Biochemical and cytokinetic modulation of L1210 and HL-60 cells by hydroxyurea and effect on 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity.
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The modulating effect of hydroxyurea (HU) on 1-beta-D-arabinofuranosylcytosine (ara-C) metabolism and cytotoxicity was evaluated in L1210 cells and the human promyelocytic leukemic cells HL-60. A dose- and time-dependent HU exposure was observed which resulted in maximum deoxycytidine 5'-triphosphate reduction, intracellular ara-C accumulation, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation, and cytotoxicity as determined by soft agar cloning. For the L1210 cells, a 5-hr pretreatment of 5 mM HU was optimum. The best result obtained with the HL-60 cells was after a 24-hr exposure of 1 mM HU. There was also a maximum incorporation of ara-C into DNA in both cells following these optimal pretreatment conditions. Cytofluorometric analysis demonstrated that this HU treatment resulted in a maximum accumulation of L1210 and HL-60 cells in the pre-S phase of the cell cycle and that after removal of the HU there was a rapid progression of the cell population through S phase. Because cytotoxicity of ara-C is considered to be predominantly from the inhibition of DNA polymerase and/or the incorporation into DNA, the cytokinetic and biochemical modulatory effects of HU must both be contributing factors to consider in achieving maximal cell kill from this drug sequence. (+info)
Modulation of 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity in L1210 cells by fluoropyrimidine pretreatment.
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The effect of pretreatment with the fluoropyrimidines 5-fluorouracil (FUra), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) on the intracellular metabolism and in vitro cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was explored in L1210 cells. A 4-hr exposure to 100 microM FUra, FUrd, or FdUrd produced greater than 3-fold increments in the intracellular accumulation of [3H]deoxycytidine, and 2-fold increments in the intracellular accumulation of [3H]ara-C were produced over a 1-hr exposure. Intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate levels in cells exposed to these 3 agents were also increased over 2-fold. L1210 cells exposed sequentially to 1 microM FUra, FUrd, or FdUrd followed by 5 microM ara-C for 1 hr resulted in synergistic cell killing measured by soft-agar cloning. Although intracellular 2'-deoxycytidine 5'-triphosphate levels were reduced in cells treated for 4 hr with FUra and FUrd, this was not associated directly with the maximum accumulation of ara-C. FdUrd treatment was as effective as FUra and FUrd at enhancing ara-C accumulation and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation and producing synergistic cytotoxicity; however, the 2'-deoxycytidine 5'-triphosphate pools were minimally affected. 5'-Deoxy-5-fluorouridine did not increase intracellular ara-C accumulation, reduce intracellular 2'-deoxycytidine 5'-triphosphate levels, or enhance ara-C cytotoxicity. This sequential drug interaction is similar to that which we have observed when methotrexate precedes ara-C and provides a rational basis from which further in vivo studies can be designed. (+info)
Relationships among Ara-CTP pools, formation of (Ara-C)DNA, and cytotoxicity of human leukemic cells.
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Cytosine arabinoside (Ara-C) is the most effective agent in the treatment of acute myelogenous leukemia. This agent incorporates in leukemic cell DNA, and the extent of this incorporation correlates with loss of clonogenic survival. The incorporated Ara-C residue behaves as a relative DNA chain terminator, and the extent of (Ara-C)DNA formation correlates with inhibition of DNA synthesis. The incorporation of Ara-C into DNA requires the formation of Ara-CTP, and previous measurements of this metabolite have also been correlated with cytotoxicity. Because it is clinically relevant to define biochemical parameters predictive of Ara-C cytotoxicity, the present studies were undertaken to determine the relationship among Ara-CTP pools, formation of (Ara-C)DNA, and loss of clonogenic survival. The results demonstrate that the incorporation of Ara-C into DNA is the single most powerful predictor of cell lethality. Furthermore, although there is a correlation between Ara-CTP pools or continuous cellular exposure to Ara-CTP and cell kill, these relationships are less significant than that obtained with formation of (Ara-C)DNA. The extent of Ara-C incorporation into DNA can be predicted by the product of the Ara-CTP level and time (T), thus supporting the concept that Ara-C incorporation is dependent on continuous exposure to the triphosphate metabolite. These findings support the formation of (Ara-C)DNA as a highly predictive parameter of lethal cellular events. (+info)
Continuous infusion high-dose cytosine arabinoside in refractory childhood leukemia.
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Ten pediatric patients with refractory leukemia received continuous infusion high-dose cytosine arabinoside (ara-C) according to one of two escalating dosage schedules: (1) a 500-mg/m2 rapid infusion loading dose followed by 3.5 g/m2 per day continuous infusion daily for four consecutive days, or (2) a 600-mg/m2 rapid infusion loading dose followed by 5.0 g/m2 per day continuous infusion daily for four consecutive days. Major toxicity at the lower dosage level was grade IV hematopoietic aplasia of three weeks' duration. At the higher dosage level, there was a prohibitive toxicity in multiple organ systems including transient noncardiogenic pulmonary edema, fungal infections, peritonitis, severe diarrhea, transaminase elevations, and one treatment-related death due to acute renal failure. In contrast to other methods of administration of high-dose ara-C, no CNS toxicity occurred. Oncolytic responses were seen in all patients and two achieved brief, partial remissions. Steady-state plasma ara-C concentrations were 13 to 40 mumol/L at the 3.5-g/m2 dosage level and 10 to 225 mumol/L at the 5-g/m2 dosage level; CSF concentrations at both dosages ranged from 2 to 5 mumol/L. Intracellular levels and ratios of 1-beta-D-arabinofuranosylcytidine-5' triphosphate and endogenous deoxycytidine 5' triphosphate in marrow blasts varied widely at steady state during infusion. No positive correlation existed between steady-state plasma ara-C levels, toxicity, oncolytic effect, or intracellular nucleotide concentration. (+info)
Quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the leukemic cells from bone marrow and peripheral blood of patients receiving 1-beta-D-arabinofuranosylcytosine therapy.
