Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system.
OBJECTIVE
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.
Okazaki
Toshihiko
T
Walther Cancer Institute, Purdue Cancer Center, West Lafayette, Indiana, USA.
Sakamuro
Daitoku
D
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015152:Blotting, Northern; D015153:Blotting, Western; D004195:Disease Models, Animal; D053222:Fas Ligand Protein / Q000378:metabolism*; D005434:Flow Cytometry; D007680:Kidney Neoplasms / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D053222:Fas Ligand Protein; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor