Mechanism of Azaline B-induced Apoptosis in Rat Testis.
- Author:
Chung PARK
1
;
Eun Jin YUN
;
Kyoung Sub SONG
;
Young Rae KIM
;
Jong Seok KIM
;
Sang Do LEE
;
Jong Il PARK
;
Jung Hwa LEE
;
Seung Keil PARK
;
Byung Doo HWANG
;
Kyu LIM
Author Information
1. Departments of Biochemistry, Chungnam National University, Korea. kyulim@cnu.ac.kr
- Publication Type:Original Article
- Keywords:
Azaline;
Fas receptor;
Fas ligand (FasL);
Apoptosis
- MeSH:
Animals;
Antigens, CD95;
Apoptosis*;
Binding Sites;
Carrier Proteins;
Deoxyribonuclease I;
DNA;
DNA Fragmentation;
Electrophoretic Mobility Shift Assay;
Fas Ligand Protein;
Follicle Stimulating Hormone;
In Situ Nick-End Labeling;
Interferon-gamma;
NF-kappa B;
Rats*;
Rats, Sprague-Dawley;
Response Elements;
RNA, Messenger;
Testis*;
Testosterone;
Weights and Measures
- From:Korean Journal of Urology
2005;46(7):750-760
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: To identify the mechanism of azaline B-dependent apoptosis, the regulation of Fas and FasL genes has been investigated. MATERIALS AND METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rats. The levels of Fas receptor (Fas) and Fas ligand (FasL) were detected by reverse transcription-polymerase chain reaction (RT- PCR). Azaline B-dependent apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) and DNA fragmentation assay. Transacting factor of FasL promoter was identified by DNase I footprinting and DNA mobility shift assay. RESULTS: The azaline B-treated testis (250microgram/kg body wt/day) had decreased to 70+/-2.5% and 38+/-1.8% of the normal testis weight at 3 and 5 days after the injection, respectively, but the weights of the testis were not changed after pretreatment of follicle-stimulating hormone (FSH) and testosterone. Apoptosis of the testis was detected by DNA fragmentation assay and TUNEL assay after the azaline B treatment. The levels of Fas and FasL mRNA were increased by the treatment of azaline B in both time- and dose-dependent manners. In DNase I footprinting assay with FasL promoter, the nuclear factor prepared from control was bound with at least four sites: SP-1 binding site at 283, NF-kappa B binding site at 219, TATA at 132 and the gamma-interferon response element (gamma-IRE) at 78. gamma-IRE was completely protected by the nuclear extract prepared from azaline B-treated rat testis. In DNA mobility shift assay, the binding activity of gamma-IRE binding protein was increased after azaline B treatment. CONCLUSIONS: These results suggest that Fas-FasL system may be important to azaline B-dependent apoptosis in rat testis and that gamma-IRE binding protein is related to the azaline B-dependent regulation of FasL gene.
