EFFECTS OF CHEMICAL CARCINOGENS ON TRANSFORMING DNA SEQUENCES AND EXPRESSION

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The aim of this project is to identify, characterize, and clone those genes that drive the development of neoplasia and whose malignant potential results from changes caused by chemical carcinogens. (1) To identify transforming genes activated by benzo[a]pyrene (BP), DNA from three BALB/3T3 cell lines transformed by BP was analyzed by DNA transfer and focus formation in the NIH/3T3 system. All three tested lines showed transforming activity that differed from each other and from the ras oncogenes by restriction endonuclease sensitivity and MspI mapping. These possibly new transforming genes are now being cloned by the sib selection protocol from Charon 4A phage genomic libraries. Three protocol cycles of selections have been carried out and the positive pools show no presence of the ras oncogenes. (2) To explore the mechanisms by which chemical carcinogens may activate proto-oncogenes, the distribution of aflatoxin Bl (AFB) adducts on genes was analyzed using purified liver nuclei and microsomes in vitro. AFB adducts were preferentially located in DNAse I hypersensitive regions of the genome. (3) To elucidate the genetic events underlying the mechanisms of tumor promotion in mouse skin, novel genes termed pro 1 and pro 2, specifying sensitivity to induction of transformation by TPA in JB-6 cells, were cloned by sib selection from a size-selected DNA library of clonal cells sensitive to promotion. By restriction mapping, heteroduplex analyses and direct hybridization, the pro genes are different from and unrelated to oncogenes or other known genes. Both pro genes have been sequenced and their functions are being investigated. Human homologs of pro genes have been isolated from the human nasopharyngeal carcinoma cell line CNE2. (4) Using the NIH/3T3 focus assay, a transforming gene was detected in human prostate carcinoma cell line PC-3 and is being cloned; these cells apparently do not contain altered ras oncogens.