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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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Enzymatic analysis of the endonucleolytic activity present in homogenates of rat parotid and ventral prostate.  SDS-zymogram (a) on a 15% polyacrylamide gel. Lane A, 140 ng protein of tissue  homogenate of rat parotid; lane B, 0.56 ng  of purified rat parotid DNase I; Lanes 0d,  1d, 3d, and 5d, 100 μg of protein from tissue homogenates from rat ventral prostate  from days 0, 1, 3, and 5, respectively.  Numbers on left margin give molecular  mass in kD. (b) Histogram of the densitometric evaluation of the corresponding  bands of gel a. Native gel electrophoresis  (c) of tissue homogenates of rat parotid  containing 0.1 μg protein and of rat ventral prostate from days 0, 1, 3, and 5 containing 30 μg of protein. (d) Densitometric evaluation of gel c. Serial dilution (e) of  the endonucleolytic activity present in day  0 and 3 homogenates using the plasmid  degradation assay. Lane 1, negative control applying 0.5 μg of Bluescript II KS(+)  plasmid on its own; lanes 2–5, day 0 undiluted containing 5 μg of protein (lane 2),  diluted 1:2 (lane 3), 1:4 (lane 4), and 1:8  (lane 5); lanes 6–9, day 3 undiluted containing 5 μg of protein (lane 6), diluted 1:2  (lane 7), 1:4 (lane 8), and 1:8 (lane 9).
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Figure 6: Enzymatic analysis of the endonucleolytic activity present in homogenates of rat parotid and ventral prostate. SDS-zymogram (a) on a 15% polyacrylamide gel. Lane A, 140 ng protein of tissue homogenate of rat parotid; lane B, 0.56 ng of purified rat parotid DNase I; Lanes 0d, 1d, 3d, and 5d, 100 μg of protein from tissue homogenates from rat ventral prostate from days 0, 1, 3, and 5, respectively. Numbers on left margin give molecular mass in kD. (b) Histogram of the densitometric evaluation of the corresponding bands of gel a. Native gel electrophoresis (c) of tissue homogenates of rat parotid containing 0.1 μg protein and of rat ventral prostate from days 0, 1, 3, and 5 containing 30 μg of protein. (d) Densitometric evaluation of gel c. Serial dilution (e) of the endonucleolytic activity present in day 0 and 3 homogenates using the plasmid degradation assay. Lane 1, negative control applying 0.5 μg of Bluescript II KS(+) plasmid on its own; lanes 2–5, day 0 undiluted containing 5 μg of protein (lane 2), diluted 1:2 (lane 3), 1:4 (lane 4), and 1:8 (lane 5); lanes 6–9, day 3 undiluted containing 5 μg of protein (lane 6), diluted 1:2 (lane 7), 1:4 (lane 8), and 1:8 (lane 9).

Mentions: Using the zymogram technique (Lacks, 1981) with the modifications described in Materials and Methods, endonucleolytic activities were detected in prostatic tissue homogenates before and after castration (Fig. 6 a). This procedure also allowed us to determine the molecular mass of the endonuclease(s) and to estimate changes of its activity after castration. Control prostates contained a single endonucleolytic entity of 32 kD and low activity (Fig. 6 a). After castration at day 1, three endonucleolytic entities of higher molecular mass were detected. However, at day 3 the pattern seemed to revert to the original one with a high activity of ∼32 kD and minor activities of higher molecular mass, whereas at day 5 only very little activity of 32 kD was detected. Densitometry of the gel revealed that the total activity had increased about sevenfold at day 3 (Fig. 6 b). The molecular mass of the endonucleolytic activity in control animals and at day 5 was ∼32 kD, almost identical to that of purified rat parotid DNase I or the activity present in rat parotid homogenate. This pattern of endonuclease isoforms was observed repeatedly using different animals (n = 6), and we assume that the shifts of apparent molecular mass at day 1 are due to increased synthesis of proforms that are processed during the following days. This analysis indicates the presence of at least four different isoforms: three at day 1 in addition to the 32-kD isoform. At day 3, the 32-kD variant predominates, although the ones with higher molecular mass were still visible. Even at day 5, isoforms of molecular mass greater than 32 kD were still detectable on the original gel. The endonucleolytic activity of all entities was inhibited by addition of either 20 mM EDTA, 20 mM EGTA, or 5 mM ZnCl2 into the reactivation buffer (not shown), indicating identical enzymatic properties.


