Cellular Phosphorylation of 1-β-d-Arabinofuranosylcytosine and 5-Azacytidine with Intact Fibrosarcoma and Leukemic Cells
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Abstract:
The phosphorylation of 1-β-d-arabinofuranosylcytosine (ara-C) and 5-azacytidine (5-aza-C) by A(T1)C1-3 hamster fibrosarcoma cells and L5178Y murine leukemic cells was studied, using intact cells. The cellular phosphorylation of both these nucleoside analogs appears to follow Michaelis-Menton kinetics. The apparent Km value for ara-C in the fibrosarcoma and leukemic cells was about 40 µm, whereas the apparent Km values for 5-aza-C in these cells were about 1.3 and 0.41 mm, respectively. Deoxycytidine and cytidine were found to be potent competitive inhibitors of the phosphorylation of ara-C and 5-aza-C, respectively. ara-C and 5-aza-C were found to be weak competitive inhibitors of the phosphorylation of deoxycytidine and cytidine, respectively. A clone isolated from the fibrosarcoma cells that was partially resistant to the cytotoxic effects of ara-C exhibited a higher Km value for both ara-C and deoxycytidine than the wild-type fibrosarcoma cells.Keywords:
Cytidine
HT1080
clone (Java method)
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The treatment of K562 human myeloblastic leukemia cells and YAC-1 murine lymphoma cells with cadeguomycin at concentrations over 0.6 microM significantly enhanced the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). The degree of potentiation depended upon the antibiotic concentration. The treatment with 75 microM cadeguomycin for 18 h increased cellular uptake of [3H]ara-C into K562 cells and formation of ara-C nucleotides, as well as incorporation into nucleic acids. The level of the diphosphate of ara-C plus the triphosphate of ara-C was approximately 10 times higher in the cadeguomycin-treated cells than in the untreated cells by 30 min of incubation with [3H]ara-C. The extracts of 15 microM cadeguomycin-treated K562 cells showed increased activity of formation of ara-C nucleotides, resulting in 4- to 5-fold higher formation of the di- and triphosphates of ara-C than the control cell extracts. Cadeguomycin did not significantly change the level of ribonucleotide and deoxyribonucleotide pool in K562 cells. The mechanism of potentiation of ara-C by cadeguomycin was discussed.
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This study attempts to determine the primary actions of 5-azacytidine (5-azaCR) on L1210 leukemia. This agent was cytotoxic toward L1210 cells growing in culture with 50 and 90% inhibition dose values of 0.019 and circa 0.15 μg/ml, respectively. 5-AzaCR inhibits the incorporation of tritiated thymidine or deoxyadenosine into DNA to a greater extent than it inhibits the incorporation of tritiated uridine into RNA. Similar results were obtained with ascitic cells isolated from leukemic mice. Equimolar amounts of cytidine reduced the inhibition of DNA synthesis, as well as the inhibition of cell growth in culture caused by 5-azaCR. Uridine, but not deoxycytidine or deoxyuridine, was also effective, but to a lesser extent than was cytidine. With cell-free extracts isolated from L1210 cells in culture, no significant effect was found on enzyme systems directly or indirectly involved in DNA synthesis.
With 5-azaCR-4-14C as the precursor, this agent was found to be phosphorylated in all leukemic tissues studied. The majority of phosphorylated products existed as the triphosphate in ascitic and cultured L1210 cells. A portion (10 to 20%) of all these phosphorylated derivatives appeared to be further reduced to deoxyribonucleoside di- and/or triphosphate forms. 5-AzaCR was also incorporated into polynucleotides in all tissues studied and incorporated into both RNA (80 to 90% total incorporated radioactivity) and DNA fractions (10 to 20%) in L1210 cells in culture. In the presence of cytidine, phosphorylation of 5-azaCR, subsequent reduction, and incorporation into polynucleotides were greatly inhibited.
A probable mechanism of action of 5-azaCR on L1210 leukemia is proposed.
