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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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Specific tests for the presence of DNase I in prostate  homogenates. (a) Ionic dependence of the endonuclease present  in prostatic homogenates using the plasmid degradation assay.  Lanes 1–3, Bluescript II KS(+) plasmid (0.5 μg) in TBE in the  presence of 5 mM ZnCl2 (lane 1), 20 mM EDTA (lane 2), or 20  mM EGTA (lane 3); lane 4, 5 μg of day 0 homogenate; lane 5,  plus 5 mM ZnCl2; lane 6, plus 20 mM EDTA; lane 7, plus 20 mM  EGTA; lane 8, day 3 homogenate (5 μg) on its own. In lanes 9–11,  the day 3 homogenate was supplemented with 5 mM ZnCl2, 20 mM  EDTA, and 20 mM EGTA, respectively. The samples were incubated for 90 min at 37°C. For details see Materials and Methods.  (b) Inhibition of the endonuclease present in rat ventral homogenate by actin:segment 1 complex. Lane 1, Bluescript II KS(+)  plasmid on its own; lane 2, day 0 homogenate (5 μg of protein);  lane 3 plus 2 μg actin:segment 1; lane 4, plus 8 μg actin:segment 1;  lanes 5–7, identical experiment using day 3 homogenate (5 μg of  protein). Note that for day 3 homogenate, complete inhibition of  endonucleolytic activity is only attained in the presence of 8 μg of  actin:segment 1. (c) Immunodepletion of the endonucleolytic activity in day 0 and 3 homogenates by immobilized anti–DNase I  (see also Materials and Methods). Lane 1, plasmid on its own;  lane 2, day 0 using 5 μg of homogenate after preincubation with  unloaded protein A–Sepharose; lane 3, day 0 homogenate after  preincubation with protein A–Sepharose loaded with antibodies  against rat DNase I; lanes 4 and 5, identical experiment using 5 μg  of day 3 homogenate (for details see Materials and Methods). (d)  Immunodepletion of DNA ladder catalyzing activity in day 0 and  3 homogenates by immobilized anti–DNase I. Lane 1, molecular  weight marker; lane 2, 2 × 105 substrate nuclei were incubated in  the presence of 20 mM EDTA and (lane 3) 5 mM CaCl2 and  MgCl2 for 24 h at 37°C. Note the absence of DNA; it was impossible to pipette the highly viscous DNA clot into the gel slot indicating the absence of endogenous endonucleases. Lanes 4–6, 40 μg  of day 0 homogenate was incubated with 2 × 105 substrate nuclei  for 24 h at room temperature: (lane 4) homogenate on its own, (lane  5) after preincubation with protein A–Sepharose alone, and (lane  6) after preincubation with protein A–Sepharose complexed with  anti–DNase I. Note that high molecular mass DNA fragments are  generated in all samples. Lanes 7–9, identical experiment using  day 3 homogenate. Note that after preincubation with immobilized anti–DNase I, ladder formation is suppressed (lane 9), although high molecular weight DNA fragments are generated.  Lane 1 gives phage λ treated with HindIII as molecular mass  marker (from top to bottom: 23,130; 9,460; 6,557; 4,322 [top four  closely spaced bands]; 2,200; and 2,027 bp).
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Figure 7: Specific tests for the presence of DNase I in prostate homogenates. (a) Ionic dependence of the endonuclease present in prostatic homogenates using the plasmid degradation assay. Lanes 1–3, Bluescript II KS(+) plasmid (0.5 μg) in TBE in the presence of 5 mM ZnCl2 (lane 1), 20 mM EDTA (lane 2), or 20 mM EGTA (lane 3); lane 4, 5 μg of day 0 homogenate; lane 5, plus 5 mM ZnCl2; lane 6, plus 20 mM EDTA; lane 7, plus 20 mM EGTA; lane 8, day 3 homogenate (5 μg) on its own. In lanes 9–11, the day 3 homogenate was supplemented with 5 mM ZnCl2, 20 mM EDTA, and 20 mM EGTA, respectively. The samples were incubated for 90 min at 37°C. For details see Materials and Methods. (b) Inhibition of the endonuclease present in rat ventral homogenate by actin:segment 1 complex. Lane 1, Bluescript II KS(+) plasmid on its own; lane 2, day 0 homogenate (5 μg of protein); lane 3 plus 2 μg actin:segment 1; lane 4, plus 8 μg actin:segment 1; lanes 5–7, identical experiment using day 3 homogenate (5 μg of protein). Note that for day 3 homogenate, complete inhibition of endonucleolytic activity is only attained in the presence of 8 μg of actin:segment 1. (c) Immunodepletion of the endonucleolytic activity in day 0 and 3 homogenates by immobilized anti–DNase I (see also Materials and Methods). Lane 1, plasmid on its own; lane 2, day 0 using 5 μg of homogenate after preincubation with unloaded protein A–Sepharose; lane 3, day 0 homogenate after preincubation with protein A–Sepharose loaded with antibodies against rat DNase I; lanes 4 and 5, identical experiment using 5 μg of day 3 homogenate (for details see Materials and