Tumors induced in mice by direct inoculation of plasmid DNA expressing both activated H-ras and c-myc.

Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes--human activated T24-H-ras and murine c-myc--and had shown that these two plasmids, pMSV-T24-H-ras and pMSV-c-myc, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression in vitro, were tested. The dual-expression plasmid, pMSV-T24-H-ras/MSV-c-myc, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 microg of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.

Oncogenicity of DNA in vivo: tumor induction with expression plasmids for activated H-ras and c-myc.

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.

Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.

VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.