The mutational spectrum of the HPRT gene from human T cells in vivo shares a significant concordant set of hot spots with MNNG-treated human cells.
The preponderance of G:C to A:T transitions in inherited and somatic human mutations has led to the hypothesis that some of these mutations arise as a result of formation of O(6)-methylguanine in DNA. To test this hypothesis, the fine structure map of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations was determined in human lymphoblastoid cells in the human hypoxanthine-guanine-phosphoribosyltransferase (HPRT) gene and compared with HPRT mutations observed in somatic T lymphocytes from normal individuals. Human TK6 cells, which are methylguanine methyltransferase deficient (MGMT(-)), were treated with the methylating agent MNNG to create a level of O(6)-methylguanine in cellular DNA equal to that found in normal human tissues. A total of 676 bp of the HPRT gene was scanned using constant denaturing capillary electrophoresis and high-fidelity PCR. MNNG induced 14 predominant hot spots, all which were G:C to A:T transitions. Thirteen of these 14 MNNG-induced hot spots were found among the in vivo set, and 10 of the MNNG-induced hot spots were among 75 putative in vivo hot spots (mutations observed two or more times in vivo). Using a hypergeometric test for concordance, the MNNG-induced hot spots were found to be a significant subset of the putative in vivo hot spots (P < 4 x 10(-7)). The set of shared hot spots comprise some 18% of the HPRT in vivo hot spot spectrum and strongly suggest that MNNG-induced hot spots in vitro share a common mutational pathway with a significant subset of somatic mutations in vivo.
Tomita-Mitchell
Aoy
A
Division of Cardiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Ling
Losee Lucy
LL
Glover
Curtis L
CL
Goodluck-Griffith
Jacklene
J
Thilly
William G
WG
D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D005091:Exons; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000172:deficiency; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology
D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase