Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

Journal Article · Wed Jun 01 00:00:00 EDT 1988 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.85.11.3875· OSTI ID:5404754

Stevens, C W; Manoharan, T H; Fahl, W E ^[1]

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1. Univ. of Wisconsin, Madison (USA)

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo({alpha})pyrene ({plus minus})anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC {yields} TA codon 12 transversions that have been commonly observed in human tumors.

• OSTI ID: 5404754
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 85:11; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BENZOPYRENE
BIOLOGICAL EFFECTS
FIBROBLASTS
CELL TRANSFORMATIONS
ONCOGENES
DNA SEQUENCING
ALKYLATING AGENTS
CHEMICAL ACTIVATION
CODONS
DNA
ELECTROPHORESIS
MAN
PHOSPHORUS 32
RECOMBINANT DNA
SKIN
ANIMAL CELLS
ANIMALS
AROMATICS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CONDENSED AROMATICS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
GENES
HYDROCARBONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
SOMATIC CELLS
STRUCTURAL CHEMICAL ANALYSIS
VERTEBRATES
550401* - Genetics- Tracer Techniques