Environmental and Molecular Mutagenesis - Legacy Tobacco ...

318 1989 EMS Abstracts 111
IMPROVERD METABOLIC CAPABILITY BY DRUG METABOLIZING GENE TRANSFECTION. Notes
R . ~Lan~enbach', C . Crespi', R . Davies', P . Smith', and K . Rudo', 'National
Tnstitu~Environment Health Sciences, Research Triangle Park, NC 27709 and
'Genetest, Woburn, MA, 01801 .
The technique of gene transfection was employed to introduce the cDNA coding
for specific cytochrome P450 enzymes into transformable C3H/10TYe mouse embryo
ce11s and mutable human AHH-1 lymphoblastoid cells . The long term objective of
this work is to enhance the metabolic capability of these cells for exploring the
role of cellular metabolism in genotoxic and nongenotoxic mechanisms of carcinogenesis
. C3H/10TS4 cells containing transfected rat cytochrome P450IIB1 (P450b)
were more sensitive to the cytotoxic effects of acetylaminofluorene and dimethylnitrosamine
than parental cells . Transfection of either human cytochrome P450IA1
(methyl cholanthrene inducible form) or cytochrome P450IIA2 increased the mutagenic
response of AHH-1 cells to aflatoxin B1 and dimethyl-/diethylnitrosamine,
respectively . The manipulation of drug metabolizing genes provides an experimental
strategy for delineating initial stages of carcinogenesis and/or mutagenesis by
toxic agents in mammalian cells .
319
RECENT DEVELOPMENTS IN THE GLYCOPHORIN A ASSAY FOR SOMATIC CELL
MUTATIONS IN HUMANS . Richard G . Langlois, William L . Bigbee, and Ronald H. Jensen,
Biomedical Sciences Div., Lawrence Livermore National Laboratory, Livermore, CA (USA) .
The glycophorin A (GPA) assay utilizes Immunofluorescent labeling and flow cytometry to
identify and enumerate rare erythrocytes which lack the expression of one allelic form of
GPA presumably due to mutations in erythroid precursor cells . Variant cells with both
hemizygous and homozygous phenotypes are observed in normal individuals at a
background frequency of about 10 per million normal cells . Transient Increases in variant
frequency (VF) were observed in cancer patients during chemotherapy, but VFs returned to
near normal values six months after therapy was completed . Significant dose-depehdent
increases in VF were observed in A-bomb survivors suggesting persistent stem cell effects
from radiation damage . Elevated spontaneous VFs have also been observed In individuats
with some cancer-prone syndromes . Hemizygous VFs were elevated about 10-fold in
individuals with ataxia telangiectasia, while approximately 60-fold elevations in both
hemizygous and homozygous VFs were observed In Individuals with Bloom's syndrome .
VFs for individuals with xeroderma pigmentosum, In contrast, consistently fell within the
range of normal individuals . A new cell labeling method has been developed to allow the
assay to be performed on a single-beam flow cytometer .Work performed under the auspices
of the U .S . Dept . of Energy by the Lawrence Livermore Nat . Lab. under Contract No. W-
7405-ENG-48 with support from the U .S. E .P .A. Grant R-811819-02-0 and the U .S .
N .I .E .H .S . Interaqemcv Agreement NIH-ES•83-14 .
320
ONCOCENE CHANCES IN CHEMICALLY-TRANSFORHED RODENT RESPIRATORY EPITHELIAL CELLS .
J .A . Lasleyl, S .J . Austinl, N .S . Schorschinskyl, D .K . Beeman2, and M .J . Mass2,
lEnvironmental Health Research and Testing, Inc ., Research Triangle Park, NC 27709
and 2Carcinogenesis and Metabolism Branch, MD-68, U .S .E .P .A ., Research Triangle
Park, NC 27711
Much evidence has accumulated linking alterations in oncogenes and the development
of cancer . However, most of these studies have utilized tumors, which are
late stages in the neoplastic process . We utilized the rat tracheal epithelial
(RTE) cell transformation system to observe the participation of oncogenes in comparatively
early stages of cell transformation in vitro . Eight cell lines and
normal cells were studied for alterations in K-ras . H-ras, and c-mvc . These lines
were transformed in culture with the carcinogens benzo(a)pyrene,
7,12-dimethylbenz(a)anthracene (DMBA), and/or the tumor promoter 12-0tetradecanoylphorbol-l3-acetate
. Southern blot hybridizations indicated no
amplification of H-ras, K-ras, or c-wc in the linas studied . We did not find
alterations in H-ras codon 61 that have been found in DMBA-induced tumors in vivo .
H-ras probing of Hpa II digests could distinguish normal from transformed cells .
One cell line had 2 types of o-wc RFLPs when digested with Bam HI or Hind III, and
also exhibited methylation changes in CCGG sequences compared with normal cells .
Northern analysis showed elevated H-ras and c-vic expression in some transformed
cells . We conclude that transformed RTE cells can demonstrate a number of oncogene
alterations, however, the relationship of each to the transformed state requires
further study . This is an abstract of a proposed presentation and does not
constitute EPA policy .
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