Androgen Ablation Leads to an Upregulation and Intranuclear Accumulation of Deoxyribonuclease I in Rat Prostate Epithelial Cells Paralleling Their Apoptotic Elimination
Androgen Ablation Leads to an Upregulation and Intranuclear Accumulation of Deoxyribonuclease I in Rat Prostate Epithelial Cells Paralleling Their Apoptotic Elimination
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雄激素消除导致大鼠前列腺上皮细胞中脱氧核糖核酸酶 I 的上调和核内积累，与其凋亡消除并行
作者：
F. Rauch;Bernhard Polzar;H. Stephan;S. Zanotti;R. Paddenberg;H. Mannherz
去势雄激素消融后，大鼠腹侧前列腺上皮细胞凋亡消失。显示凋亡染色质降解的细胞数量随着时间的增加而增加，直到去势后第3天，这一点通过DNA片段的原位末端标记得到证实。凋亡染色质降解是由Ca2+， Mg2+依赖性内切酶催化的。最近，有证据表明脱氧核糖核酸酶I （DNase I）与凋亡内切酶相同或非常密切相关（Peitsch, m.c.， B. Polzar， H. Stephan， T. Crompton， H.R. MacDonald， H.G. Mannherz, and J. Tschopp. 1993）。EMBO[欧元。摩尔。杂志。器官。[j] . 12:371-377。因此，采用免疫组织化学和生化技术，从蛋白、酶活性和基因转录水平分析雄激素消融前后大鼠腹侧前列腺中DNase I的表达。在雄激素消融前，仅在少数单个上皮细胞中检测到DNase I免疫反应性。去势后，在上皮细胞内观察到DNase I免疫反应性的时间依赖性增加。大约12小时后，在大量上皮细胞的顶端区域首次出现。至去势后第3天，细胞内DNase I抗原性持续升高，细胞核逐渐呈DNase I阳性。在第5天，几乎所有的上皮细胞核都被抗DNase I染色。DNase I的免疫反应性特别集中在表现出细胞凋亡形态学迹象的细胞中，如核断裂，并且在许多情况下在凋亡小体中持续存在。采用斑点法、Northern印迹法和RNase保护法检测对照动物的dna酶I基因转录本。在雄激素消融后，总可提取RNA中DNase I基因转录本的数量在第5天没有变化或仅略有下降。通过原位杂交证实了它们在上皮细胞内的排他性定位。去势前，DNase - 1基因转录本均匀分布在所有上皮细胞中。第3天，DNase - 1特异性mRNA在凋亡形态细胞中高浓度表达。使用酶谱技术，在去势前的组织匀浆中检测到约32 kD的单一核内溶活性。雄激素消融术后，到第3天，内核溶解活性增加了约4 - 7倍。然而，在第五天，它已经下降到原来的水平。在第1天，表达了三个更高分子质量的新的核内溶变异体。在第3天，主要的核内溶活性显示出32 kD的表观分子质量。去势前后前列腺匀浆中存在的内切酶的酶分析表明，它们的性质与dna酶i相同。它们被二价阳离子、Zn2+离子和单体肌动蛋白的螯合剂抑制。通过固定大鼠腮腺dna酶I特异性抗体实现免疫清除。在制备凝胶电泳的第0天和第3天匀浆中富集内切酶后，Western blotting显示针对变性dna酶I的多克隆抗体在32-kD条带上染色。因此，这些数据表明，雄激素消融导致大鼠腹侧前列腺中具有与DNase I相同性质的核内溶解活性的翻译上调，随后在注定凋亡消除的上皮细胞中发生细胞内保留和最终核易位。
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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影响因子：
3.9
作者：
BARRY, MA;EASTMAN, A
通讯作者：
EASTMAN, A
去势雄激素消除后大鼠前列腺叶特异性凋亡细胞死亡。
DOI：
10.1210/endo.136.10.7664656
发表时间：
1995
期刊：
Endocrinology.
影响因子：
--
作者：
Banerjee,PP;Banerjee,S;Tilly,KI;Tilly,JL;Brown,TR;Zirkin,BR
通讯作者：
Zirkin,BR
影响因子：
4.8
作者：
KYPRIANOU, N;ISAACS, JT
通讯作者：
ISAACS, JT
DOI：
--
发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Lacks,SA
通讯作者：
Lacks,SA