Apoptosis: a general comment.
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Cell Survival↗
Biomedical subjects
R Buttyan
Publications and source records attributed to R Buttyan.
Sulfated glycoprotein-2 induced endogenous resistance to ischemia and reperfusion injury in the seminiferous tubules.
Evidence of increased resistance of Sertoli cells to ischemia and reperfusion injury was presented by numerous histological, morphological, and quantitative studies. In situ hybridization techniques and immunocytochemical studies demonstrated intense expression of sulfated glycoprotein-2 (SGP-2) in Sertoli cells. We propose that protective effects of SGP-2 are the core of the differential tolerance of ischemia by the various testicular cells and the morphology of the postischemic testis. We believe that suppression of ischemic damage selectively in Sertoli cells is the consequence of the ability to produce SGP-2, an endogenous inhibitor of ischemic injury. This hypothetical function of SGP-2 is supported by its immunosuppressive properties and its structural and functional identity to several types of human complement cytolysis inhibitors.
Animals↗
Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.
The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions from the urethra, the entire length of the ductal system can be divided into three segments, i.e. the proximal, intermediate, and distal segments. The present study was carried out to examine the pattern of localization of sulfated glycoprotein-2 (SGP-2), a marker protein associated with programmed cell death, in various regions of the prostatic ductal system under normal conditions and during castration-induced regression. SGP-2 has been considered an androgen-repressed gene product in the rat prostate and has previously been known as castration-induced protein or TRPM-2. In the normal rat prostate, immunoreactive SGP-2 was localized in epithelial cells lining the proximal segment in which signs of programmed cell death were apparent. Cells lining the distal and intermediate segments were, however, devoid of SGP-2. This observed regional variation in SGP-2 localization did not support an earlier hypothesis which stated that SGP-2 was constitutively expressed by all prostatic epithelial cells in the normal rat prostate. After castration in adult rats, there was a shift in the location of cells containing SGP-2 from the proximal segment toward the distal segment. Therefore, there is a regional variation in the distribution of SGP-2 in the rat prostate both before and after castration in the host. These findings are likely to be associated with a regional variation in cellular responsiveness to androgen stimulation and androgen depletion in the prostatic ductal system. Results also support the view that SGP-2 localization is associated with an early manifestation of programmed cell death in the rat prostate.
Androgens↗
Chromosomal assignment of the human homologue encoding SGP-2.
Although originally characterized as a constitutively expressed gene product in mammalian Sertoli cells, sulfated glycoprotein-2 (SGP-2) has gained widespread attention due to its remarkably rapid and sizable induction in numerous types of mammalian cells undergoing apoptosis, or programmed death. In order to identify the chromosomal assignment for the human homologue of SGP-2, we performed Southern blot analysis of Bgl II restricted genomic DNA extracted from a panel of cloned hamster-human hybrid cell lines and screened for the presence of restriction fragments homologous to SGP-2. The results of this analysis indicate that the human homologue of SGP-2 resides on chromosome 8.
Animals↗
Characterization of the products of a gene expressed during androgen-programmed cell death and their potential use as a marker of urogenital injury.
Regression of the rat ventral prostate gland following castration is accompanied by the induced expression of messenger RNA encoding Testosterone Repressed Prostate Message-2 (TRPM-2). Subsequent studies have shown that this gene is also induced during renal injury. In each of these tissues, the TRPM-2 RNA products are expressed by cells undergoing programmed death as a result of the hormonal stimuli or the traumatic insult. In an attempt to characterize this gene and its products, we partially sequenced complementary DNAs for TRPM-2 isolated from a recombinant library constructed using RNA of a hydronephrotic kidney. The sequence of these clones showed close homology with the sulfated glycoprotein-2 (SGP-2/clusterin) gene, expressed constitutively by mammalian Sertoli cells. Antibody recognition studies confirm this homology. Antiserum made against rat clusterin recognized TRPM-2 encoded polypeptides in extracts of regressing rat ventral prostate glands. Western blot analysis allowed us to demonstrate large increases in the concentration of these proteins in extracts of regressing ventral prostate gland and in rat serum and urine during the acute period of prostatic regression. These results indicate that proteins are synthesized from the large amount of TRPM-2 RNA produced by dying prostate cells and imply that these proteins are shed into the serum and urine. Based on the intense synthesis of TRPM-2 gene products by dying cells in the urogenital tract and the ability to assay for these products in serum and urine, we suggest that an assay for TRPM-2 products might allow us to monitor the extent of cellular damage associated with specific urogenital disease states.
Animals↗
Immediate induction of c-fos and c-myc transcripts following unilateral nephrectomy.
Unilateral nephrectomy in the rat results in contralateral compensatory renal growth. The immediate molecular events which occur in the remaining renal unit were studied by examining RNA isolated from these kidneys. Northern blot analysis was used to analyze the expression of the growth-related (c-fos and c-myc) genes during the 1-hour period following unilateral nephrectomy. Both c-fos and c-myc band transcripts were detected within 15 min after nephrectomy. The immediate, transient induction of these genes in the remaining renal unit mimics that seen during proliferative stimulation of cultured cells and may play a role in modulating the eventual compensatory growth response.
