D. Wilkinson et al., HPRT MUTANTS IN A TRANSPLANTABLE MURINE TUMOR ARISE MORE FREQUENTLY IN-VIVO THAN IN-VITRO, British Journal of Cancer, 72(5), 1995, pp. 1234-1240
A model system was developed to allow investigation of the frequency a
t which clastogenic and/or mutagenic events occur in situ in a transpl
antable murine fibrosarcoma tumour (MC1A-C1) compared with in vitro cu
lture. 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 rev
ertants to hypoxanthine/aminopterin/thymidine resistance (HAT(R)) were
isolated and found to be approximately 1000 times more sensitive than
the parental tumour to induction of 6-TG(R) mutants by cobalt-60 gamm
a-rays. This sensitivity is expected for a heterozygous marker; these
revertants may therefore possess only one functional hprt locus but tw
o or more active X chromosomes. A clone with a stable hprt gene was id
entified 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 c
ould be estimated by culturing explanted tumour cells in the presence
of 6-TG, using G418 selection to distinguish tumour from host cells. T
he frequency of mutants in MN-II cells grown as tumours was found to b
e 3.4-fold higher than in tissue culture for an equivalent period of t
ime. These data provide the first direct evidence for the existence of
mutagenic factors in a tumour environment that might contribute to tu
mour progression.