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XB Kong, WP Tong and TC Chou
Laboratory of Biochemical Pharmacology, Memorial Sloan-Kettering Cancer Center, Cornell University Graduate School of Medical Sciences, New York, New York 10021.
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)
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