Abstract
Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.
Highlights
Cancer (Blattner et al, 1979)
Susceptibility to a wide range of cancers is consistent with such a mechanism, since mutation introduced by malfunctioning topoisomerases could affect many genomic sites. For these reasons we have studied DNA topoisomerase activity in non-cancerous fibroblasts from members of a LiFraumeni family
The solution was phenol-chloroformed, ethanol precipitated and the DNA analysed by gel electrophoresis and densitometry as above. Both the family's fibroblast lines and the NIH-3T3 transfectants have a similar perturbation of the dose response relationship for cytoplasmic catenation activity (Figure 1), with higher thresholds, but steeper slopes and significantly (P
Summary
Cancer (Blattner et al, 1979). the mechanism of oncogenesis ( unlikely to be identical in each pedigree (Little et al, 1987)) may be relevant to many forms of non-familial neoplasms. Radiation resistance has been demonstrated in the non cancerous skin fibroblasts from family members (Bech-Hansen et al, 1981), but this finding is not common to all Li-Fraumeni families (Little et al, 1987). The ostensibly normal non-cancerous radioresistant cells were found to have an apparent activation of the c-raf-1 gene and a 3-8-fold elevation in the expression of c-myc (Chang et al, 1987). The transfer of either the family's c-raf-J gene, the genes of other serine/threonine kinases or ras, into murine cells conveyed the radioresistant phenotype, but the myc, fes and abl oncogenes failed to do so (Chang et al, 1987; Pirollo et al, 1989; Sklar, 1988)
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