Methods). (d) Immunodepletion of DNA ladder catalyzing activity in day 0 and 3 homogenates by immobilized anti–DNase I. Lane 1, molecular weight marker; lane 2, 2 × 105 substrate nuclei were incubated in the presence of 20 mM EDTA and (lane 3) 5 mM CaCl2 and MgCl2 for 24 h at 37°C. Note the absence of DNA; it was impossible to pipette the highly viscous DNA clot into the gel slot indicating the absence of endogenous endonucleases. Lanes 4–6, 40 μg of day 0 homogenate was incubated with 2 × 105 substrate nuclei for 24 h at room temperature: (lane 4) homogenate on its own, (lane 5) after preincubation with protein A–Sepharose alone, and (lane 6) after preincubation with protein A–Sepharose complexed with anti–DNase I. Note that high molecular mass DNA fragments are generated in all samples. Lanes 7–9, identical experiment using day 3 homogenate. Note that after preincubation with immobilized anti–DNase I, ladder formation is suppressed (lane 9), although high molecular weight DNA fragments are generated. Lane 1 gives phage λ treated with HindIII as molecular mass marker (from top to bottom: 23,130; 9,460; 6,557; 4,322 [top four closely spaced bands]; 2,200; and 2,027 bp).

Mentions: A number of specific tests for DNase I were performed comparing prostatic homogenates (5 μg) of day 0 and 3. Using the plasmid degradation assay, the ionic requirements of the prostatic endonuclease were further investigated (Fig. 7 a). It can be seen that the endonucleolytic activity present in the homogenate of control and day 3 prostates was active in the presence of 2 mM CaCl2 and 2 mM MgCl2 but completely inhibited after addition of 5 mM ZnCl2, 20 mM EDTA, or 20 mM EGTA. This ion dependence parallels the known behavior of the Ca2+, Mg2+-dependent apoptotic endonuclease or DNase I.


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)

Specific tests for the presence of DNase I in prostate  homogenates. (a) Ionic dependence of the endonuclease present  in prostatic homogenates using the plasmid degradation assay.  Lanes 1–3, Bluescript II KS(+) plasmid (0.5 μg) in TBE in the  presence of 5 mM ZnCl2 (lane 1), 20 mM EDTA (lane 2), or 20  mM EGTA (lane 3); lane 4, 5 μg of day 0 homogenate; lane 5,  plus 5 mM ZnCl2; lane 6, plus 20 mM EDTA; lane 7, plus 20 mM  EGTA; lane 8, day 3 homogenate (5 μg) on its own. In lanes 9–11,  the day 3 homogenate was supplemented with 5 mM ZnCl2, 20 mM  EDTA, and 20 mM EGTA, respectively. The samples were incubated for 90 min at 37°C. For details see Materials and Methods.  (b) Inhibition of the endonuclease present in rat ventral homogenate by actin:segment 1 complex. Lane 1, Bluescript II KS(+)  plasmid on its own; lane 2, day 0 homogenate (5 μg of protein);  lane 3 plus 2 μg actin:segment 1; lane 4, plus 8 μg actin:segment 1;  lanes 5–7, identical experiment using day 3 homogenate (5 μg of  protein). Note that for day 3 homogenate, complete inhibition of  endonucleolytic activity is only attained in the presence of 8 μg of  actin:segment 1. (c) Immunodepletion of the endonucleolytic activity in day 0 and 3 homogenates by immobilized anti–DNase I  (see also Materials and Methods). Lane 1, plasmid on its own;  lane 2, day 0 using 5 μg of homogenate after preincubation with  unloaded protein A–Sepharose; lane 3, day 0 homogenate after  preincubation with protein A–Sepharose loaded with antibodies  against rat DNase I; lanes 4 and 5, identical experiment using 5 μg  of day 3 homogenate (for details see Materials and Methods). (d)  Immunodepletion of DNA ladder catalyzing activity in day 0 and  3 homogenates by immobilized anti–DNase I. Lane 1, molecular  weight marker; lane 2, 2 × 105 substrate nuclei were incubated in  the presence of 20 mM EDTA and (lane 3) 5 mM CaCl2 and  MgCl2 for 24 h at 37°C. Note the absence of DNA; it was impossible to pipette the highly viscous DNA clot into the gel slot indicating the absence of endogenous endonucleases. Lanes 4–6, 40 μg  of day 0 homogenate was incubated with 2 × 105 substrate nuclei  for 24 h at room temperature: (lane 4) homogenate on its own, (lane  5) after preincubation with protein A–Sepharose alone, and (lane  6) after preincubation with protein A–Sepharose complexed with  anti–DNase I. Note that high molecular mass DNA fragments are  generated in all samples. Lanes 7–9, identical experiment using  day 3 homogenate. Note that after preincubation with immobilized anti–DNase I, ladder formation is suppressed (lane 9), although high molecular weight DNA fragments are generated.  Lane 1 gives phage λ treated with HindIII as molecular mass  marker (from top to bottom: 23,130; 9,460; 6,557; 4,322 [top four  closely spaced bands]; 2,200; and 2,027 bp).
