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Influence of TRP53 Status on FAS Membrane Localization, CFLAR (c-FLIP) Ubiquitinylation, and Sensitivity of GC-2spd (ts) Cells to Undergo FAS-Mediated Apoptosis¹

College of Pharmacy,³ Division of Pharmacology and Toxicology Cell and Molecular Biology Graduate Program,⁴ The University of Texas at Austin, Austin, Texas 78712-0125

ABSTRACT

Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 32°C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (37°C) while real-time PCR results indicated an absence of Cflar(L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 37°C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L).

apoptosis, CFLAR (c-FLIP), FAS, GC-2 cells, signal transduction, testis, toxicology, TRP53, ubiquitinylation

² Correspondence: John H. Richburg, The University of Texas at Austin, College of Pharmacy, PHR 5.218D, 1 University Station, A1915, Austin, TX 78712-0125. FAX: 512 471 5002; john_richburg{at}mail.utexas.edu

¹ Supported, in part, by grants from the National Institute of Environmental Health Sciences/NIH (ES09145), NIEHS toxicology training grant (for C.M., T32 ES007247), and NIEHS Center Grant (P30 ES07784). Assistance was provided to Y.C. and Y.Y. by the University of Texas at Austin's Center for Molecular and Cellular Toxicology.

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