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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 hybridization of rat ventral prostates using DNase I–specific sense and antisense probes. Sections were treated as given  in Materials and Methods. Using the sense probe: (a) No staining. (b–g) Antisense probe. (b and c) Before; (d) 2 d after; (e and f) 3 d after; and (g) 5 d after castration. Note cytoplasmic positive reaction at supranuclear and apical position (blue) in (b–d). At day 3 (e and f),  a large number of apoptotic cells are detectable that exhibit strong staining of their fragmented nuclei, (arrows). At day 5, most nuclei  are negative; only weak cytoplasmic, supranuclear reaction is obtained. Bars, 10 μm.
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Figure 4: In situ hybridization of rat ventral prostates using DNase I–specific sense and antisense probes. Sections were treated as given in Materials and Methods. Using the sense probe: (a) No staining. (b–g) Antisense probe. (b and c) Before; (d) 2 d after; (e and f) 3 d after; and (g) 5 d after castration. Note cytoplasmic positive reaction at supranuclear and apical position (blue) in (b–d). At day 3 (e and f), a large number of apoptotic cells are detectable that exhibit strong staining of their fragmented nuclei, (arrows). At day 5, most nuclei are negative; only weak cytoplasmic, supranuclear reaction is obtained. Bars, 10 μm.

Mentions: In situ hybridization using DNase I–specific antisense and sense RNA was used to localize its gene transcripts and to analyze its time-dependent variation after castration. Sections of the ventral prostate before and after castration were stained using sense and antisense probes. The sense probe did not show any labeling at all when used on sections of control prostates and at any other time point after androgen ablation (Fig. 4 a). In control prostates, a specific staining (blue stain) of exclusively epithelial cells was obtained using the antisense probe (Fig. 4, b and c). Within the epithelial cells, the staining was most prominent at supranuclear and apical localization that was frequently found to be interrupted by a narrow unstained region (Fig. 4, c and d). From ultrastructural studies, it is known that the rough endoplasmic reticulum is interrupted and localized in supranuclear and apical positions (Aumüller at al., 1980). Using the antisense probe, the homogeneous staining of the epithelial cells remained until day 2 (Fig. 4 d). At day 3, a considerable number of apoptotic cells were detected whose fragmented nuclei were in many instances strongly labeled (Fig. 4, e and f, black stain). The intensity of the specific stain decreased at day 5 (Fig. 4 g) and was further reduced at day 7 (not shown).


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 hybridization of rat ventral prostates using DNase I–specific sense and antisense probes. Sections were treated as given  in Materials and Methods. Using the sense probe: (a) No staining. (b–g) Antisense probe. (b and c) Before; (d) 2 d after; (e and f) 3 d after; and (g) 5 d after castration. Note cytoplasmic positive reaction at supranuclear and apical position (blue) in (b–d). At day 3 (e and f),  a large number of apoptotic cells are detectable that exhibit strong staining of their fragmented nuclei, (arrows). At day 5, most nuclei  are negative; only weak cytoplasmic, supranuclear reaction is obtained. Bars, 10 μm.
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Related In: Results  -  Collection

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

Figure 4: In situ hybridization of rat ventral prostates using DNase I–specific sense and antisense probes. Sections were treated as given in Materials and Methods. Using the sense probe: (a) No staining. (b–g) Antisense probe. (b and c) Before; (d) 2 d after; (e and f) 3 d after; and (g) 5 d after castration. Note cytoplasmic positive reaction at supranuclear and apical position (blue) in (b–d). At day 3 (e and f), a large number of apoptotic cells are detectable that exhibit strong staining of their fragmented nuclei, (arrows). At day 5, most nuclei are negative; only weak cytoplasmic, supranuclear reaction is obtained. Bars, 10 μm.
Mentions: In situ hybridization using DNase I–specific antisense and sense RNA was used to localize its gene transcripts and to analyze its time-dependent variation after castration. Sections of the ventral prostate before and after castration were stained using sense and antisense probes. The sense probe did not show any labeling at all when used on sections of control prostates and at any other time point after androgen ablation (Fig. 4 a). In control prostates, a specific staining (blue stain) of exclusively epithelial cells was obtained using the antisense probe (Fig. 4, b and c). Within the epithelial cells, the staining was most prominent at supranuclear and apical localization that was frequently found to be interrupted by a narrow unstained region (Fig. 4, c and d). From ultrastructural studies, it is known that the rough endoplasmic reticulum is interrupted and localized in supranuclear and apical positions (Aumüller at al., 1980). Using the antisense probe, the homogeneous staining of the epithelial cells remained until day 2 (Fig. 4 d). At day 3, a considerable number of apoptotic cells were detected whose fragmented nuclei were in many instances strongly labeled (Fig. 4, e and f, black stain). The intensity of the specific stain decreased at day 5 (Fig. 4 g) and was further reduced at day 7 (not shown).

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