The major objective of this research is to utilize human hybrid cells to investigate mechanisms of radiation-induced neoplastic transformation. To this end, a unique quantitative assay for in vitro neoplastic transformation has been developed using HeLa x skin fibroblast hybrids. This assay takes advantage of the expression of the HeLa tumor-associated antigen, recently identified as intestinal alkaline phosphatase (IAP). This antigen is suppressed in the non-tumorigenic hybrids but is re- expressed in both spontaneous and radiation-induced tumorigenic segregant, and can be used as a marker for neoplastic transformation. In the proposed studies for the next funding period, this system will be applied to quantitative studies dealing with the influence of chemical agents which modify gap junction intercellular communication (GJIC) on radiation-induced transformation frequency in an attempt to assess the importance of this phenomenon as a modulator of neoplastic transformation in this system. Apart from the application to quantitative studies, the HeLa x skin fibroblast system affords a unique opportunity to investigate the molecular and cellular changes underlying neoplastic transformation, particularly with respect to the role of tumor-suppressor gene inactivation. Chromosome 11 has been identified as carrying the putative HeLa suppressor gene which negatively regulates both tumorigenicity and the expression of IAP. Thus, radiation-induced tumorigenic cell lines will be subjected to Southern analysis using informative RFLP's for chromosome 11. This analysis should, firstly, establish whether damage to chromosome 11 correlates with induction of tumorigenicity, and secondly, by establishment of a common deleted region, the locus of the putative suppressor gene should be able to be established. Radiation- induced tumorigenic cell lines have been shown a wide range of levels of expression of IAP. They therefore afford a unique opportunity to study the role of damage to the negative regulator of IAP in neoplastic transformation in this system. Finally, it has been shown that segregation of the tumorigenic phenotype is associated with loss of gap junction intercellular communication, and loss of expression of the gap junction protein, connexin 43. Thus, we propose to examine the extent of GJIC, level of connexin 43 expression and level of expression of IAP in radiation-induced tumorigenic cells in order to establish the role of loss of GJIC in the acquisition of tumorigenicity by these cells. Furthermore, in an attempt to clarify whether loss of GJIC and GJ gene expression is a cause or an effect of acquisition of the tumorigenic phenotype, we propose to compare the effects of connexin 43 transfection on the one hand, and microcell transfer of chromosome 11 on the other, into tumorigenic hybrids on the endpoints of GJIC, connexin 43 expression, IAP expression, and tumorigenicity.
| Redpath, J L; Antoniono, R J (1995) Reduced temperature (22 degrees C) results in enhancement of cell killing and neoplastic transformation in noncycling HeLa x skin fibroblast human hybrid cells irradiated with low-dose-rate gamma radiation. Radiat Res 144:102-6 |
