Isolation of transformation-suppressor genes by using a cDNA-expression library having sense and anti-sense inserts.

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1. We used artificial overexpression of cDNAs synthesized from NIH3T3 cell mRNA to induce reversion of NIH3T3 cells transformed by the activated Harvey-ras oncogene. The revertants were screened by observation under microscope for "flat" appearance. Among about 100 revertant cell lines obtained, we have chosen one revertant, Rev6-4, which showed marked decrease in its ability to form colonies in soft agar and to form tumors in nude mice, and isolated an incorporatcd cDNA from its genomic DNA by molecular cloning after PCR (polymerase chain reaction) amplification.2. Analysis of the CDNA isolated from Rev6-4 showed that the CDNA encoded P68, which had been shown to have ATP-dependent RNA helicase activity, and that the cDNA appeared to be over-expressed in an anti-sense manner in Rev6-4 cells. Concominantly, a decrease in the amount of endogenous P68 mRNA was observed. We have confirmed that the cloned Rev6-4 CDNA could induce decrease in colony formation in soft agar, in tumor formation in nude mice and in saturation density when overexpressed in the ras-transformed NIH3T3 cells in the anti-sense manner.3. We have isolated a full-length cDNA encoding P68. When the cDNA was over-expressed in NIH3T3 cells in a sense direction, formation of foci which appeared morphologically-transformed was observed.However, cells in the foci could not form tumors when injected into nude mice. Presently, we do not know if the P68 gene is an oncogene whose product is acting in downstream of ras. We are currently analysing a physiological function of P68 by using antibodies against it.4. We have isolated cDNAs from two other revertants and are currently analysing their structure and examining for their ability to induce reversion.