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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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Dot and Northern  blots as well as RNase protection of a radioactively labeled probe specific for rat  parotid DNase I. Dot blots (a  and b) using total RNA purified from day 0, 1, 3, and 5  prostates at two dilutions. (a)  Hybridized with rat parotid  DNase I–specific cDNA and  (b) with a probe specific for  β-actin. Amount of RNA applied: Upper rows, 5 μg/dot;  lower rows, 1.25 μg/dot.  Northern blot (c) using 20 μg  of total RNA from day 0, 1,  3, and 5 prostates (lanes 0, 1, 3,  and 5) and 5 μg total RNA  from rat parotid gland (lane P)  hybridized with the DNase  I–specific cDNA. RNase protection (d) of a DNase I–specific  probe by DNase I–specific  mRNA present in the total  RNA of day 0, 1, 3, and 5  prostates. Total RNA prepared from rat prostates before and after castration  (100 μg each) and from rat  parotid gland (5 and 1 μg)  were hybridized with the labeled antisense probe as detailed in Materials and Methods. After ribonuclease treatment, the samples were run on 5% polyacrylamide/8 M urea gel and autoradiographed. Lane 0, before; lanes 1, 3, and 5, days 1, 3, and 5 after castration; lanes P and P′, RNA isolated from parotid gland, 5 μg and  1 μg, respectively, as positive control and to qualitatively compare the concentrations of DNase I–specific mRNA in prostate to parotid  gland; lane M, 5 × 103 cpm of antisense probe as standard (651 bp).
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Figure 5: Dot and Northern blots as well as RNase protection of a radioactively labeled probe specific for rat parotid DNase I. Dot blots (a and b) using total RNA purified from day 0, 1, 3, and 5 prostates at two dilutions. (a) Hybridized with rat parotid DNase I–specific cDNA and (b) with a probe specific for β-actin. Amount of RNA applied: Upper rows, 5 μg/dot; lower rows, 1.25 μg/dot. Northern blot (c) using 20 μg of total RNA from day 0, 1, 3, and 5 prostates (lanes 0, 1, 3, and 5) and 5 μg total RNA from rat parotid gland (lane P) hybridized with the DNase I–specific cDNA. RNase protection (d) of a DNase I–specific probe by DNase I–specific mRNA present in the total RNA of day 0, 1, 3, and 5 prostates. Total RNA prepared from rat prostates before and after castration (100 μg each) and from rat parotid gland (5 and 1 μg) were hybridized with the labeled antisense probe as detailed in Materials and Methods. After ribonuclease treatment, the samples were run on 5% polyacrylamide/8 M urea gel and autoradiographed. Lane 0, before; lanes 1, 3, and 5, days 1, 3, and 5 after castration; lanes P and P′, RNA isolated from parotid gland, 5 μg and 1 μg, respectively, as positive control and to qualitatively compare the concentrations of DNase I–specific mRNA in prostate to parotid gland; lane M, 5 × 103 cpm of antisense probe as standard (651 bp).

Mentions: The results obtained by in situ hybridization indicated the presence of DNase I gene transcripts in rat ventral prostates even before castration. Total RNA was isolated from ventral prostates before and at different time points after castration. Using the cDNA of rat parotid DNase I as probe on dot blots of total RNA of control and day 1, 3, and 5 prostates, the presence of DNase I gene transcripts was verified (Fig. 5 a). Similar results were obtained by Northern blotting (Fig. 5 c). One single mRNA band was obtained for days 0, 1, 3, and 5 that was identical in length to the one present in rat parotid RNA (1, 1 kb). Both assays indicated a slight decrease in the concentration of the DNase I–specific gene transcripts after androgen withdrawal. Furthermore, RNase protection analysis was used to attempt a more reliable, relative quantification of the DNase I gene transcript using total prostatic RNA (see Materials and Methods). A DNase I–specific signal was obtained for rat prostate that was identical in size to the one for rat parotid gland, although with an ∼20-fold lower intensity (Fig. 5 d). Again, a clear decrease in the concentration of DNase I gene transcript was seen during the 5 d after castration.


