Environmental and Molecular Mutagenesis - Legacy Tobacco ...

324
DEVELOPMENT OF A PROCEDURE FOR QUANTITATION OF MUTATIONS AT THE NGPRT LOCUS IN
CHINESE HAMSTER OVARY (CHO) CELLS WITH EXPHESSION AND SELECTION IN SEMISOLID CULTURE
NEDIUM . P .S . Lee, K .Y . Henry, and C .J . Rudd, SRI International, Menlo Park, CA (USA) .
We are developing a modified procedure for quantitation of mutations at the HGPRT
locus to reduce the cost and potential errors associated with the suhculturing of
cells during the expression period . In established procedures, CH0 cells usually
require subculturing every 2 to 3 days for 7 days prior to treatment with the selective
agent, 6-thioguanine (TG) . Colonies of TG-resistant CH0 cells can be selected
either attached to tissue culture dishes or suspended in semisolid medium . In our
laboratory, the cloning efficiencies of unselected cells and the numbers of mutant
colonies were comparable under the two conditions . Using a modified procedure, we
eliminated the need for subculturing during the expression period by growing lower
densities of cells in semisolid medium after the induction of mutations . Mutant
colonies were selected after 4 to 7 days by adding TG (final concentration 30 uM) as
an overlay with additional medium . The colonies were counted after a total of 14 or
more days incubation . The recovery of HGPRT- cells in reoonstruetion experiments was
equivalent in the presence or absence of wild-type (HGPRT+) cells under these conditions
. Small HGPRT- colonies could be distinguished from non-viable microcolonies of
HGPRT• cells by staining with Thiozolyl Blue (MTT, 2 .5 mg/dish) . By growing the
cells in semisolid medium for the duration of the experiment, olonal populations were
maintained . Therefore, with the modified procedure, the number of TG-reslstant colonies
should more closely represent the number of cells with a mutation at the HGPRT
locus at the beginning of the expression period . This inereased accuracy, and the
savings in labor and materials costs, are clear advantages of the modified protocol .
325 THE ELECTROPHORETIC SPECIFIC LOCUS TEST IN THE MOUSE AND ANIMAL NOOELS
OF HUMAN DISEASE
Susan E . Lewis
Research Triangle Institute . P . 0 . Box 12194, Research Triangle Park . NC 27709
The mutagenicity of x-rays and a variety of chemicals have now been studied
in the electrophoretic specific locus system . Comparisons of mutation frequencies
in the treated groups in the electrophoretic specifiE locus tests . with those in
the visible specific locus test . will be made . Electrophoretic screeniny has
identified a number of electrophoretically detectable mutations, both spontaneous
and induced by various agents . The underlying molecular basis for, and the physiological
impact of selected mutations are discussed . Although animals homoZyflous
for null alleles at many of the loci under study appear to be perfectly
healthy . certain others are either not viable or impaired . Mouse models of human
diseases have been recovered in the course of this and other biochemical screening
programs . These are discussed as to their potential utility in experimental therapeutics
and the relative susceptibility of the relevant loci to mutagenic change .
(Supported in part by Contract No . N01-ES-2-5012 . NIEHS .)
326
Hypothesis : Modification of Oncogene Expression as a Major
Mechanism of Action of "Nongenotoxic" Carcinogens
A . P . Li, Monsanto Company Environmental Health Laboratory, 645
ewstead, St . Louis, MO 63110 .
Recent findings in cancer research have consistently associated
mutations in oncogenes with carcinogenesis, therefore supporting
the relevance of genetic damage in this process . However,
findings in genetic toxicology now point out that a large variety
of carcinogens, especially hepatocarcinogens, do not have
genotoxicity readily measured using conventional assays (e .g .
Ames test) . Examination of the biological effects of these
"nongenotoxic" carcinogens include the following : a . Cell
proliferation, either via a mitogenic effect (e .g . phenobarbital)
or cytotoxicity-induced compensatory cell division (e .g . CC14) ;
b . Enzyme induction, such as P450 (e .g . PCBs) or peroxisome
induction (e .g . clofibrate) ; c . Alterations in DNA methylation
(e .g . choline-deficient diet) . I hypothesize that these events
share one common phenomenon which is key to carcinogenesis :
Induction of oncogene expression . Cell proliferation is known to
lead to sequential expression of several oncogenes which are not
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
1989 EMS Abstracts 113
Notes