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Androgen ablation leads to an upregulation and intranuclear accumulation of deoxyribonuclease I in rat prostate epithelial cells paralleling their apoptotic elimination.

Rauch F, Polzar B, Stephan H, Zanotti S, Paddenberg R, Mannherz HG - J. Cell Biol. (1997)

Bottom Line: After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5.At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology.The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Ruhr-University, Bochum, Germany.

ABSTRACT
After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.

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In situ end-labeling  of rat ventral prostate before  and after castration. (a) Positive control of rat prostate at  day 0. The section was pretreated with purified bovine  pancreatic DNase I to fragment the chromatin of all nuclei. (b–e) In situ end-labeling after castration: (b) 12 h;  (c) 24 h; (d) cluster of cells;  and (e) single cell at day 3.  Bars, 100 μm.
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Figure 1: In situ end-labeling of rat ventral prostate before and after castration. (a) Positive control of rat prostate at day 0. The section was pretreated with purified bovine pancreatic DNase I to fragment the chromatin of all nuclei. (b–e) In situ end-labeling after castration: (b) 12 h; (c) 24 h; (d) cluster of cells; and (e) single cell at day 3. Bars, 100 μm.

Mentions: Cells containing fragmented DNA can be identified by in situ end-labeling of the free 3′-OH ends generated during the internucleosomal DNA-degradation using labeled dUTP and terminal deoxynucleotidyl transferase (Gavrieli et al., 1992; Wijsman et al., 1993; Polzar et al., 1994). Applying this technique on sections of rat prostate at different time intervals after castration, we found that the number of labeled cells increased up to day 3 (Fig. 1, b–e) in agreement with previous quantitative data showing that most of the cells are eliminated between day 3 and 5 (Kyprianou et al., 1988; Aumüller et al., 1995; Banerjee et al., 1995; Tenniswood et al., 1995). The strongly stained nuclei were of varying size (Fig. 1 d) or fragmented (Fig. 1 c). The fate of the apoptotic cells seemed to differ: In many instances, we observed positively end-labeled cells located underneath epithelial cells of normal appearance (Fig. 1 d). In some cases, the apoptotic cells were found to be extruded into the lumenal space (see also Fig. 3 b).


Androgen ablation leads to an upregulation and intranuclear accumulation of deoxyribonuclease I in rat prostate epithelial cells paralleling their apoptotic elimination.

Rauch F, Polzar B, Stephan H, Zanotti S, Paddenberg R, Mannherz HG - J. Cell Biol. (1997)

In situ end-labeling  of rat ventral prostate before  and after castration. (a) Positive control of rat prostate at  day 0. The section was pretreated with purified bovine  pancreatic DNase I to fragment the chromatin of all nuclei. (b–e) In situ end-labeling after castration: (b) 12 h;  (c) 24 h; (d) cluster of cells;  and (e) single cell at day 3.  Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139838&req=5

Figure 1: In situ end-labeling of rat ventral prostate before and after castration. (a) Positive control of rat prostate at day 0. The section was pretreated with purified bovine pancreatic DNase I to fragment the chromatin of all nuclei. (b–e) In situ end-labeling after castration: (b) 12 h; (c) 24 h; (d) cluster of cells; and (e) single cell at day 3. Bars, 100 μm.
Mentions: Cells containing fragmented DNA can be identified by in situ end-labeling of the free 3′-OH ends generated during the internucleosomal DNA-degradation using labeled dUTP and terminal deoxynucleotidyl transferase (Gavrieli et al., 1992; Wijsman et al., 1993; Polzar et al., 1994). Applying this technique on sections of rat prostate at different time intervals after castration, we found that the number of labeled cells increased up to day 3 (Fig. 1, b–e) in agreement with previous quantitative data showing that most of the cells are eliminated between day 3 and 5 (Kyprianou et al., 1988; Aumüller et al., 1995; Banerjee et al., 1995; Tenniswood et al., 1995). The strongly stained nuclei were of varying size (Fig. 1 d) or fragmented (Fig. 1 c). The fate of the apoptotic cells seemed to differ: In many instances, we observed positively end-labeled cells located underneath epithelial cells of normal appearance (Fig. 1 d). In some cases, the apoptotic cells were found to be extruded into the lumenal space (see also Fig. 3 b).

Bottom Line: After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5.At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology.The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Ruhr-University, Bochum, Germany.

ABSTRACT
After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.

Show MeSH
Related in: MedlinePlus