this cell line.

Augmentation by aphidicolin of 1-β- d-arabinofuranosylcytosine-induced c- jun and NF-κB activation in a human myeloid leukemia cell line: Correlation with apoptosis

1-β- d-arabinofuranosylcytosine (ara-C) (2 μM) can induce apoptosis in a human myeloid leukemia cell line, U937, after 4 h of incubation. Pretreatment of cells with aphidicolin (2 μM) augments ara-C-induced apoptosis, since it was first observed at 0.4 μM ara-C and became more intense at 2 and 10 μM. Although aphidicolin itself had a marginal effect on c- jun expression, it significantly augmented ara-C induced c- jun upregulation by shortening the lag time and lowering ara-C concentrations necessary for the induction of detectable c- jun transcripts. Aphidicolin and ara-C acted synergistically to increase NF-κB DNA binding activity as determined by an electrophoretic mobility shift assay. Expression of c- myc was slightly increased through the DNA degradative phase, and was then downregulated. Thus, the activation of NF-κB and c- jun expression seems to be well correlated with the potentiation by aphidicolin of ara-C-induced apoptosis.

In vitro effects of bryostatin 1 on the metabolism and cytotoxicity of 1-β- d-arabinofuranosylcytosine in human leukemia cells

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-β- d-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 μM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 × 10 6 cells/ mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 μM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 solpmol 10 6 ), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10–100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 μM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth. These findings suggest that the novel agent bryostatin 1 induces biochemical perturbations in leukemic cells that favor ara-C activation, particularly in high-density cells exhibiting impaired ara-C nucleotide formation. They also raise the possibility that pharmacologie agents acting through second messenger pathways may modulate the metabolism of ara-C, and potentially other nucleoside analogs.

90 METABOLISM OF GUANINE ARABINOSIDE AND CYTOSINE ARABINOSIDE IN CELLS FROM PATIENTS WITH LEUKEMIA

Guanine arabinoside (araG) and cytosine arabinoside (araC) are nucleoside analogs which elicit cytotoxicity through their corresponding 5′-triphosphate, metabolites. Although less potent than araC, araG is of interest as an antileukemic agent due to its selective toxicity to cultured T compared to B lymphoblasts. In order to determine whether araG would exhibit a similar selectivity of action in vivo, we have examined the metabolism of this drug in mononuclear cells from patients with leukemia. Peripheral blood was diluted and incubated with 100 μM araG or 10 μM araC for 4 hours. The highest level of araGTP accumulation occurred in leukocytes from patients with T-cell ALL (median value 187 pmol/107 cells). Cells from patients with AML or non-T-, non-B cell. ALL accumulated lower levels of araGTP (72 and 86 pmol/107 cells, respectively), and cells from patients with CLL accumulated the lowest amount of araGTP (31 pmol/107 cells). In contrast, accumulation of araCTP was similar in cells from patients with T-cell ALL, AML and non-T, non-B-cell ALL. The amount of araG or araC remaining in plasma after 4 hours varied and was not related to the cellular accumulation of the corresponding nucleotides. These results suggest that araG may act as a selective chemotherapeutic agent in patients with T-cell ALL.

Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine

0.3–1.0 σmM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1–2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 μM araC-treated cells. The cell cycle activated by caffeine in 0.3 μM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 μM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.

Potentiation of 1-β- d-arabinofuranosylcytosine in hepatoma cells by 2′-deoxyadenosine or 2′-deoxyguanosine

A 2-hr pretreatment with 2′-deoxyadenosine (AdR), 2′-deoxyguanosine (GdR), or thymidine (TdR) synergized the growth-inhibitory effect of subsequent arabinosylcytosine (Ara-C) treatment in rat hepatoma cell lines N 1S 1 and 3924A and in the rat fibroblast line BF5. In addition, the sequence of 100 μM AdR or GdR for 2 hr followed by 15 μM Ara-C for 6 hr resulted in reduction of N 1S 1 and 3924A colony formation to 13 per cent of control values, representing synergistic killing when compared with treatment by any of these agents alone. Measurements in N 1S 1 hepatoma cells showed that AdR caused increased dATP (221 per cent) and decreased dCTP and dTTP (23 and 41 per cent respectively) in comparison with untreated controls. GdR caused increases in dGTP (351 per cent control) and dATP (191 per cent control) and decreased dCTP (36 per cent control) and dTTP (37 per cent control). Pretreatment with AdR (but not GdR) caused increased cellular Ara-CTP concentration. Pretreatment with AdR (100 μM) for 2hr increased the incorporation of [ 3H]Ara-C into DNA by 2-fold, and 2-hr pretreatment with GdR (100 μm) increased [ 3H]Ara-C incorporation into DNA 3-fold. AdR also increased incorporation of [ 3H]Ara-C into RNA, but GdR did not have this effect. In all three cell lines, AdR treatment decreased the concentrations of cellular UTP and CTP. Of the three deoxynucleosides, AdR, GdR and TdR, the most potent synergism with Ara-C was given by GdR. It was concluded that for all three nucleosides the potentiation was primarily a consequence of decreased cellular pools of dCTP.