Korean J Urol.  2003 May;44(5):436-445.

Apoptosis of Renal Cell Carcinoma Cells by Expression of FADD (Fas-Associated Death Domain)

Affiliations
  • 1Departments of Urology, Seoul National University College of Medicine, Korea. urology@snu.ac.kr
  • 2Departments of Urology, Seoul Municipal Boramae Hospital, Seoul, Korea.

Abstract

PURPOSE: The Fas-associated death domain (FADD) constitutes a novel protein that specifically associates with the cytoplasmic death domain of Fas, and induces apoptosis. FADD is composed of a death effector domain (DED) and a death domain. In this study, we evaluated the in vivo antitumor effect of the FADD, or FADD-DED, gene in renal cell carcinoma cells, using a plasmid vector expressing the human FADD and FADD-DED genes.
MATERIALS AND METHODS
The cDNA of the human FADD and FADD-DED genes were amplified by RT-PCR, and cloned to the pCR(R) 3.1. The expressions of the cloned FADD and FADD-DED (pCR(R)3.1-FADD and pCR3.1-FADD-DED) were observed by Western blot analysis. The efficacy of the growth inhibition by the cloned FADD and FADD-DED genes was tested, in vitro, on A498 and Caki-1 human renal cell carcinoma cell lines using the MTT assay. To evaluate the apoptosis, DNA fragmentation and caspase-3 assays were performed.
RESULTS
Expressions of the FADD protein, and the FADD-DED, of the transfected A498 and Caki-1 cells had increased by 48 and 24 hours, respectively, compared with the control cell lines. The cytotoxicity of the pCR3.1-FADD and pCR3.1-FADD-DED on the A498 and Caki-1 cells significantly increased compared to the empty vector. The increased cytotoxicity of the FADD- or FADD-DED-transfected cell lines was associated with enhanced apoptosis, as assessed by DNA fragmentation and caspase-3 assays.
CONCLUSIONS
Our results showed that the cloned FADD or FADD-DED expression plasmid vector efficiently inhibited the growth of A498 and Caki-1 human renal cell carcinoma cell lines. These data suggest that the exogenous FADD or FADD-DED expressions may have therapeutic applications in renal cell carcinomas.

Keyword

FADD; DED; Carcinoma; renal cell; Apoptosis

MeSH Terms

Apoptosis*
Blotting, Western
Carcinoma, Renal Cell*
Caspase 3
Cell Line
Clone Cells
Cytoplasm
DNA Fragmentation
DNA, Complementary
Humans
Plasmids
Caspase 3
DNA, Complementary
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