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Title: 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

Abstract

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 codonmore » 12 transversions that have been commonly observed in human tumors.« less

Authors:
; ;  [1]
  1. Univ. of Wisconsin, Madison (USA)
Publication Date:
OSTI Identifier:
5404754
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America; (USA)
Additional Journal Information:
Journal Volume: 85:11; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Stevens, C W, Manoharan, T H, and Fahl, W E. 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. United States: N. p., 1988. Web. doi:10.1073/pnas.85.11.3875.
Stevens, C W, Manoharan, T H, & Fahl, W E. 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. United States. https://doi.org/10.1073/pnas.85.11.3875
Stevens, C W, Manoharan, T H, and Fahl, W E. 1988. "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". United States. https://doi.org/10.1073/pnas.85.11.3875.
@article{osti_5404754,
title = {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},
author = {Stevens, C W and Manoharan, T H and Fahl, W E},
abstractNote = {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.},
doi = {10.1073/pnas.85.11.3875},
url = {https://www.osti.gov/biblio/5404754}, journal = {Proceedings of the National Academy of Sciences of the United States of America; (USA)},
issn = {0027-8424},
number = ,
volume = 85:11,
place = {United States},
year = {Wed Jun 01 00:00:00 EDT 1988},
month = {Wed Jun 01 00:00:00 EDT 1988}
}