Purpose: Changes in wild-type p53 protein function occur in the majority of human tumors, and may alter genomic stability and the cellular response to ionizing radiation. Whether oncoproteins can render tumor cells both radioresistant and metastatic, may have implications for clinical strategies designed to improve local tumor control. In the studies reported here, we tested the hypothesis that acquired radioresistance correlates with metastatic potential within a large panel of transformed rat embryo cell (REF) lines following transfection with activated H-ras, mutant p53, and HPV16-E7 alleles.
Methods and materials: Rat embryo cells (REF cells) were transfected using the calcium-phosphate technique with an activated H-ras gene alone, or in combination with human papillomavirus HPV16-E7 and/or human or murine mutant p53 sequences. Other rat embryo cell clones expressing transfected HPV-E7 and activated ras sequences subsequently acquired endogenous p53 gene mutations during culture in vitro. The relative expression of p21ras and p53 protein for each REF transformant was determined by Western blot analysis following transfection. REF clones were phenotypically characterized at early passage (i.e., passages 5-7) and late passage (i.e., passages 10-20) for their: (a) relative tumor growth rate, and (b) their ability to undergo spontaneous metastasis following intramuscular injection into the hind legs of SCID mice. In vivo phenotypic end points were then compared to previously measured parameters of in vitro radiosensitivity for each cell line. Additionally, the expression of the cellular protease, plasminogen activator, was determined for a number of metastatic and nonmetastatic cell lines.
Results: We found no evidence that selected oncogene-transfected REF transformants that were radioresistant in culture had a greater spontaneous metastatic potential than nonradioresistant REF transformants. Neither the level of expression of the p21ras protein nor that of the p53 protein was correlated with the spontaneous metastatic phenotype when tested at early passage. The metastatic phenotype appeared to be independent of p53 genotype. The majority of metastatic REF clones tested (7 out of 9 clones) expressed plasminogen activator following oncogene transfection, in contrast to nonmetastatic REF transformed cell lines.
Conclusions: Our results suggest that (a) intrinsic radioresistance does not correlate with spontaneous metastatic potential in oncogene-expressing REF transformant cell lines, and (b), novel clinical strategies designed to overcome oncogene-mediated radioresistance could potentially impact on overall survival, as gains in local tumor control may not be offset by a greater risk of distant metastasis.