X-ray induction of methotrexate resistance due to dhfr gene amplification.
The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.
Hahn
P
P
Department of Radiology, State University of New York Health Science Center, Syracuse 13210.
Nevaldine
B
B
Morgan
W F
WF
D000818:Animals; D001375:Azaguanine / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002478:Cells, Cultured; D002875:Chromosomes / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004351:Drug Resistance / Q000235:genetics / Q000528:radiation effects; D004586:Electrophoresis / Q000379:methods; D005784:Gene Amplification / Q000187:drug effects / Q000528:radiation effects*; D008727:Methotrexate / Q000494:pharmacology*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011838:Radiation-Sensitizing Agents; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D001549:Benzamides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011838:Radiation-Sensitizing Agents; C025160:3-aminobenzamide; D013762:Tetrahydrofolate Dehydrogenase; D001375:Azaguanine; D008727:Methotrexate