Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system

Toshihiko Okazaki, Daitoku Sakamuro

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)581-588
Number of pages8
JournalJournal of Cancer Research and Clinical Oncology
Volume133
Issue number9
DOIs
StatePublished - Sep 1 2007
Externally publishedYes

Fingerprint

Apoptosis
Ligands
Carcinoma
Kidney
CD95 Antigens
Temperature
myc Genes
In Vitro Techniques
Luciferases
Oncogenes
Proline
Adenoviridae
Northern Blotting
Transcriptional Activation
Neoplasms
Cell Cycle
Cell Death
Western Blotting
Epithelial Cells
Alleles

Keywords

  • Apoptosis
  • Fas ligand
  • Fas receptor
  • Renal carcinoma
  • Transactivation
  • c-Myc oncogene
  • p53 tumor suppressor

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

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title = "Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system",
abstract = "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.",
keywords = "Apoptosis, Fas ligand, Fas receptor, Renal carcinoma, Transactivation, c-Myc oncogene, p53 tumor suppressor",
author = "Toshihiko Okazaki and Daitoku Sakamuro",
year = "2007",
month = "9",
day = "1",
doi = "10.1007/s00432-007-0201-5",
language = "English (US)",
volume = "133",
pages = "581--588",
journal = "Journal of Cancer Research and Clinical Oncology",
issn = "0171-5216",
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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

PY - 2007/9/1

Y1 - 2007/9/1

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

VL - 133

SP - 581

EP - 588

JO - Journal of Cancer Research and Clinical Oncology

JF - Journal of Cancer Research and Clinical Oncology

SN - 0171-5216

IS - 9

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