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Figure 7: Specific tests for the presence of DNase I in prostate homogenates. (a) Ionic dependence of the endonuclease present in prostatic homogenates using the plasmid degradation assay. Lanes 1–3, Bluescript II KS(+) plasmid (0.5 μg) in TBE in the presence of 5 mM ZnCl2 (lane 1), 20 mM EDTA (lane 2), or 20 mM EGTA (lane 3); lane 4, 5 μg of day 0 homogenate; lane 5, plus 5 mM ZnCl2; lane 6, plus 20 mM EDTA; lane 7, plus 20 mM EGTA; lane 8, day 3 homogenate (5 μg) on its own. In lanes 9–11, the day 3 homogenate was supplemented with 5 mM ZnCl2, 20 mM EDTA, and 20 mM EGTA, respectively. The samples were incubated for 90 min at 37°C. For details see Materials and Methods. (b) Inhibition of the endonuclease present in rat ventral homogenate by actin:segment 1 complex. Lane 1, Bluescript II KS(+) plasmid on its own; lane 2, day 0 homogenate (5 μg of protein); lane 3 plus 2 μg actin:segment 1; lane 4, plus 8 μg actin:segment 1; lanes 5–7, identical experiment using day 3 homogenate (5 μg of protein). Note that for day 3 homogenate, complete inhibition of endonucleolytic activity is only attained in the presence of 8 μg of actin:segment 1. (c) Immunodepletion of the endonucleolytic activity in day 0 and 3 homogenates by immobilized anti–DNase I (see also Materials and Methods). Lane 1, plasmid on its own; lane 2, day 0 using 5 μg of homogenate after preincubation with unloaded protein A–Sepharose; lane 3, day 0 homogenate after preincubation with protein A–Sepharose loaded with antibodies against rat DNase I; lanes 4 and 5, identical experiment using 5 μg of day 3 homogenate (for details see Materials and Methods). (d) Immunodepletion of DNA ladder catalyzing activity in day 0 and 3 homogenates by immobilized anti–DNase I. Lane 1, molecular weight marker; lane 2, 2 × 105 substrate nuclei were incubated in the presence of 20 mM EDTA and (lane 3) 5 mM CaCl2 and MgCl2 for 24 h at 37°C. Note the absence of DNA; it was impossible to pipette the highly viscous DNA clot into the gel slot indicating the absence of endogenous endonucleases. Lanes 4–6, 40 μg of day 0 homogenate was incubated with 2 × 105 substrate nuclei for 24 h at room temperature: (lane 4) homogenate on its own, (lane 5) after preincubation with protein A–Sepharose alone, and (lane 6) after preincubation with protein A–Sepharose complexed with anti–DNase I. Note that high molecular mass DNA fragments are generated in all samples. Lanes 7–9, identical experiment using day 3 homogenate. Note that after preincubation with immobilized anti–DNase I, ladder formation is suppressed (lane 9), although high molecular weight DNA fragments are generated. Lane 1 gives phage λ treated with HindIII as molecular mass marker (from top to bottom: 23,130; 9,460; 6,557; 4,322 [top four closely spaced bands]; 2,200; and 2,027 bp).
Mentions: A number of specific tests for DNase I were performed comparing prostatic homogenates (5 μg) of day 0 and 3. Using the plasmid degradation assay, the ionic requirements of the prostatic endonuclease were further investigated (Fig. 7 a). It can be seen that the endonucleolytic activity present in the homogenate of control and day 3 prostates was active in the presence of 2 mM CaCl2 and 2 mM MgCl2 but completely inhibited after addition of 5 mM ZnCl2, 20 mM EDTA, or 20 mM EGTA. This ion dependence parallels the known behavior of the Ca2+, Mg2+-dependent apoptotic endonuclease or DNase I.

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