Animals↗
Gene activity during the early phase of androgen-stimulated rat prostate regrowth.
Androgenic steroids regulate the proliferation rate of normal and malignant prostate cells. In order to investigate the molecular basis of this control, we utilized Northern and Western blot techniques to measure changes in the expression of individual genes during the early phase of prostate regrowth. Vestigial ventral prostate glands of 7-day castrated rats showed increased numbers of replicating cells within 12 h of continuous pharmacological testosterone replacement as demonstrated by flow cytometric procedures. The period prior to the onset of proliferative enhancement was accompanied by the sequential induction of a variety of transcripts encoding gene products often associated with cell growth. Within 1 h of treatment, mature mRNA transcripts for c-fos were induced 6-fold, returning to control levels by 2 h. Other genes showed transiently elevated transcript levels after 2 h (c-Ha-ras, c-Ki-ras) or after 8 h (c-myc,c-myb, beta-actin, and Mr 70,000 heat shock protein) of testosterone replacement. Expression of the polypeptide encoded by c-Ha-ras was coordinately enhanced (2-fold) during this period, but not to the levels of the transcript (20-fold induction). Transcripts encoding basic fibroblast growth factor were increased 16 h and later, subsequent to the earlier enhancement in the proliferation rate. By placing these genes in a defined temporal order with regard to their expression in response to testosterone, we have constructed a map of gene activity during early prostate regrowth. This map shows a number of genes, the products of which might participate in the mechanism by which androgens induce mitogenesis of prostate cells.
Animals↗
Gene expression in response to acute unilateral ureteral obstruction.
Acute unilateral ureteral obstruction results in differential growth characteristics of both the ipsilateral and contralateral kidney. The obstructed kidney undergoes cellular atrophy following an initial phase of interstitial proliferation while the contralateral kidney hypertrophies. To evaluate the molecular events occurring in both kidneys after obstruction, we examined the expression of growth related (c-fos, c-myc, cH-ras, HSP 70), cell maintenance (beta-actin), and cellular damage (TRPM-2) genes at the mRNA level. In the contralateral kidney an early and transitory induction of c-fos and c-myc expression occurred while a bimodal induction was noted in the obstructed kidney. The patterns of cH-ras, HSP 70 and actin expression also differed in both kidneys. Induction of TRPM-2 was noted only in the obstructed kidney. Rapid gene activation is evident in both the contralateral and obstructed kidney following unilateral ureteral obstruction. The patterns of expression are distinct and may reflect the cellular response to stress (cell death and stromal proliferation) in the obstructed kidney versus a response to a systemic stimulus resulting in cellular hypertrophy in the contralateral kidney.
Acute Disease↗
Induction of the TRPM-2 gene in cells undergoing programmed death.
RNA and protein products encoded by the testosterone-repressed prostate message-2 gene (TRPM-2) are induced to high levels, coordinate with the onset of cell death, in numerous rodent models of inducible tissue damage. These models include cell death initiated by hormonal stimuli (prostate regression), pressure insult (renal atrophy after ureteral obstruction), developmental stimuli (necrosis of interdigital tissue), and cytotoxic injury (chemotherapeutic regression of a tumor). Sequence analysis of cDNA encoding TRPM-2 revealed its close homology with a product referred to as SGP-2 or clusterin expressed constitutively by Sertoli cells; however, the immunologically related polypeptides expressed in regressing tissues differ in molecular mass from the forms secreted by the testis. Although the function(s) of the products encoded by the TRPM-2 gene remains unclear, their presence provides a remarkable and early indicator of programmed cell death in many types of mammalian cells.
Animals↗
Gene expression during the early phases of regression of the androgen-dependent Shionogi mouse mammary carcinoma.
The effects of castration on gene expression were measured in the androgen-dependent Shionogi mouse mammary carcinoma. From 0 to 144 h after castration, polyadenylated RNA from the tumors was analyzed by Northern blotting for the expression of genes associated with cell maintenance (beta-actin and alpha-tubulin), cell growth and differentiation (c-fos and c-myc), and cell stress and death (heat shock 70 and TRPM-2). During the first 48-72 h after castration, the tumor continued to increase its mass but thereafter (72-144 h) began to regress. Throughout, the concentration of polyadenylated RNA recovered and the expression of both beta-actin and alpha-tubulin remained relatively constant, implying that in the surviving cells there are no major decreases in RNA synthesis. By comparison, c-myc exhibited a small increase in relative expression during the entire period examined, whereas c-fos displayed a transient peak at 12 h after castration, suggesting that this gene is acutely sensitive to androgen withdrawal. Heat shock protein 70 displayed a pattern similar to that of c-fos; however, the transient rise in the level of heat shock protein 70 expression could be induced by sham operations alone. The concentration of the transcripts encoding TRPM-2 rose only when tumor regression was most evident (72-144 h after castration). Thus, gene expression in the Shionogi carcinoma following castration can be grouped according to those genes which show little or no response, those which are acutely sensitive, and those which show a late effect and are more closely affiliated with cell death.
