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Department of Anatomy and Embryology, Ruhr-University, Bochum, Germany
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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