Hprt mutants in a transplantable murine tumour arise more frequently in vivo than in vitro.
A model system was developed to allow investigation of the frequency at which clastogenic and/or mutagenic events occur in situ in a transplantable murine fibrosarcoma tumour (MC1A-C1) compared with in vitro culture. The marker selected for detecting these events was the X-linked hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. We found that the hprt gene in MC1A-C1 was not suitable for this purpose, most likely because multiple active copies were present. To circumvent the problem, HPRT- [6-thioguanine (6-TG)-resistant] clones were isolated by inactivating all hprt genes with methylnitrosourea. Spontaneous revertants to hypoxanthine/aminopterin/thymidine resistance (HATR) were isolated and found to be approximately 1000 times more sensitive than the parental tumour to induction of 6-TGR mutants by cobalt-60 gamma-rays. This sensitivity is expected for a heterozygous marker, these revertants may therefore possess only one functional hprt locus but two or more active X chromosomes. A clone with a stable hprt gene was identified and a neo gene was introduced. The resulting cell line (MN-11) could be grown as a subcutaneous tumour in syngeneic C57BL/6 animals. The frequency of mutations arising in vivo in the marker hprt gene could be estimated by culturing explanted tumour cells in the presence of 6-TG, using G418 selection to distinguish tumour from host cells. The frequency of mutants in MN-11 cells grown as tumours was found to be 3.4-fold higher than in tissue culture for an equivalent period of time. These data provide the first direct evidence for the existence of mutagenic factors in a tumour environment that might contribute to tumour progression.
Wilkinson
D
D
Ottawa Regional Cancer Centre, Ontario, Canada.
Sandhu
J K
JK
Breneman
J W
JW
Tucker
J D
JD
Birnboim
H C
HC
D000818:Animals; D004249:DNA Damage; D005354:Fibrosarcoma / Q000473:pathology*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D012680:Sensitivity and Specificity; D013866:Thioguanine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014960:X Chromosome
D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine