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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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Partial purification  of DNase I from rat ventral  prostate before and after castration using preparative gel  electrophoresis in the presence of SDS. (a and b) Plasmid degradation assays to  localize endonucleolytic activity in fractions collected  from Prep Cell. 0.25 μg of  the circular pUC 18-plasmid  was applied to a 1% agarose  gel on its own (negative control) or after incubation for  16 h at 37°C with 1 μg of prostatic homogenate (positive  control). Numbers above  lanes indicate the number of  fractions collected. 10 μl of  each fraction was incubated  with 0.25 μg of plasmid. For  details see Materials and  Methods. (a) Representative  fractions collected from day  0 and (b) day 3 homogenate.
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Figure 8: Partial purification of DNase I from rat ventral prostate before and after castration using preparative gel electrophoresis in the presence of SDS. (a and b) Plasmid degradation assays to localize endonucleolytic activity in fractions collected from Prep Cell. 0.25 μg of the circular pUC 18-plasmid was applied to a 1% agarose gel on its own (negative control) or after incubation for 16 h at 37°C with 1 μg of prostatic homogenate (positive control). Numbers above lanes indicate the number of fractions collected. 10 μl of each fraction was incubated with 0.25 μg of plasmid. For details see Materials and Methods. (a) Representative fractions collected from day 0 and (b) day 3 homogenate.

Mentions: Using the affinity-purified polyclonal antibody, attempts were made to identify DNase I in prostate homogenates by Western blotting. Most probably because of the low abundance of this enzyme, no positive reaction was obtained when using whole tissue homogenates. Therefore, the endonucleolytic activity was partially purified and enriched from tissue homogenates of control and day 3 prostates by preparative SDS-PAGE applying 10 mg of protein. The fractions collected were analyzed for endonuclease activity by the plasmid degradation assay (Fig. 8, a and b). Analysis of the activity containing fractions by SDSPAGE indicated a molecular mass range between 32 and 35 kD (Fig. 9, a and c) in agreement with the zymogram analysis. These fractions most probably represent a mixture of different prostatic proteins of this molecular mass range. Fractions containing the highest endonucleolytic activity of control and day 3 eluate were subjected to SDSPAGE followed by standard Western blotting. A band of ∼32 kD apparent molecular mass was stained by the polyclonal, affinity-purified antibody (Fig. 9, b and d). A much stronger immunoreaction was observed for the day 3 fraction. Densitometry indicated a fivefold increase in intensity at day 3 using the constant amount of purified rat parotid DNase I applied as internal standard (not shown). In agreement with the histochemical data, this result indicates that the amount of DNase I gene product was increased in day 3 homogenate. No staining was observed of fractions void of endonucleolytic activity (Fig. 9, b and d lanes 0′ and 3′). In this experiment we used purified rat parotid DNase I as control, which for unknown reasons always gave a higher apparent molecular mass (∼33 kD) than when present in whole tissue extracts (see also Fig. 6).


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)

Partial purification  of DNase I from rat ventral  prostate before and after castration using preparative gel  electrophoresis in the presence of SDS. (a and b) Plasmid degradation assays to  localize endonucleolytic activity in fractions collected  from Prep Cell. 0.25 μg of  the circular pUC 18-plasmid  was applied to a 1% agarose  gel on its own (negative control) or after incubation for  16 h at 37°C with 1 μg of prostatic homogenate (positive  control). Numbers above  lanes indicate the number of  fractions collected. 10 μl of  each fraction was incubated  with 0.25 μg of plasmid. For  details see Materials and  Methods. (a) Representative  fractions collected from day  0 and (b) day 3 homogenate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139838&req=5

Figure 8: Partial purification of DNase I from rat ventral prostate before and after castration using preparative gel electrophoresis in the presence of SDS. (a and b) Plasmid degradation assays to localize endonucleolytic activity in fractions collected from Prep Cell. 0.25 μg of the circular pUC 18-plasmid was applied to a 1% agarose gel on its own (negative control) or after incubation for 16 h at 37°C with 1 μg of prostatic homogenate (positive control). Numbers above lanes indicate the number of fractions collected. 10 μl of each fraction was incubated with 0.25 μg of plasmid. For details see Materials and Methods. (a) Representative fractions collected from day 0 and (b) day 3 homogenate.
Mentions: Using the affinity-purified polyclonal antibody, attempts were made to identify DNase I in prostate homogenates by Western blotting. Most probably because of the low abundance of this enzyme, no positive reaction was obtained when using whole tissue homogenates. Therefore, the endonucleolytic activity was partially purified and enriched from tissue homogenates of control and day 3 prostates by preparative SDS-PAGE applying 10 mg of protein. The fractions collected were analyzed for endonuclease activity by the plasmid degradation assay (Fig. 8, a and b). Analysis of the activity containing fractions by SDSPAGE indicated a molecular mass range between 32 and 35 kD (Fig. 9, a and c) in agreement with the zymogram analysis. These fractions most probably represent a mixture of different prostatic proteins of this molecular mass range. Fractions containing the highest endonucleolytic activity of control and day 3 eluate were subjected to SDSPAGE followed by standard Western blotting. A band of ∼32 kD apparent molecular mass was stained by the polyclonal, affinity-purified antibody (Fig. 9, b and d). A much stronger immunoreaction was observed for the day 3 fraction. Densitometry indicated a fivefold increase in intensity at day 3 using the constant amount of purified rat parotid DNase I applied as internal standard (not shown). In agreement with the histochemical data, this result indicates that the amount of DNase I gene product was increased in day 3 homogenate. No staining was observed of fractions void of endonucleolytic activity (Fig. 9, b and d lanes 0′ and 3′). In this experiment we used purified rat parotid DNase I as control, which for unknown reasons always gave a higher apparent molecular mass (∼33 kD) than when present in whole tissue extracts (see also Fig. 6).

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