3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

Abstract

3'-Azido-3'-deoxythymidine (AZT), a nucleoside analogue used for the treatment of acquired immunodeficiency syndrome (AIDS), induced a significant dose-related increase in the thymidine kinase (Tk) mutant frequency (MF) in L5178Y/Tk(+/-) 3.7.2C mouse lymphoma cells. Treatment with 1 mg/ml (3,742 muM) AZT for 24 hr resulted in a MF of 407 x 10(-6) compared to a control MF of 84 x 10(-6). The MFs of the large and small colony mutants resulting from AZT exposure were 142 x 10(-6) and 265 x 10(-6), respectively. One hundred and fifty mutants from the 1 mg/ml (3,742 muM) AZT-treated culture and sixty-nine mutants from independent untreated cultures were isolated and analyzed. LOH analysis using a heteromorphic microsatellite locus located in the Tk gene was performed to determine the presence or absence of the Tk(+) allele. Eight other microsatellite markers spanning the entire mouse chromosome 11 also were examined for heterozygosity to determine the extent of LOH. In addition, Tk gene dosage analysis was conducted using Real-Time PCR in those mutants showing LOH at the Tk locus. The presence of only one Tk allele based on Real-Time PCR indicated that the mutant resulted from deletion while the presence of two alleles was consistent with a recombination event. More mutants from the AZT-treated culture showed Tk LOH than did independent mutants from the untreated cultures (91% vs. 64%) and the induced mutants also showed distinct chromosome 11 LOH patterns. The mutation spectrum of mutants from AZT-treated cells was also significantly different from that of spontaneous mutants. More deletions and fewer intragenic mutations were observed in the mutants from the AZT-treated culture than independent mutants from the untreated control. Our data indicate that AZT primarily induced LOH mutations in L5178Y mouse lymphoma cells and a large number of LOH mutations resulted from deletions.