Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR.
OBJECTIVE
To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and (60)Co gamma-rays.


METHODS
Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and (60)Co gamma-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 micro g/ml, P<0.01; in the group with dosage of (60)Co gamma-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 micro g/ml, P<0.05; in the group of (60)Co gamma-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).


CONCLUSIONS
The spectra of spontaneous mutations differs completely from that induced by EUN or (60)Co gamma-ray. Although both ENU and gamma-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.
Liu
Sheng-Xue
SX
Department of Healath Toxicology, Preventive Medical College, Third Military Medical University, Chongqing 400038, China.
Cao
Jia
J
An
Hui
H
Shun
Hua-Min
HM
Yang
Lu-Jun
LJ
Liu
Yong
Y
D000477:Alkylating Agents / Q000494:pharmacology*; D005038:Ethylnitrosourea / Q000494:pharmacology*; D005720:Gamma Rays; D018922:HL-60 Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D000477:Alkylating Agents; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea