Induction of deoxycytidine kinase by 5-azacytidine in an HL-60 cell line resistant to arabinosylcytosine

Induction of 2′-deoxycytidine kinase (dCK) by 5-azacytidine (5-Aza-C) in a dCK-deficient HL-60 cell line resistant to 1-β-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 μM 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 μM for HL-60 cells, 12.5 μM for HL-60/Ara-C cells, and 0.55 μM for HL-60/Ara-C cells pretreated with 5-Aza-C for 40 hr. The K_m and V_max of dCyd for HL-60 dCK were similar to those for 5-Aza-C-induced HL-60/Ara-C dCK. The restriction enzymes HpaII, which cleaves CCGG sequences but cannot cleave at sites methylated at the internal cytosines (5′-C^MeCGG), and MspI, 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 MspI to a greater extent than that of HL-60/Ara-C cells. After exposure to 1 μM 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, DMAs from 5-Aza-C-treated resistant cells were cleaved to a greater extent by MspI than by HpaII, suggesting that internal cytosine-residue methylation was relatively uninhibited. The data given above suggest that 1) dCK in HL-60/Ara-C cells was induced by 5-Aza-C and the resistance to Ara-C in the cells was partially reversed by 5-Aza-C, 2) the induced enzyme was similar in terms of its kinetic properties to the wild-type enzyme, 3) DNA from HL-60/Ara-C cells may have more methylation at the external cytosine residues of 5′-CCGG sequences than DNA from wild-type HL-60 cells, and 4) the DNA methylation at external cytosine residues was inhibited (i.e., hypomethylation state) when dCK expression was induced by 5-Aza-C treatment.