TY - JOUR
T1 - Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system
AU - Okazaki, Toshihiko
AU - Sakamuro, Daitoku
N1 - Funding Information:
Acknowledgments We thank Drs. D. Green, S. Nagata, B. Vogel-stein, W. El-Deiry, and S. Kaufmann for their plasmid DNAs. We also thank the previous and current members of the Sakamuro laboratory for their suggestion and assistance. This work was supported by grants from the US Army New Investigator Prostate Cancer Research Program (DAMD 17-02-10131), the Wendy Will Case Cancer Fund, the Expedition Inspiration Fund for Breast Cancer Research, the V Foundation for Cancer Research, the Walther Cancer Institute/Purdue Cancer Center Cancer Research Grant, and the Louisiana Cancer Research Consortium Immediate Response Program grant (to D.S.).
PY - 2007/9
Y1 - 2007/9
N2 - Purpose: The Fas/CD95/APO-1 ligand (FasL) is a death cytokine that binds to cell surface Fas/CD95/APO-1 receptor, yet a possible role of FasL expression in p53-dependent apoptosis is not fully understood in many human malignancies, including renal carcinoma. Methods: By Northern blot and Western blot analyses, we determined the effect of p53 on the FasL and Fas receptor expression. To do this, we employed an in vitro renal carcinoma model system that was previously established by stably co-transfecting a temperature-sensitive mutant allele of the p53 tumor suppressor (ts-p53) with either the c-Myc oncogene or adenovirus E1A oncogene in baby rat kidney (BRK) epithelial cells. The ts-p53 is activated only at a permissive temperature. The transactivation activity of p53 was assessed by luciferase reporter assays. The sub-G1 cell population in the cell cycle representing apoptotic cell death was measured by flow cytometric analysis. Results: We found that the level of endogenous FasL, but not Fas receptor, was increased at a permissive temperature with delayed kinetics when compared with p21WAF1 expression, but was coincident with p53-induced apoptosis, whereas an apoptosis-defective mutant p53, which lacks the PxxP region (P: Proline, x: any amino acid), failed to induce FasL expression and hence apoptosis. Notably, p53-induced apoptosis was completely blocked by overexpressing a dominant negative inhibitor of the FADD/Mort-1, a pro-apoptotic adaptor that lies immediately downstream of the FasL/Fas receptor. Conclusions: These results suggest that the FasL is a critical downstream effector of p53-dependent apoptosis in a cultured BRK renal carcinoma model system.
AB - Purpose: The Fas/CD95/APO-1 ligand (FasL) is a death cytokine that binds to cell surface Fas/CD95/APO-1 receptor, yet a possible role of FasL expression in p53-dependent apoptosis is not fully understood in many human malignancies, including renal carcinoma. Methods: By Northern blot and Western blot analyses, we determined the effect of p53 on the FasL and Fas receptor expression. To do this, we employed an in vitro renal carcinoma model system that was previously established by stably co-transfecting a temperature-sensitive mutant allele of the p53 tumor suppressor (ts-p53) with either the c-Myc oncogene or adenovirus E1A oncogene in baby rat kidney (BRK) epithelial cells. The ts-p53 is activated only at a permissive temperature. The transactivation activity of p53 was assessed by luciferase reporter assays. The sub-G1 cell population in the cell cycle representing apoptotic cell death was measured by flow cytometric analysis. Results: We found that the level of endogenous FasL, but not Fas receptor, was increased at a permissive temperature with delayed kinetics when compared with p21WAF1 expression, but was coincident with p53-induced apoptosis, whereas an apoptosis-defective mutant p53, which lacks the PxxP region (P: Proline, x: any amino acid), failed to induce FasL expression and hence apoptosis. Notably, p53-induced apoptosis was completely blocked by overexpressing a dominant negative inhibitor of the FADD/Mort-1, a pro-apoptotic adaptor that lies immediately downstream of the FasL/Fas receptor. Conclusions: These results suggest that the FasL is a critical downstream effector of p53-dependent apoptosis in a cultured BRK renal carcinoma model system.
KW - Apoptosis
KW - Fas ligand
KW - Fas receptor
KW - Renal carcinoma
KW - Transactivation
KW - c-Myc oncogene
KW - p53 tumor suppressor
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U2 - 10.1007/s00432-007-0201-5
DO - 10.1007/s00432-007-0201-5
M3 - Article
C2 - 17505842
AN - SCOPUS:34447513709
SN - 0171-5216
VL - 133
SP - 581
EP - 588
JO - Journal of Cancer Research and Clinical Oncology
JF - Journal of Cancer Research and Clinical Oncology
IS - 9
ER -