Cell Growth & Differentiation, Vol 2, Issue 1 1-6, Copyright © 1991 by American Association of Cancer Research
Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland
R Zhang, JD Haag and MN Gould
Department of Human Oncology, University of Wisconsin-Madison 53792.
Mammary carcinogenesis is a multistep process consisting minimally of
initiation and promotion/progression stages. The rate-limiting stage in the
carcinogenesis process is undetermined but can in part be addressed by
estimating the frequency of initiation, a heritable early event. Here, we
use an in vivo limiting dilution transplantation assay to estimate
initiation frequency in a rat mammary epithelial stem-like cell population
that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU)
administered i.v. We estimate that this dose resulted in the killing of 65%
of exposed mammary cells. Known numbers of cells surviving NMU exposure
were grafted into fat-pads of recipient rats in which the cells grew and
differentiated into structurally and functionally normal mammary glands.
Recipient rats were hormonally manipulated to provide maximal promotion of
initiated cells. Mammary carcinomas developing at graft sites were
quantitated over a 2-year period. Based on these results, we estimate that
at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated.
Seventeen % of these graft site carcinomas had an activated H-ras oncogene
with a G to A mutation in codon 12. This suggests that at least 1 mammary
cell in 43,000 was mutated in this fashion by in situ exposure to NMU.
These data suggest that cH-ras represents approximately 1 of 5 of the
initiation events produced by NMU exposure of rat mammary glands.