The preponderance of evidence suggests that human cancer can be the result of mutation. Appropriate candidates for causative mutant DNA sequences (human oncogenes) sometimes can be isolated from human tumors by the capacity of the sequence either to establish precrisis rat cells or to convert NIH3T3 mouse cells into focus-forming or anchorage-independent transformant clones. Yet the great majority of human tumors and tumor cell lines have not yielded oncogenes in this classic DNA transfection assay. We hypothesize that oncogenes exist in some or all of these tumors as well. We propose a research plan to isolate and characterize such novel human oncogenic sequences. First we will transform early passage (P 5) human cells. In preliminary experiments we have found that infection of 6th passage human cells with origin-minus SV40 plasmid generates clonable transformed foci, at an acceptable efficiency. By infecting early passage cells we circumvent the issue of establishment or the lack of it in human cell transformation. Second, we will determine whether the two oncogene complementation groups derived from primary rat cell transfections are valid for early passage normal human cells as well. Third, we will determine which parts or mutants of the SV40 early region correspond to the complementation groups of the human cell assay. The results from these three sets of experiments will provide us with the experience for the fourth and final step. This will be the transfection of human tumor DNA from tissues and cell lines into early passage normal human cells. We intend to recover oncogenes from the DNA of human tumors and tumor cell lines which cannot transform NIH3T3 cells. Tumor DNA preparations will be transfected alone into human cells, or co-transfected with oncogenes of each complementation group determined in step 2 of this proposal. Human tumor DNA preparations must first be linked to a marker DNA sequence because we are transfecting human DNA into human cells. The marker will be PSV-2 minus the bacterial XPRT gene. This marker will be followed by blot- hybridization through several rounds of DNA transfection, isolation of DNA from transformants, and re-transfection. The DNA tertiary transformants will be cloned into lambda phage or cosmid vectors.
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| Chen, S; Levesque, P; Pomert, E et al. (1987) Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen. J Virol 61:3521-7 |