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A method for the detection and quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the active metabolite cells and leukemic cells of the peripheral blood from patients receiving ara-C therapy is described. ara-CTP is separated from normal cellular nucleotides by high-pressure liquid chromatography and is quantitated by its absorbance of ultraviolet light at 280 nm with a lower limit of sensitivity of 25 pmol/2 x 10(7) cell equivalents. During separate courses of continuous infusion of different therapeutic doses of ara-C, ara-CTP accumulated in the leukemic bone marrow cells of a patient with acute myelogenous leukemia in proportion to the dose of ara-C. Continuous infusion of ara-C (90 mg/sq m/day) resulted in plateau levels of ara-CPT in peripheral blast cells after 24 hr (115 pmol/1 x 10(7) cell equivalents). A priming dose of ara-C(125 to 250 mg/sq m) followed by a 1-hr infusion of an equal dose of ara-C to patients with acute myelogenous leukemia facilitated the determination of ara-CTP retention in bone marrow and peripheral blood leukemic cells in vivo. This procedure should be useful for extended studies of the biochemical pharmacology of ara-CTP in vivo. (+info)
Altered sensitivity to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate of DNA polymerase alpha from leukemic blasts of acute lymphoblastic leukemia.
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DNA polymerase alpha from the leukemic cells of acute lymphoblastic leukemia (ALL) was found to be more resistant to the inhibition by 1-beta-D-arabinofuranosylcytosine 5'-triphosphate than that from acute myeloblastic leukemia (AML). Apparent Ki values for 1-beta-D-arabinofuranosylcytosine 5'-triphosphate of DNA polymerase alpha from eight patients with ALL [26.7 +/- 7.1 (S.D.) microM] were 5 times higher than those from nine patients with AML [5.2 +/- 1.3 microM]. In contrast, apparent Km values for a normal substrate deoxycytidine 5'-triphosphate of DNA polymerase alpha preparations from either AML and ALL were almost identical (9.4 to 10.9 microM). Likewise, apparent Ki values for another arabinoside analog, 9-beta-D-arabinofuranosyladenine 5'-triphosphate, of DNA polymerase alpha from blasts of seven patients with ALL (16.9 +/- 6.9 microM) were significantly higher than those from patients with AML (3.8 +/- 0.5 microM). These results indicate that DNA polymerase alpha from ALL blast cells has a decreased affinity to the arabinoside analogs of deoxynucleotide triphosphate. The sensitivity of DNA polymerase alpha of blast cells to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate may be one of the determinants of the clinical response to 1-beta-D-arabinofuranosylcytosine treatment. (+info)
Effect of liposomally trapped antitumour drugs on a drug-resistant mouse lymphoma in vivo.
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A TLX-5 mouse lymphoma which was resistant to 1-beta-D-arabinofuranosyl cytosine (AraC) was used in vivo to study the possibility of using liposomes as drug-delivery vehicles in order to overcome drug resistance.The effects of free drugs (AraC, AraCTP and methotrexate) and the liposomally associated drugs on the survival time of tumour-bearing mice were determined.As a more sensitive measure of cell survival, (125)IUdR was incorporated into the DNA of the ascites TLX-5 cells before i.p. injection. Cell survival and the cytotoxic effects of the drugs on the tumour cells were determined by using a double-headed gamma counter to measure the retention of the (125)I label.Both AraC and AraCTP, either as the free drugs or liposomally associated, had no effects on the tumour. Due to the lack of response of tumour cells to these drugs, further studies were initiated with free and liposomally associated methotrexate (MTX), a drug to which the cells were known to be sensitive. It was found that the liposomally associated MTX, at a 5-10-fold lower dose than the free drug, was (a) more effective in prolonging the survival of tumour-bearing mice and (b) as effective as the free drug in killing tumour cells (as measured by the (125)I retention).In vivo MTX was more effective in the liposomally associated form, whereas liposomally entrapped AraC and AraCTP were ineffective. It is proposed that in vivo liposomally associated drugs may be acting not by actively localizing in the tumour cells, but by the liposomes providing a slow-release drug depot, improving the pharmacokinetic properties of MTX. (+info)
Liposomally trapped AraCTP to overcome AraC resistance in a murine lymphoma in vitro.
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Two cell lines, one sensitive and one resistant to the cytotoxic effects of cytosine arabinoside (AraC) were studied in vitro as a drug-resistance model. The sensitivity of these cell lines, to the effects of free and liposomally trapped AraC and AraCTP as well as empty liposomes alone and mixed with free drug, was studied. This was done by following the inhibition of [3H]-dT incorporation into cellular DNA during exposure to the various drugs and liposomes. Some of the liposomal-lipid compositions inhibited [3H]-dT incorporation at very low concentrations, which made them unsuitable for further study. Liposomes composed of a 7:2:1 molar ratio of phosphatidylcholine:cholesterol:phosphatidic acid were selected as a suitable non-inhibitory carrier. Sensitivity of the two cell lines to free AraC differed by 3 logs, when compared in the [3H]-dT-incorporation assay. The resistant cell line was studied further, and was found to be up to 2 logs more sensitive to AraCTP when given in liposomes than to either the free drug alone or mixed with empty liposomes. It appears from these studies that liposomes are able to help overcome drug resistance in this cell line in vitro. (+info)