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)

Dot and Northern  blots as well as RNase protection of a radioactively labeled probe specific for rat  parotid DNase I. Dot blots (a  and b) using total RNA purified from day 0, 1, 3, and 5  prostates at two dilutions. (a)  Hybridized with rat parotid  DNase I–specific cDNA and  (b) with a probe specific for  β-actin. Amount of RNA applied: Upper rows, 5 μg/dot;  lower rows, 1.25 μg/dot.  Northern blot (c) using 20 μg  of total RNA from day 0, 1,  3, and 5 prostates (lanes 0, 1, 3,  and 5) and 5 μg total RNA  from rat parotid gland (lane P)  hybridized with the DNase  I–specific cDNA. RNase protection (d) of a DNase I–specific  probe by DNase I–specific  mRNA present in the total  RNA of day 0, 1, 3, and 5  prostates. Total RNA prepared from rat prostates before and after castration  (100 μg each) and from rat  parotid gland (5 and 1 μg)  were hybridized with the labeled antisense probe as detailed in Materials and Methods. After ribonuclease treatment, the samples were run on 5% polyacrylamide/8 M urea gel and autoradiographed. Lane 0, before; lanes 1, 3, and 5, days 1, 3, and 5 after castration; lanes P and P′, RNA isolated from parotid gland, 5 μg and  1 μg, respectively, as positive control and to qualitatively compare the concentrations of DNase I–specific mRNA in prostate to parotid  gland; lane M, 5 × 103 cpm of antisense probe as standard (651 bp).
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Related In: Results  -  Collection

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Figure 5: Dot and Northern blots as well as RNase protection of a radioactively labeled probe specific for rat parotid DNase I. Dot blots (a and b) using total RNA purified from day 0, 1, 3, and 5 prostates at two dilutions. (a) Hybridized with rat parotid DNase I–specific cDNA and (b) with a probe specific for β-actin. Amount of RNA applied: Upper rows, 5 μg/dot; lower rows, 1.25 μg/dot. Northern blot (c) using 20 μg of total RNA from day 0, 1, 3, and 5 prostates (lanes 0, 1, 3, and 5) and 5 μg total RNA from rat parotid gland (lane P) hybridized with the DNase I–specific cDNA. RNase protection (d) of a DNase I–specific probe by DNase I–specific mRNA present in the total RNA of day 0, 1, 3, and 5 prostates. Total RNA prepared from rat prostates before and after castration (100 μg each) and from rat parotid gland (5 and 1 μg) were hybridized with the labeled antisense probe as detailed in Materials and Methods. After ribonuclease treatment, the samples were run on 5% polyacrylamide/8 M urea gel and autoradiographed. Lane 0, before; lanes 1, 3, and 5, days 1, 3, and 5 after castration; lanes P and P′, RNA isolated from parotid gland, 5 μg and 1 μg, respectively, as positive control and to qualitatively compare the concentrations of DNase I–specific mRNA in prostate to parotid gland; lane M, 5 × 103 cpm of antisense probe as standard (651 bp).
Mentions: The results obtained by in situ hybridization indicated the presence of DNase I gene transcripts in rat ventral prostates even before castration. Total RNA was isolated from ventral prostates before and at different time points after castration. Using the cDNA of rat parotid DNase I as probe on dot blots of total RNA of control and day 1, 3, and 5 prostates, the presence of DNase I gene transcripts was verified (Fig. 5 a). Similar results were obtained by Northern blotting (Fig. 5 c). One single mRNA band was obtained for days 0, 1, 3, and 5 that was identical in length to the one present in rat parotid RNA (1, 1 kb). Both assays indicated a slight decrease in the concentration of the DNase I–specific gene transcripts after androgen withdrawal. Furthermore, RNase protection analysis was used to attempt a more reliable, relative quantification of the DNase I gene transcript using total prostatic RNA (see Materials and Methods). A DNase I–specific signal was obtained for rat prostate that was identical in size to the one for rat parotid gland, although with an ∼20-fold lower intensity (Fig. 5 d). Again, a clear decrease in the concentration of DNase I gene transcript was seen during the 5 d after castration.

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