involves extrapolation of data derived from sensitive and quantitative experimental systems

We undertook a program to establish model systems to quantify the oncogenic activity of DNA in vivo. From such data, it was hoped that estimates of risk could be made. Because the major source of the oncogenic activity in neoplastic cells would be activated cellular oncogenes, we have used cellular oncogenes rather than viral oncogenes for these studies. In our initial study, we generated expression plasmids for the T24 version of the human H-ras gene and the mouse c-mycgene, as these genes were known to transform primary rodent cells in vitro into cells that could form tumors in immunocompromised mice. The chosen promoter for these genes was the murine sarcoma virus 59 long terminal repeat, and termination signals were the bovine growth hormone poly site followed by the MSV 39 LTR. Inoculation of these plasmids by the subcutaneous route into adult and newborn NIH Swiss and C57BL/6 mice established that 1) tumors could be induced by direct introduction of DNA, 2) both oncogenes were required to induce tumors, 3) newborns were more susceptible than adults, and 4) NIH Swiss mice were more susceptible than C57BL/6 mice. The majority of tumors appeared between 4 and 9 weeks after inoculation, and cell lines established from the tumors expressed both the H-Ras and c-Myc oncoproteins. Analysis of the integration patterns of the DNA from tumor-cell lines demonstrated that most, if not all, of the tumors induced by the oncogenes were clonal. However, tumors were induced only at the highest dose of DNA with lower doses being insufficient. To increase the sensitivity of the assay, several modifications to the original system were investigated. Because both oncogenes were required in the same cell for tumor induction, it was reasoned that placing both oncogenes on the same molecule would result in increased efficiency of tumor induction; this expectation was confirmed, as 1 mg of the dual-expression plasmid pMSV-T24-Hras/MSV-c-myc was found to be oncogenic in newborn NIH Swiss mice. In addition, because uptake of DNA was likely a ratelimiting step, we investigated whether transfection facilitators, compounds that increase DNA uptake in vitro, would increase the efficiency of tumor induction. Surprisingly, no transfection facilitator had any effect on tumor induction by DNA. Furthermore, because we had found differences in the susceptibility of mouse Silmitasertib msds strains as part of our exploratory studies, we evaluated various mouse strains, both immune competent and immune defective. In this paper, we report that the CD3 epsilon transgenic mouse, which is deficient in both T-cell and NK-cell functions, is the most sensitive mouse strain identified to date for the detection of oncogenic activity of DNA; amounts of DNA as low as 25 ng of the plasmid.

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