The long-term objectives are to develop highly effective therapy for the treatment of colon carcinoma. This may be achieved by 1) identifying new molecular targets that may regulate cell death and be amenable to therapeutic exploitation, 2) understanding the specific signalling pathways involved in the regulation of cell death and apoptosis, and 3) developing new therapeutic strategies based upon specific molecular characteristics. The proposal will elucidate the role of the Fas death receptor signalling pathway in the regulation of thymineless death in colon carcinoma cells both in vitro and in vivo. In cultured cells we will test the hypotheses: 1) thymidylate synthase- deficient (TS-) cells are sensitive to thymineless stress by signalling via the Fas death receptor, in contrast to other modes of DNA damage which induce apoptosis by Fas-independent mechanisms, 2) JNK/SAPK are involved in activation of FasL, and NF-kappaB and/or AP-1 are involved in the transcriptional regulation of FasL in response to thymineless stress, 3) Fas- mediated apoptosis is potentiated by cytokines and FUra/LV- induced cytotoxicity is potentiated by cytokines in a Fas- dependent manner in human colon carcinoma cell lines. The second focus is to develop improved therapeutic strategies by elucidating the role of modulation of the Fas signalling pathway in enhancing sensitivity to FUra/LV. We will test the hypotheses that: 1) modulation of Fas expression in cultured cell lines using the cytokines recombinant human IFN-gamma and/or TNF-alpha, that can exert synergistic effects on upregulating Fas expression and activating NF-kappaB, potentiate FUra/LV-induced cytotoxicity in a Fas-dependent manner and exert greater than additive effects, 2) modulating expression of FasL using tetracycline regulatable FasL expression, or elevation of membrane FasL using KB8301, a metalloproteinase inhibitor that prevents cleavage of FasL from the cell surface, sensitizes cells to apoptosis or FUra/LV-induced apoptosis, and 3) effective drug combinations and exposures, additionally defined by pharmacokinetic analyses in mice, demonstrate enhanced curative potential of human colon adenocarcinoma xenografts. It is anticipated that the studies proposed will identify new therapeutic targets related to signalling via the Fas death receptor, and be of significance in the identification and development of highly effective regimens for the successful treatment of colon carcinoma in man.
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