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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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Immunohistochemical staining of rat ventral prostate with anti–DNase I. Rat ventral prostates were treated and stained with  polyclonal affinity-purified anti–DNase I as described in Materials and Methods. (a) Before castration: A single positively stained cell  (arrow) can be seen within the secretory epithelium. (b) 6 h after castration: Again, a single positively stained cell (arrow) can be seen.  The cytoplasm of some cells exhibits weak DNase I immunoreactivity. (c) 12 h after and (d) 3 d after castration; note that many, but not  all nuclei are positively stained. (e) 5 d after castration; note that most nuclei are positively stained. (f) 7 d after castration. Here the number of positively stained nuclei is decreased; many are DNase I negative. Identical magnification for all pictures. Bar, 100 μm.
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Figure 2: Immunohistochemical staining of rat ventral prostate with anti–DNase I. Rat ventral prostates were treated and stained with polyclonal affinity-purified anti–DNase I as described in Materials and Methods. (a) Before castration: A single positively stained cell (arrow) can be seen within the secretory epithelium. (b) 6 h after castration: Again, a single positively stained cell (arrow) can be seen. The cytoplasm of some cells exhibits weak DNase I immunoreactivity. (c) 12 h after and (d) 3 d after castration; note that many, but not all nuclei are positively stained. (e) 5 d after castration; note that most nuclei are positively stained. (f) 7 d after castration. Here the number of positively stained nuclei is decreased; many are DNase I negative. Identical magnification for all pictures. Bar, 100 μm.

Mentions: In agreement with previous data (Kyprianou et al., 1988), we found that the wet weight of the rat ventral prostate continuously decreased to about one fifth of its control value within the first 7 d after castration. Histological analysis showed a considerable reduction in the height of the secretory epithelium and the size of the secretory follicles at days 5 and 7 (see Fig. 2, e and f). In this study, we only used the ventral prostate, which was previously shown to be most affected by androgen withdrawal (Kyprianou et al., 1988; Furuya et al., 1994; Banerjee et al., 1995).


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)

Immunohistochemical staining of rat ventral prostate with anti–DNase I. Rat ventral prostates were treated and stained with  polyclonal affinity-purified anti–DNase I as described in Materials and Methods. (a) Before castration: A single positively stained cell  (arrow) can be seen within the secretory epithelium. (b) 6 h after castration: Again, a single positively stained cell (arrow) can be seen.  The cytoplasm of some cells exhibits weak DNase I immunoreactivity. (c) 12 h after and (d) 3 d after castration; note that many, but not  all nuclei are positively stained. (e) 5 d after castration; note that most nuclei are positively stained. (f) 7 d after castration. Here the number of positively stained nuclei is decreased; many are DNase I negative. Identical magnification for all pictures. Bar, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Immunohistochemical staining of rat ventral prostate with anti–DNase I. Rat ventral prostates were treated and stained with polyclonal affinity-purified anti–DNase I as described in Materials and Methods. (a) Before castration: A single positively stained cell (arrow) can be seen within the secretory epithelium. (b) 6 h after castration: Again, a single positively stained cell (arrow) can be seen. The cytoplasm of some cells exhibits weak DNase I immunoreactivity. (c) 12 h after and (d) 3 d after castration; note that many, but not all nuclei are positively stained. (e) 5 d after castration; note that most nuclei are positively stained. (f) 7 d after castration. Here the number of positively stained nuclei is decreased; many are DNase I negative. Identical magnification for all pictures. Bar, 100 μm.
Mentions: In agreement with previous data (Kyprianou et al., 1988), we found that the wet weight of the rat ventral prostate continuously decreased to about one fifth of its control value within the first 7 d after castration. Histological analysis showed a considerable reduction in the height of the secretory epithelium and the size of the secretory follicles at days 5 and 7 (see Fig. 2, e and f). In this study, we only used the ventral prostate, which was previously shown to be most affected by androgen withdrawal (Kyprianou et al., 1988; Furuya et al., 1994; Banerjee et al., 1995).

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