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)

Enzymatic analysis of the endonucleolytic activity present in homogenates of rat parotid and ventral prostate.  SDS-zymogram (a) on a 15% polyacrylamide gel. Lane A, 140 ng protein of tissue  homogenate of rat parotid; lane B, 0.56 ng  of purified rat parotid DNase I; Lanes 0d,  1d, 3d, and 5d, 100 μg of protein from tissue homogenates from rat ventral prostate  from days 0, 1, 3, and 5, respectively.  Numbers on left margin give molecular  mass in kD. (b) Histogram of the densitometric evaluation of the corresponding  bands of gel a. Native gel electrophoresis  (c) of tissue homogenates of rat parotid  containing 0.1 μg protein and of rat ventral prostate from days 0, 1, 3, and 5 containing 30 μg of protein. (d) Densitometric evaluation of gel c. Serial dilution (e) of  the endonucleolytic activity present in day  0 and 3 homogenates using the plasmid  degradation assay. Lane 1, negative control applying 0.5 μg of Bluescript II KS(+)  plasmid on its own; lanes 2–5, day 0 undiluted containing 5 μg of protein (lane 2),  diluted 1:2 (lane 3), 1:4 (lane 4), and 1:8  (lane 5); lanes 6–9, day 3 undiluted containing 5 μg of protein (lane 6), diluted 1:2  (lane 7), 1:4 (lane 8), and 1:8 (lane 9).
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Figure 6: Enzymatic analysis of the endonucleolytic activity present in homogenates of rat parotid and ventral prostate. SDS-zymogram (a) on a 15% polyacrylamide gel. Lane A, 140 ng protein of tissue homogenate of rat parotid; lane B, 0.56 ng of purified rat parotid DNase I; Lanes 0d, 1d, 3d, and 5d, 100 μg of protein from tissue homogenates from rat ventral prostate from days 0, 1, 3, and 5, respectively. Numbers on left margin give molecular mass in kD. (b) Histogram of the densitometric evaluation of the corresponding bands of gel a. Native gel electrophoresis (c) of tissue homogenates of rat parotid containing 0.1 μg protein and of rat ventral prostate from days 0, 1, 3, and 5 containing 30 μg of protein. (d) Densitometric evaluation of gel c. Serial dilution (e) of the endonucleolytic activity present in day 0 and 3 homogenates using the plasmid degradation assay. Lane 1, negative control applying 0.5 μg of Bluescript II KS(+) plasmid on its own; lanes 2–5, day 0 undiluted containing 5 μg of protein (lane 2), diluted 1:2 (lane 3), 1:4 (lane 4), and 1:8 (lane 5); lanes 6–9, day 3 undiluted containing 5 μg of protein (lane 6), diluted 1:2 (lane 7), 1:4 (lane 8), and 1:8 (lane 9).
Mentions: Using the zymogram technique (Lacks, 1981) with the modifications described in Materials and Methods, endonucleolytic activities were detected in prostatic tissue homogenates before and after castration (Fig. 6 a). This procedure also allowed us to determine the molecular mass of the endonuclease(s) and to estimate changes of its activity after castration. Control prostates contained a single endonucleolytic entity of 32 kD and low activity (Fig. 6 a). After castration at day 1, three endonucleolytic entities of higher molecular mass were detected. However, at day 3 the pattern seemed to revert to the original one with a high activity of ∼32 kD and minor activities of higher molecular mass, whereas at day 5 only very little activity of 32 kD was detected. Densitometry of the gel revealed that the total activity had increased about sevenfold at day 3 (Fig. 6 b). The molecular mass of the endonucleolytic activity in control animals and at day 5 was ∼32 kD, almost identical to that of purified rat parotid DNase I or the activity present in rat parotid homogenate. This pattern of endonuclease isoforms was observed repeatedly using different animals (n = 6), and we assume that the shifts of apparent molecular mass at day 1 are due to increased synthesis of proforms that are processed during the following days. This analysis indicates the presence of at least four different isoforms: three at day 1 in addition to the 32-kD isoform. At day 3, the 32-kD variant predominates, although the ones with higher molecular mass were still visible. Even at day 5, isoforms of molecular mass greater than 32 kD were still detectable on the original gel. The endonucleolytic activity of all entities was inhibited by addition of either 20 mM EDTA, 20 mM EGTA, or 5 mM ZnCl2 into the reactivation buffer (not shown), indicating identical enzymatic properties.

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