Increased methotrexate‐induced DNA strand breaks and cytotoxicity following mutational loss of thymidine kinase

Abstract

M T X for 16 hr, TK-cells showed a significantly higher number of DNA strand breaks. Concomitahtly, DNA fragmentation at the nucleosomal linker region was detected more prominently in TK-cells. Although MTX tended to decrease l T P pools similarly in all 3 cell types, the initial l T P level in TK-cells was only about one-fifth of that found in the wild type. These results indicate that the thymidine salvage pathway has a pivotal role in mediating MTX-induced toxicity and DNA lesions.Methotrexate (MTX) limits the intracellular supply of reduced folates (tetrahydrofolate) through inhibition of dihydrofolate reductase (DHFR) (Werkheiser, 1963;Jolivet et al., 1983). Consequently, cells are depleted of both thymidylate and purine nucleotides (Bokkerink et al., 1988), since tetrahydrofolates act as co-factors in their biosynthesis. Therefore, the growth-inhibitory effects of MTX can be explained by thymine-less and/or purine-less mechanisms. In fact, MTXinduced cytotoxicity is reversed by the simultaneous addition of thymidine and purine bases such as hypoxanthine at appropriate concentrations (Hakala and Taylor, 1969). However, there is some controversy as to whether the thymine-less or the purine-less state is more relevant to the cytotoxicity.The accumulation of DNA strand breaks following MTX treatment has been reported in Ehrlich ascites tumor cells (Li and Kaminskas, 1984). Several groups have confirmed this observation in other cell lines, such as human colon adenocarcinorna cells (Lonn and Lonn, 1988) and NIH/3T3 cells (Lorico et al., 1988). Although the biochemical mechanism remains to be elucidated, one attractive hypothesis involves the activation of endonuclease through imbalances in deoxyribonucleoside triphosphate (dNTP) pools (Yoshioka et al., 1987). Similar DNA lesions had been induced during treatment with 5-fluorodeoxyuridine (Yoshioka et al., 1987), which inhibits thymidylate synthetase following phosphorylation into its mononucleotide.This report presents the results of biochemical genetic studies of the role of a thymidine or purine salvage pathway in MTX-induced DNA strand breaks. MATERIAL AND METHODS Cell linesA human promyelocytic leukemia cell line, HL-60, came from the Japanese Cancer Resources Bank (Tokyo, Japan). Cells were grown in suspension culture in RPMI-I640 medium supplemented with 10% heat-inactivated dialyzed fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 p g h l ) . Dialysis of the serum is necessary to minimize the influence of several nucleosides or nucleobases contained in the fetal bovine serum. The cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO,. HGPRT-deficient (HGPRT-) or TK-deficient (TK-) HL-60 cells were selected in our laboratory on the basis of their resistance to either 8-azaguanine or bromodeoxyuridine after mutagenesis with ethylmethane sulfonate (Kubota et al., 1983). The drug-resistant population was cloned by limiting dilution (0.5 cell/well) in 96-well tissue culture trays. The clones analyzed in...

pH Effects and Cooperativity among Key Titratable Residues for Escherichia coli Glycinamide Ribonucleotide Transformylase

“…Cell division requires purines through the de novo purine biosynthesis and salvage pathways. , In normal cells, purines are mainly supplied through the salvage pathway . However, in some cancer cell lines, purines are primarily supplied by the de novo purine biosynthesis pathway. , Glycinamide ribonucleotide transformylase (GAR Tfase) is one of the regulatory enzymes in the de novo purine biosynthesis pathway that transfers the formyl- group from 10-formyl tetrahydrofolic acid (10f-THF) to the substrate β-glycinamide ribonucleotide (GAR). Many inhibitors such as lometrexol, AG2034, and pemetrexed inhibit the GAR Tfase enzyme and prevent cancerous cells from undergoing uncontrolled cell proliferation .…”