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Interferon-–Induced Sensitization of Colon Carcinomas to ZD9331 Targets Caspases, Downstream of Fas, Independent of Mitochondrial Signaling and the Inhibitor of Apoptosis Survivin

Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee

We have demonstrated previously a Fas-dependent component in thymineless death of human colon carcinoma cells. Importantly, the cytotoxic effects of thymidine deprivation induced by 5-fluorouracil (FUra) combined with leucovorin (LV) was enhanced by IFN-, and the synergism was shown to be dependent on Fas, FUra-induced DNA damage, and independent of p53. Subsequently we examined the potential for synergistic interactions between IFN- and the specific thymidylate synthase inhibitor, ZD9331. IFN- sensitized colon carcinomas to ZD9331-induced apoptosis and loss in clonogenic survival, also dependent on ZD9331-induced DNA damage, independent of p53. Synergism occurred in HCT116, demonstrating previously RNA-mediated FUra/LV cytotoxicity that could not be potentiated by IFN-. Manipulation of the Fas death receptor pathway from the level of the receptor (Nok1/Nok2, Fas overexpression, and DN-FADD) to the mitochondria (Bcl-xL and Bcl-2) did not modulate ZD9331 ± IFN--induced cytotoxicity in HT29, with the exception that Nok1/Nok2-blocking antibodies partially protected HT29 from the cytotoxic activity of ZD9331 alone. However, IFN- alone (but not ZD9331) up-regulated the expression of caspases -3, -4, -7, and -8, and in combination with ZD9331 demonstrated enhanced caspase activation and cleavage of poly(ADP-ribose) polymerase that was not prevented by overexpression of Bcl-2. Additionally, IFN- increased the activity of the proteasome in HT29, leading to selective down-regulation of the antiapoptotic protein survivin, whereas simultaneously increasing Fas expression. However, reduction in the survivin:Fas ratio by transfection of survivin small interfering RNA and/or overexpression of Fas did not affect sensitivity of HT29 to ZD9331 ± IFN-. Data demonstrate that IFN- combined with ZD9331 is synergistic in additional cell lines that demonstrate RNA-mediated FUra/LV cytotoxicity, and that a major target of interaction is at the level of caspases, downstream of Fas, and independent of involvement of either the mitochondria or survivin.

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				J. I. Geller, K. Szekely-Szucs, I. Petak, B. Doyle, and J. A. Houghton P21Cip1 Is a Critical Mediator of the Cytotoxic Action of Thymidylate Synthase Inhibitors in Colorectal Carcinoma Cells Cancer Res., September 1, 2004; 64(17): 6296 - 6303. [Abstract] [Full Text] [PDF]