Inactivity of acrylonitrile epoxide to modify a Ha-ras DNA in a non-focus transfection-transformation assay.
The transforming potential of acrylonitrile epoxide (ANO) was tested in a modified NIH3T3 transfection-transformation assay. This involves a new ras construct obtained by ligating a human c-Ha-ras-1 proto-oncogene to the pSV2neo mammalian vector. The new plasmid was allowed to react with ANO or an established carcinogen in vitro, and the modified ras DNA transfected into NIH 3T3 cells. The transfectants are subjected to triple selections: G418 (neomycin) resistance, low serum growth, and limit dilutions. The end points are scored by cell growth kinetics and monolayer saturation density. In using this protocol, the EJ tumor ras plasmid was the positive control, and anti-benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (anti-BPDE) and N-methyl-N-nitrosourea were found to be positive in yielding transformants. Although ANO-modified ras gave rise to two G418R clones, both were scored negative due to their normal growth rate and monolayer density similar to the negative controls. Southern blot analysis of anti-BPDE transformant DNA revealed a fragment of 411 bp, indicating a ras mutation at codon 11 or 12. However, both the ANO clones showed the wild-type band of 355 bp by the same method.
Yuan
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Department of Chemistry, University of Louisville, KY 40292.
Wong
J L
JL
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D015139:Blotting, Southern; D002273:Carcinogens; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000032:analysis / Q000187:drug effects*; D005027:Ethylene Oxide / Q000031:analogs & derivatives* / Q000633:toxicity; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D008770:Methylnitrosourea / Q000633:toxicity; D010957:Plasmids; D000090063:Proto-Oncogene Mas; D014162:Transfection; D014170:Transformation, Genetic
D002273:Carcinogens; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C033154:2-cyanoethylene oxide; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D008770:Methylnitrosourea; D004247:DNA; D005027:Ethylene Oxide