Search

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

Human p53 protein was found to be functional in fission yeast in terms of growth repression and checkpoint control. Expression of wild-type p53 or the hot spot mutant p53His273 results in dramatic morphological changes and loss of viability of recipient yeast cells. Overexpression of cdc25C phosphatase, the mitotic activator of cdc2, results in suppression of a p53-induced growth arrest. In order to understand the interplay between p53 and cdc25C in mammalian cells we isolated and sequenced cdc25C cDNA from the epidermoid carcinoma cell line A431, which is known to carry the p53His273 mutation. Two different transcripts of the human cdc25C gene were detected by RT-PCR analysis - one full-length transcript and a shortened version (cdc25Cdm) that carries two deletions in the 5'-region of the gene. In normal human skin fibroblasts only one full-length cdc25C transcript was detected. The two different transcripts code for proteins with a molecular weight of 55 kDa and 46 kDa, respectively. Both cdc25C cDNAs from A431 cells were found to complement a conditional lethal cdc25.22 mutant strain as well as a cdc25 deletion strain of Schizosaccharomyces pombe indicating that functional proteins were translated. Expression of cdc25Cdm variant leads to a stronger uncoupling of DNA replication from mitosis than expression of cdc25C suggesting that the deletion within the amino-terminus of cdc25C leads to a protein which might contribute some potential for oncogenic transformation. As with cdc25C, uncoupling of the DNA synthesis checkpoint by cdc25Cdm was reversed by coexpression of wild-type p53.

Human p53 is a growth suppressor which not only functions in mammalian cells but also in fission yeast. It was previously shown that the cell cycle regulating phosphatase cdc25C suppresses the p53 induced growth arrest in fission yeast. In the present study we analysed the mechanism of this suppression. We found that cdc25C directly interacts with p53. By using different deletion mutants the binding region was narrowed down on the polypeptide chain of p53 to amino acids 287-340. To test the functional significance we analysed the effect of this interaction on the DNA binding activity of p53. As shown by band shift experiments binding of cdc25C to p53 does not modify the DNA binding activity of p53. Our data suggest that the observed suppression of the p53 induced growth arrest by cdc25C might be achieved by direct binding of cdc25C to the C-terminus of p53.

We produced a rabbit monoclonal antibody (MAb) against human CDC25C phosphatase. The antibody reacts with a minimal epitope between amino acids 291-295 in the highly conserved C-terminal region of CDC25C. The antibody recognizes denatured CDC25C of recombinant and mammalian origin in Western blot analysis. The corresponding rabbit polyclonal serum is able to immunoprecipitate the native protein, but this ability has been lost during the selection procedure. Although the production of the rabbit MAb requires more effort and patience than the mouse MAb technology, it offers a true alternative in case of antigens that are not immunogenic in mice.