Cellular metabolism of 1-beta-D-arabinofuranosyl-5-azacytosine and incorporation into DNA and RNA of human lymphoid CEM/0 and CEM/dCk(-) cells.

1-beta-D-arabinosyl-5-azacytosine (ara-AC) is a relatively new antitumor agent under clinical investigation, which has the 2'-beta arabinosyl configuration found in the tumoricidal drug ara-C and the nitrogen substitution in the 5-position of the pyrimidine ring found in 5-azacytidine (5-aza-C). The present study examined the cellular metabolism and the effect on DNA methylation of ara-AC in human CCRF/CEM cells sensitive and resistant to ara-C. The triphosphate anabolite of the drug, ara-ACTP, was the major anabolite in the CEM cellular extracts, peaking at 50.6 +/- 23 microM 4 h after incubation with IC50 concentrations (0.25 microM) of [3H]ara-AC. The mono- and diphosphate anabolites accumulated 10-fold lower cellular concentrations than ara-ACTP. The nucleoside triphosphate (NTP) pools and, especially, cellular ATP declined significantly by 9 h after the initiation of drug treatment and remained depleted for the 24-h treatment. The drug anabolite was gradually incorporated into both RNA and DNA, peaking in CEM/0 at 3.44 and 0.14 nmol/10(7) cells, respectively. The DNA methylation levels in these cells declined rapidly after treatment with ara-AC, attaining a nadir plateau at 29% of control methylation value. The deoxycytidine kinase (dCK) mutant CEM cell line [CEM/dCk(-)] neither activated ara-AC at appreciable levels nor induced DNA hypomethylation at low concentrations (0.25-1 microM). However, the drug was activated at 0.2-1 microM extracellular concentrations of ara-AC, probably by an as yet unknown nucleoside kinase at approximately 10% of the amount in CEM/0 cells. Ara-AC appears to mediate its cytotoxic action through the accumulation of its triphosphate anabolite, ara-ACTP, and the subsequent incorporation into nucleic acids. DNA methylation may also contribute to its cytotoxicity.

Cellular metabolism of 5,6-dihydro-5-azacytidine and its incorporation into DNA and RNA of human lymphoid cells CEM/O and CEM/dCk(-).

5,6-Dihydro-5-azacytidine (DHAC) is a hydrolytically stable analog of 5-azacytidine (5-aza-C) that has antileukemic activity against experimental leukemias and, like 5-aza-C, causes DNA hypomethylation. We report the cellular metabolism of DHAC and its incorporation into nucleic acids in the CCRF/CEM/O and deoxycytidine kinase mutant CCRF/CEM/dCk(-) human lymphoid cell lines. The cells were incubated with their respective IC50 concentrations for 24 h, then aliquot samples were removed at predetermined intervals and extracted for nucleotides. The acid-soluble extracts of the cells were assayed on HPLC for nucleotides of DHAC. The major anabolite of [3H]DHAC, [3H]DHACTP, peaked at 110.3 +/- 30.7 microM in CEM/O and at 96.3 +/- 41.9 microM in CEM/dCk(-) cells at 9 and 12 h, respectively. The intracellular concentrations of the deoxyribonucleoside triphosphate, [3H]DHAdCTP, peaked at 13.5 +/- 7.7 microM at 4 h in CEM/O and at 80.8 +/- 13.8 microM at 12 h, a 6-fold greater cellular concentration, in the dCk mutant cell line. The amount of DHAC anabolites incorporated into CEM/O nucleic acids reached a plateau in RNA at 552.6 +/- 7.8 pmol/10(7) cells and in DNA at 64.55 +/- 10.0 pmol/10(7) cells. In CEM/dCk(-) cells, DHAC anabolites reached a plateau in RNA and DNA at 4,256.3 +/- 631.0 and 395.5 +/- 145.4 pmol/10(7) cells, respectively. Thus, with equitoxic treatments of DHAC, the incorporation of its analog anabolites into RNA and DNA was 8- and 6-fold greater in CEM/dCk(-) cells. DNA methylation levels were depressed equally despite a 6-fold greater incorporation of the analog in DNA in the CEM/dCk(-) cells indicating that hypomethylation may be saturated after DHAC treatment. The DNA methylation levels reached a nadir of 0.19% and 0.20% methyl-C (percentage of methylation) in the two cell lines at 6 and 12 h after the beginning of drug treatment and remained relatively constant for the duration of the 24-h treatment. A curve-linear relationship was obtained between the DNA methylation levels in both cell lines and the amounts of DHAC anabolite incorporated into DNA.

Pharmacodynamic and DNA methylation studies of high-dose 1-beta-D-arabinofuranosyl cytosine before and after in vivo 5-azacytidine treatment in pediatric patients with refractory acute lymphocytic leukemia.

The primary development of clinical resistance to 1-beta-D-arabinofuranosyl cytosine (ara-C) in leukemic blast cells is expressed as decreased cellular concentrations of its active anabolite. Correlations exist between the cellular concentrations of 1-beta-D-arabinofuranosyl cytosine 5'-triphosphate (ara-CTP) in leukemic blast cells and inhibition of DNA synthetic capacity with the clinical response to high-dose cytosine arabinoside (HDara-C). 5-Azacytidine (5-Aza-C) and its congeners are potent DNA hypomethylating agents, an action closely associated with the reexpression of certain genes such as that for deoxycytidine kinase (dCk) in ara-C-resistant mouse and human leukemic cells. Reexpression of dCk could increase the cellular ara-CTP concentrations and the sensitivity to ara-C. A total of 17 pediatric patients with refractory acute lymphocytic leukemia (ALL) received a continuous infusion of 5-Aza-C at 150 mg/m2 daily for 5 days after not responding to (13/17) or relapsing from (4/17) an HDara-C regimen (3 g/m2 over 3 h, every 12 h, x 8 doses). Approximately 3 days after the end of the 5-Aza-C infusion, the HDara-C regimen was given again with the idea that the induced DNA hypomethylation in the leukemic cells may have increased the dCk activity and that a reversal of the tumor drug resistance to ara-C could have occurred. Deoxycytidine kinase (expressed as cellular ara-CTP concentrations) in untreated blasts, DNA synthetic capacity (DSC), and the percentage of DNA methylcytidine levels were determined before and after 5-Aza-C administration. Cellular ara-CTP was enhanced to varying degrees in 15 of 16 patients after 5-Aza-C treatment. The average cellular concentration of ara-CTP determined in vitro by the sensitivity test was 314 +/- 390 microM, 2.3-fold higher than the average value before 5-Aza-C treatment. In 12 patients in whom the DNA methylation studies were completed before and after 5-Aza-C treatment, the average DNA hypomethylation level was 55.6% + 15.8% of pretreatment values (n = 13; mean +/- SD). DSC showed a profound decline in 2/9 evaluable patients who achieved a complete response (CR) after this regimen. The data suggest that treatment with a cytostatic but DNA-modulatory regimen of 5-Aza-C causes DNA hypomethylation in vivo, which is associated with dCk reexpression in the patients' leukemic blasts. The partial reversal of drug resistance to ara-C by 5-Aza-C yielded two CRs in this poor-prognosis, multiply relapsed patient population with refractory ALL.(ABSTRACT TRUNCATED AT 400 WORDS)