Heritable alterations at the adenine phosphoribosyltransferase (APRT) locus in human lymphoblastoid cell lines

Abstract

Human lymphoblastoid cell lines derived from WI-L2 exhibit unexpected frequencies of diaminopurine (DAP) resistant mutants. The background mutant fractions of 10 −7 to 10 −8 in untreated cultures are much lower than the frequencies expected for loss of a heterozygous autosomal locus (10 −5 to 10 −6), yet much higher than expected for a homozygous locus (10 −10 to 10 −12). We used aminopterin, adenine and thymidine (AAT) to select DAP-senitive (DAP S) revertants from one resistant line. The background frequency of DAP R in these revertant cell lines ranged from 3.5 to 6.5 × 10 −4, approximately the square root of 10 −7. Thus these data suggest that both alleles of aprt are inactivated at similarly high frequencies. They also indicate that the DAP S revertants were heterozygotes ( aprt +/−) or hemizygotes ( aprt +/0) and that WI-L2 was homozygous ( aprt +/+). Mutational dose-response studies with X-rays, ethyl methanesulfonate (EMS), and ICR-191 were conducted in 4 of these revertant cell lines. EMS and ICR-191, which induce mainly point mutations, did not induce an increase in mutant fraction. A dose of 200 cGy X-rays, however, induced a frequency of 10 −3. Treatment of DAP R cells with 5-azacytidine induced a significant increase in reversion to DAP S. Southern blot analysis of the aprt gene after digestion with MspI or HpaII also suggests that differential methylation changes may play a major role in the generation of DAP sensitivity and resistance.