Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR

摘要

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and ~(60)Co γ-rays. METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and ~(60)Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique. RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 g/ml, P<0.01; in the group with dosage of ~(60)Co γ-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P<0.05; in the group of ~(60)Co γ-ray with 1-4 Gy, P<0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %, P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gamma-ray-induced mutants, 88.1 % in ENU-induced mutants). CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or ~(60)Co γ-ray. Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.