L1210 cells
Cytidine
Thymidine
Polynucleotide
Deoxyuridine
Deoxyadenosine
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1601 1-beta-D-arabinofuranosylcytosine ( cytarabine; ara-C ) is the most active agent in myeloid leukemia. 5-aza-2′-deoxycytidine ( 5aza-dC) is a cytosine analog that inhibits DNA methylation and also has activity in myeloid leukemia. In order to investigate the possible therapeutic combination of these agents, we studied the interaction of ara-C and 5aza-dC on human Acute Myeloid Leukemia (HL-60, ML-1), and Acute Lymphoblastic Leukemia (RAJI) cell lines. We initially examined inhibition of cell growth. The combination of 5aza-dC 1uM + ara-C 0.01 uM, 0.1uM, or 1uM showed antagonism on day 3 or day 5 compared to either agent alone in HL60, ML-1 and RAJI. For example, 5aza-dC 1uM alone caused 43% growth inhibition at day 3 in HL60, ara-C 1uM alone caused 78% growth inhibition, while the combination showed 58% inhibition. Antagonism was also observed when 5aza-dC 1uM was given first followed by ara-C (0.1uM, 1uM) on day 3 or day 5 in HL60, ML-1 and RAJI. In searching for a possible mechanism for this antagonism, we initially examined reactivation of silenced genes. RIL and P57 were activated 5-fold or greater by 5aza-dC in HL60 and ML-1, but this activation was almost completely inhibited by the addition of ara-C to 5aza-dC. We next looked at DNA methylation of Alu repetitive elements in these cells as a surrogate for global methylation. In HL60, 5aza-dC induced Alu demethylation by 18% after 3 days and 30% after 5 days. With co-administration of ara-C at 0.1uM, Alu demethylation was reduced to 4.1% on day 3 and 5.0 % on day 5. Increasing ara-C to 1uM, 5aza-dC further reduced demethylation to 0.9% on day 3 or 3.9% on day 5. Similar results were seen in ML-1. These data show that ara-C administered with 5aza-dC or shortly after inhibits demethylation and gene reactivation, accounting for the observed antagonism. This is likely related to the fact that 5aza-dC and ara-C compete for deoxycytidine kinase for incorporation into DNA. Our results suggest that combination of 5aza-dC + ara-C must be designed with a large interval between the 2 drugs.
HL60
Demethylating agent
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The 2-fluoro derivative of 9-beta-D-arabinofuranosyladenine (2-F-ara-A) and its soluble 5'-formate and 5'-phosphate derivatives were therapeutically effective against the parent leukemia L1210 (L1210/0). 2-F-ara-A and 9-beta-D-arabinofuranosyladenine 5'-formate were inactive aginst a 1-beta-D-arabinofuranosylcytosine-resistant subline (L1210/ara-C) that was deficient in deoxycytidine kinase. Deoxycytidine prevented 2-F-ara-A-induced inhibition of proliferation of L1210/0 cells in culture and alleviated 2-F-ara-a inhibition of DNA synthesis. After treatment of mice with 9-beta-D-arabinofuranosyladenine 5'-formate, intracellular levels of the 5'-triphosphate of 9-beta-D-arabinofuranosylfluoroadenine in leukemia cells were more than 10 times higher in L1210/0 cells than in L1210/ara-C cells. Similar results were obtained in this line of leukemia cells from mice treated with the 5'-monophosphate of 9-beta-D-arabinofuranosyl-2-fluoroadenine. Thus, L1210/ara-C cells deficient in deoxycytidine kinase activity were also deficient in capacity to phosphorylate 2-F-ara-A. Kinase activity from L1210/0 cells for deoxycytidine and for 2-F-ara-A coeluted from calcium phosphate cellulose and from diethylaminoethyl cellulose columns and had similar mobility on gel electrophoresis. Deoxyadenosine kinase was clearly separated from deoxycytidine kinase. Deoxycytidine competed with 2-F-ara-A for phosphorylation by the partially purified enzyme from L1210 cells. These results indicate that 2-F-ara-A is phosphorylated to the 5'-monophosphate by deoxycytidine kinase of leukemia L1210 cells.
