Our previous experiments had shown that two cooperating oncogenes are needed to induce tumors in vivo
. The results we present here show that, as expected, having both oncogenes on the same plasmid induces tumors more efficiently than having the two cooperating oncogenes on separate plasmids. The lowest amount of the dual-expression plasmid pMSV-T24-H-ras
that was able to induce tumors in newborn NIH Swiss mice was 1 µg. With adult mice, the lowest amount was 5 µg. This result agrees with our earlier finding with the separate expression plasmids 32
, where newborn mice were found to be more sensitive to DNA-mediated tumor induction than adult mice.
Although the incidence of tumors induced by the three versions of the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc generally reflected the amount of DNA inoculated, there was no strict dose-response relationship, a result that was probably due to the relatively low number of animals used per group. This conclusion is supported if all three versions of the pMSV-T24-H-ras/MSV-c-myc plasmid are considered to be functionally indistinguishable (their in vitro transformation activities are the same) and the results for each DNA dose in Table are combined. In this case, the incidence becomes: 20% for 20 µg, 21% for 10 µg, 13% for 5 µg, and 0% for 1 µg in the adult mouse, and 77% for 20 µg, 27% for 10 µg, 36% for 5 µg, and 7% for 1 µg in the newborn mouse. While there is still some variability, there is evidence of a dose-response relationship. Additional studies with larger numbers of mice are planned to address this experimental variability.
Establishing the clonality of the tumors was considered to be important. If the tumors were polyclonal, then they would be derived from the transformation of multiple normal mouse cells, and thus the efficiency of tumor induction by DNA would be higher than if the tumors were monoclonal and derived from a single cell transformed by DNA. With one possible exception, the integration patterns of the plasmid DNA in the tumors were consistent with a single cell being converted into a tumor cell. The exception was the D004-I (Fig. A and B, lanes h), where the Southern analysis showed multiple Bgl
II fragments containing T24-H-ras
sequences, indicating that multiple plasmid sequences were taken up and integrated in cells in the tumor. Whether the oncogenes in D004-I were taken up by a single cell, in which case the tumor would be clonal, or by multiple cells, in which case the tumor would not be clonal, remains to be determined. In our earlier study 32
, a tumor-cell line whose DNA gave rise to multiple restriction fragments that hybridized to the ras
probes was shown, by single-cell cloning, to be clonal.
This conclusion, that the tumors were mostly, if not always, clonal would only be true if there were no bias in which of the cells in the tumor gave rise to the established cell lines in culture. While the tumors were not analyzed in this study, we have shown in other studies that oncogene-containing cells in tumor-cell lines accurately reflect the population of oncogene-containing cells in the original tumors (unpublished observations).
One of the important practical findings from this study was that there could have been a selective loss of tumor cells when the tumor tissue was dispersed by trypsin digestion, thus resulting in the outgrowth of normal cells and the loss of tumor cells, as seen with two lines established from tumors induced in adult mice (Fig. ). Whether tumor cells in these tumors are more sensitive to trypsin digestion than normal untransformed cells is not known, but we have recently found that cutting up the tumors into small pieces and allowing tumor cells to grow out from these explants in culture has resulted in a higher success rate in establishing tumor-cell lines.
An unexpected finding was that reagents that increase DNA uptake in vitro did not increase the efficiency of tumor induction DNA in vivo. In fact, the transfection facilitators tested appeared to have a negative effect. One possible explanation for this finding was that the facilitators stimulated the immune system and this immune activation caused a rejection of the tumor in the mouse. Because the cellular arm of the immune system is believed to be involved in tumor rejection, we assessed whether transfection facilitators would be effective in a mouse strain that is defective in T-cell function, the nude mouse. Although the numbers were small, no differences were found in the efficiency of DNA-induced tumor formation between the nude mice and their immune-competent littermates. This could indicate either that some part of the immune system other than the cellular arm is being stimulated or that DNA uptake is not limiting. Additional experiments will be necessary to explain the lack of activity of the transfection reagents in DNA-induced tumor formation.
The DNA oncogenicity assay described in this paper might provide a new way of measuring the activity of cellular or viral oncogenes in vivo
without having to create cell lines expressing the oncogenes and then testing the tumorigenicity of these cells in either syngeneic animals or immune-deficient animals. For example, transfection of both activated H-ras
into normal rodent fibroblasts cells can convert these cells into cells that can form tumors in vivo 35-37
. However, direct inoculation of oncogenes into animals would circumvent the in vitro
step, potentially revealing hitherto unknown activities of these oncogenes. Direct inoculation of oncogenes might also be used to evaluate potential differences in the induction of tumors by DNA in vivo
compared with the formation of tumors by the injection of cells transformed in vitro
This study confirms results of our earlier experiments showing that activated oncogenes from neoplastic cells are capable of inducing normal cells to become tumorigenic after direct injection into an animal 32
, a finding that argues that the potential risk from the residual DNA in vaccines produced in neoplastic cells can no longer be considered hypothetical. However, because of the relative sizes of the oncogene expression plasmid and the mammalian genome, if 1 µg of the plasmid induces tumors, then 100 µg to 1 mg of mammalian DNA would be required. We are currently evaluating different strains of mice, such as mice with various degrees of immune deficiencies and mice predisposed to tumor development in response to carcinogens, for their sensitivity to DNA oncogenic activity in order to optimize assay sensitivity and more clearly define levels of risk posed by residual DNA in vaccines.