Accumulation of DNA strand breaks and methotrexate cytotoxicity.

Abstract

There was a progressive formation of strand breaks in mature DNA of Ehrlich ascites tumor cells that were treated with methotrexate. Cells were labeled with [14C~thyMi-dine before incubation with methotrexate, and DNA strand breaks were measured by alkaline and by neutral filter elution methods. DNA single-strand breaks accumulated in a linear fashion as a function of time during the first 10 hr of incubation with 2 ,uM methotrexate. Thereafter, the accumulation of DNA strand breaks deviated from linearity because of progressive cell death. The extent of DNA strand breakage in cells that had been incubated with methotrexate for 24 hr was as high as in cells that had been irradiated with 300 rads. DNA strand breaks persisted in cells that were incubated, after exposure to methotrexate, in medium containing thymidine, hypoxanthine, and nonessential amino acids, indicating that these strand breaks were poorly repaired. Cell death commenced after 10 hr of incubation with methotrexate and continued during the following 3-4 days. These findings suggest that cell death was due to a lethal accumulation of DNA strand breaks. The formation of DNA strand breaks is probably due to inefficient DNA repair, resulting from the inhibition of syntheses of thymidylate and of purine nucleotides. The accumulation of DNA strand breaks was minimal in growth-arrested cells, which are resistant to methotrexate toxicity.Cytotoxicity of the antifolate methotrexate (MTX) is generally thought to be related to the inhibition of DNA synthesis, although the precise mechanism of cell death is not known (1). Multiplying cells are sensitive to MTX, especially cells in the S phase of the cell cycle (2), while resting cells are not (2-4). MTX restricts the synthesis of thymidylate and of purine nucleotides by inhibiting dihydrofolate reductase and, to a lesser extent, thymidylate synthetase (1). Hence, MTX cytotoxicity has been postulated to occur both by "thymineless death" and "purineless death" mechanisms (5). DNA synthesis is inhibited rapidly in cells that are incubated with micromolar concentrations of MTX. In CCRF-CEM human lymphoblastoid cells, DNA synthetic rates decrease by 80% within 30 min and then remain stable for at least 6 hr (6). The newly synthesized DNA accumulates in 80S fragments and does mature to bulk-size DNA upon reversal of the MTX block, at least within the first hr of MTX treatment (6). An abnormal progression of DNA synthesis, with accumulation of low molecular weight DNA fragments, also was demonstrated with the antifolate metoprine (7). In addition, there was suggestive evidence that newly formed DNA may be partially degraded, perhaps as a result of misincorporation of dUMP and the subsequent repair reactions (7). Misincorporation of dUMP also has been demonstrated in cells treated with MTX (8), and it was hypothesized that futile repair processes could result in extensive DNA degradation, by analogy to thymineless death in bacteria (9-15). (16). Ehrlich ascites tumor cells were grown in continuous spinner cu...

Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

“…We extended these observations by showing that there is a similar reduction in the frequency of cells with chromosome aberrations in cell populations recovering from treatment with these drugs. DNA strand breaks have been shown to accumulate during prolonged inhibition of DNA synthesis with MTX, and this effect (as well as cytotoxicity) is strongly reduced in growth-arrested cells (19). Similar results were reported for HU treatment by Coyle and Strauss (7), who also found that, in contrast to HU, the mild cytotoxicity of cycloheximide (which was very similar to the level reported here) was not associated with extensive DNA strand breakage.…”

Gene Amplification in a p53-Deficient Cell Line Requires Cell Cycle Progression under Conditions That Generate DNA Breakage

“…By utilizing a selective agent other than PALA, we were able to determine that the failure to stop cell cycle progression under suboptimal growth conditions was an important step in the generation of amplified sequences. PALA and MTX can cause DSBs, but only when the treated cells progress through the cell cycle in the presence of the drug (6,23,43,55). In cells lacking the MTX-induced growth arrest, MTX functions both to initiate the amplification process by generating DSBs and to select for those cells with increased DHFR copy number, analogous to the action of PALA in p53-deficient cells.…”

Mechanism of cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cells in vitro and modulation of the drug activity by folic or folinic acid

“…For other antifolates such as methotrexate (MTX), it has been proposed that cytotoxicity is due to the formation of DNA breaks, presumably caused by uracil misincorporation into DNA and/or activation of endonucleolytic enzymes (Li & Kaminskas, 1984;Lorico et al, 1988). Since MTX inhibits both thymidine and purine synthesis, it has been suggested that the DNA fragmentation is triggered by the block of DNA synthesis.…”