Deoxycytidine kinase
L1210 cells
Deoxyadenosine
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Induction of 2'-deoxycytidine kinase (dCK) by 5-azacytidine (5-Aza-C) in a dCK-deficient HL-60 cell line resistant to 1-beta-D-arabinofuranosylcytosine (Ara-C) (HL-60/Ara-C) was examined by measurement of the incorporation of [3H]deoxycytidine ([3H] dCyd) into cellular DNA, the kinetic properties of purified dCK, the cytotoxic potency (IC50 values), and the DNA methylation patterns of 5-Aza-C-treated and untreated cells. Following a 72-hr exposure to 1 microM 5-Aza-C, the incorporation of [3H]dCyd into DNA was increased 6-fold and the total dCK activity was increased 12-fold, with a peak for both on day 6. The onset of dCK induction was dependent on the length of exposure time. The IC50 values for cell growth inhibition by Ara-C on day 3 were 0.08 microM for HL-60 cells, 12.5 microM for HL-60/Ara-C cells, and 0.55 microM for HL-60/Ara-C cells pretreated with 5-Aza-C for 40 hr. The Km and Vmax of dCyd for HL-60 dCK were similar to those for 5-Aza-C-induced HL-60/Ara-C dCK. The restriction enzymes Hpall, which cleaves CCGG sequences but cannot cleave at sites methylated at the internal cytosines (5'-CMeCGG), and Mspl, which cleaves sequences with internal methylated cytosine but cannot cleave at sites methylated at external cytosines (5'-MeCCGG), were used for DNA methylation pattern determination. The newly synthesized DNA of HL-60 wild-type cells was cleaved by Mspl to a greater extent than that of HL-60/Ara-C cells. After exposure to 1 microM 5-Aza-C for 40 hr, methylation patterns of newly synthesized DNA reverted in HL-60/Ara-C cells to a clevage pattern similar to that in HL-60 wild-type cells. When compared with untreated control, DNAs from 5-Aza-C-treated resistant cells were cleaved to a greater extent by Mspl than by Hpall, suggesting that internal cytosine-residue methylation was relatively uninhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
Deoxycytidine kinase
Cytosine
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Cytidine
Deoxycytidine kinase
Deamination
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Summary The effect of 1-β-d-arabinofuranosylcytosine 5′-monophosphate (ara-CMP) against leukemia L1210 was very similar to that of 1-β-d-arabinofuranosylcytosine (ara-C): equimolecular daily doses of the two drugs produced nearly the same increases in survival time. The nucleotide seemed to be slightly less active and less toxic. Efflux of ara-CMP from L1210 ascites cells which were incubated with tritiated ara-C was very small compared to the rate of entry of the nucleoside. This finding and the cross-resistance to ara-CMP of a subline of L1210 resistant to ara-C indicate that ara-CMP does not enter the cells intact but is dephosphorylated prior to uptake.
L1210 cells
Efflux
Mode of Action
Neoplasm
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Kinetics of 5-aza-2'-deoxycytidine phosphorylation in mouse spleen and L1210 leukemic cell extracts.
Out of different nucleosides and deoxynucleosides testes for their ability to block the phosphorylation of labeled 5-aza-2'-deoxycytidine in the presence of ATP and the cell-free extract from mouse spleen only deoxycytidine and cytosine arabinoside depressed the reaction significantly. With the same system the phosphate donor specificity for 5-aza-2'-deoxycytidine and deoxycytidine was determined. All of the 5'-triphosphates used were less efficient donors with respect to the analogue than to the natural substrate. The apparent Michaelis constants for the phosphorylation of 5-aza-2'-deoxycytidine and deoxycytidine were 2.9 and 2.5 x 10(-5) M, respectively. Using the extract from L1210 leukemic cells the Km constant for 5-aza-2'-deoxycytidine was 6.4 x 10(-5) M and that for deoxycytidine 2.7 x 10(-5) M. The Ki constants with the spleen extract were 1.2 x 10(-3) M for 5-aza-2'-deoxycytidine and 5.8 x 10(-6) M for deoxycytidine. Both compounds acted as competitive inhibitors of one another.
Deoxycytidine kinase
Cytosine
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