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Abstract
Backgrounds
Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated.
Methods
Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay.
Results
The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor (P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration.
Figures and Tables
Fig. 1
Regression and regrowth of the rat ventral prostate gland in response to androgen manipulation. The ventral prostate obtained of 48 hours (A) or 5 days (B) after castration are shown, respectively The other assays were performed as described in Materials and Methods. Cast, castration; T, testosterone; FSH, follicle stimulating hormone.
Fig. 2
Time course of the castration-dependent changes of prostatein C1 and TRPM-2 mRNA in the ventral prostate. At the indicated time, ventral prostate was removed from groups of rats which were either sham operated or castrated, and total RNA was prepared. Total RNA (30 µg) was analyzed by Northern blot hybridization using prostatein C1 and TRPM-2 cDNA probe as described in Materials and Methods.
Fig. 3
Terminal deoxynucleotidyl transferase dependent dUTP nick end labeling (TUNEL) assay after castration. They were visualized by diaminobenzidine (DAB) and other cells were counter stained with methyl green as described in Materials and Methods. A, sham operation; B, castration; C, testosterone supplementation. Magnification: ×400.
Fig. 4
Testosterone suppression of castration-induced apoptotic DNA fragmentation in rat ventral prostate. Before castration, testosterone (5 mg/day) were subcutaneously injected as 24 hrs intervals. After 48 hours, the rats were killed. a, DNA was isolated from ventral prostate, labeled on 3'-ends with [α-32P]-dideoxy-ATP, and analyzed by electrophoresis in 2% agarose gel. Autography and β-counting of low-molecular weight (≤15 kb) DNA fractions were followed. The amount of [α-32P]-dideoxy-ATP incoporated into DNA fractions from different groups were compared with that of intact animals at each time point an arbitrary unit 1.0. Cast, castration; Sham, sham operation; T, testosterone.
Fig. 5
Time course of castration-dependent change of FasL mRNA in rat ventral prostate. The FasL mRNA and protein levels were measured by RT-PCR and Western blot, respectively as described in Materials and Methods. PCR products were saperated on 1.2% agarose gel containing ethidium bromide. Animals were killed at indicated time after castration A, RT-PCR; B, Western blot.
Fig. 6
Effect of testosterone and FSH on castration-induced FasL gene expression. Before castration, testosterone (5 mg/day) and FSH (20 IU/day) were subcutaneously injected as 24 hrs intervals. The FasL mRNA and protein levels were measured by RT-PCR as described in Materals and Methods. PCR products were separated on 1.2% agarose gel containing ethidium bromide. A, RT-PCR; B, Western blot. T, testosterone; AzB, azaline B.
Fig. 7
Time course of castration-dependent change of Fas mRNA in rat ventral prostate. The Fas mRNA levels and protein were measured by RTPCR and Western blot, respectively, as described in Materials and Methods. PCR products were separated on 1.2% agarose gel containing ethidium bromide. Animals were killed at indicated time after castration A, RT-PCR; B, Western blot.
Fig. 8
Effect of testosterone and FSH on castration- induced Fas gene expression. Before castration, testosterone (5 mg/day) and FSH (20 IU/day) were subcutaneously injected at 24 hrs intervals. The Fas mRNA and protein levels were measured by RT-PCR and Western blot, respectively as described in Materials and Methods. PCR products were separated on 1.2% agarose gel containing ethidium bromide. A, RT-PCR; B, Western blot.
Fig. 9
The pattern of protein-binding site on the minimal FasL promoter region. The FasL promoter fragment (Hind III-Xho I) was labeled with [γ-32P]- dATP in the presence of T4 polynucleotide kinase at the -318 bp position of the coding, Upper strand. The labeled probe was incubated with 80 µg nuclear extracts from rat testis, and the mixtures were digested with 2 µL of DNase I (4,000 units/mL) for 30 minutes on the ice. After extraction with phenol/chloroform, the DNAs were resolved by electrophoresis on a 5% polyacrylamide gel in 8 M urea and the labeled DNA bands were visualized by autoradiography. The lane indicated by A+G is the wild type probe digested at adenine and guanine residues. The other assays were performed as described in Materials and Methods. A, DNase I footprinting; B, the sequences of 5'-flanking region of FasL gene. The arrow denoted the hypersensitive site. □, no protection; ■, protection.
Fig. 10
Effect of castration on binding activity of nuclear extracts of prostate to SP-1 motif. Nuclear extracts from rat ventral prostate of sham and castrated were incubated with 32P-labeled double strand oligonucleotides corresponding to -293~-266 bp from the transcription initiation site. DNA-protein complexes were resolved native 4% polyacrylamide gel and visualized by autoradiography. The other assays were performed as described in Materials and Methods. Free did not contain nucear extracts. FD, Free DNA probe; Comp, competitor.
Fig. 11
Effect of testosterone and FSH on castration-induced Bcl-2 gene expression. Before castration and azaline B administration, testosterone (5 mg/day) and FSH (20 IU/day) were subcutaneously injected at 24 hrs intervals. The bcl-2 mRNA and protein levels were measured by RT-PCR and Western blot, respectively, as described in Materials and Methods. PCR products were separated on 1.2% agarose gel containing ethidium bromide. A, RT-PCR; B, Western blot.
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