Upregulation of O6-alkylguanine-DNA-alkyltransferase expression and the presence of double minute chromosomes in alkylating agent selected Chinese hamster cells.
Chinese hamster V79 lung fibroblasts are sensitive to the toxic effects of chloroethylating agents such as mitozolomide (Mz) and express very low levels (less than 2 fmol/mg) of the DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (ATase). These cells were subjected to selection by treatment with serially increasing doses of Mz. After each dose, the surviving population was expanded and ATase activity was determined in cell extracts. ATase specific activity increased stepwise and in cells surviving selection at 120 micrograms/ml Mz had reached 430 fmol/mg protein: polyacrylamide gel electrophoresis and fluorography showed the size of the ATase as 25 kDa. Cytological examination of these cells showed the presence of double minute (DM) chromosomes (mean approximately 3/cell) but no obvious homogeneously staining regions. In cells grown in continuous culture without further selection no marked decrease in ATase activity or DM frequency was observed. Karyotype analysis and DNA profiling confirmed that the parent and selected cells were of the same origin with, in the latter case, the probable loss or gain of a single restriction endonuclease site. No major differences were seen in the intensity of hybridization signals following Southern analyses of DNA from control and Mz selected cells using the human ATase cDNA as a probe. These results indicate that the ATase gene is not amplified in the Mz selected cells and suggest that increased ATase activity is a consequence only of increased transcription or translation of the ATase gene.[1]References
- Upregulation of O6-alkylguanine-DNA-alkyltransferase expression and the presence of double minute chromosomes in alkylating agent selected Chinese hamster cells. Morten, J.E., Bayley, L., Watson, A.J., Ward, T.H., Potter, P.M., Rafferty, J.A., Margison, G.P. Carcinogenesis (1992) [Pubmed]
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