Animals↗
Calcium channel antagonists delay regression of androgen-dependent tissues and suppress gene activity associated with cell death.
Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding c-fos. Since c-fos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.
Androgens↗
Gene expression in renal growth and regrowth.
To elucidate the molecular events associated with postnatal and compensatory renal growth, the expression of growth/differentiation genes (c-fos, c-myc, c-H-ras, c-K-ras), a stress-related gene (HSP70) and a structural gene (collagen type IV, alpha 1 and 2) were examined. Northern analysis of messenger ribonucleic acid from the newborn mouse reveals high levels of expression of HSP70, c-H-ras, and c-K-ras during the first week of life. By day 40 HSP70-related and c-H-ras expression decreases somewhat, c-K-ras remains unchanged and collagen type IV, which encodes for the renal glomerular basement membrane, expression decreases significantly. During compensatory renal growth increased expression of HSP70, c-H-ras and c-K-ras occurs. The results seem to indicate that growth/differentiation genes may be necessary for continued cell growth (hypertrophy) in postnatal and compensatory renal growth, and that collagen type IV formation continues up through week 2 of postnatal growth consistent with the interval of glomerular basement membrane formation.
Animals↗
Cascade induction of c-fos, c-myc, and heat shock 70K transcripts during regression of the rat ventral prostate gland.
Castration of an adult male rat results in the rapid regression of the ventral prostate gland. Most of the acinar epithelial cells lining the ducts of the gland will die during the first 5 days after androgen withdrawal. The molecular events which accompany the death of these cells were studied by examining RNA isolated from ventral prostate glands of normal and from a sequential 24-h series of castrate rats. These RNAs were analyzed by Northern blot methods to quantify the expression of growth-related (c-fos, c-myc, and heat shock 70K) and cell maintenance (alpha-tubulin) genes during prostatic regression. Each of these genes showed a bimodal pattern of expression. Within the first few days after castration, transcript levels decline; whereas later, during the most active period of cell death, their expression was transiently induced. Levels of mature c-myc, and alpha-tubulin transcripts increased approximately 6- to 8-fold on the third day after castration, while transcripts encoding hsp 70-related genes increased greater than 6-fold on the fourth day after castration. Further analysis of RNA extracted from regressing ventral prostate glands at sequential 12-h intervals after castration showed that the c-fos gene was induced at 36 h, earlier than c-myc, alpha-tubulin, and hsp 70.(ABSTRACT TRUNCATED AT 250 WORDS)
Androgens↗
Enhanced expression of the c-myc protooncogene in high-grade human prostate cancers.
We examined a series of 29 surgical specimens of benign and malignant human prostate tissue for the expression of both the cHa-ras and c-myc protooncogenes. Northern blots were prepared using polyadenylated mRNA extracted from nine prostatic adenocarcinomas, 19 benign hypertrophied prostates (BPH) and one normal prostate. When the Northern blots were hybridized to a probe for cHa-ras, only one specimen of BPH showed an appreciable amount of the 1.2-kb transcript homologous to cHa-ras. Upon reprobing these blots with c-myc, six cancers showed a considerable amount of a 2.4-kb transcript homologous to c-myc. Three other cancers and all the benign tissue showed little or no detectable 2.4-kb c-myc transcript. On retrospective analysis, the cancers with elevated c-myc transcripts were found to have a Gleason score of 5 and above (poorly differentiated tumors), while the cancers with little or no c-myc transcripts were all of Gleason score 4 and lower. Finally, we compared our ability to detect c-myc transcripts in mRNA extracted from a surgically derived prostate tumor with mRNA extracted from the same tumor subject to a sham electrocautery procedure, as would occur during transurethral resection. The electrocautery procedure decreased both the intensity and the integrity of the c-myc signal in mRNA from the tumor. Thus, our exclusive use of surgically derived prostate tumors may be the reason we are able to detect an elevation in the expression of c-myc mRNA in high-grade tumors.
Adenocarcinoma↗
Prediction of transcriptional activity based on gene association with the nuclear matrix.
In this report, we show that androgen-dependent genes expressed in rat ventral prostate, C1, C2 and C3, are enriched in residual DNA extracted from the nuclear matrix of ventral prostate. The enrichment is androgen-dependent and is likely to be related to their transcriptional activity. While this finding corroborates other data demonstrating that transcriptionally active genes can be protected from nuclease digestion by their association with the nuclear matrix, we have extended this analysis to show that nuclear matrix protection experiments can be used both to analyze tissue-specific expression among members of a highly related gene family and also to predict transcriptional activity of genes in particular tissues.
Androgen-Binding Protein↗
Alterations of physical and biochemical parameters of the R3327-CP rat prostate adenocarcinoma following hormonal manipulation of the host.
Several physical and biochemical parameters of a rapidly growing, hormonally responsive, poorly differentiated strain of Dunning R3327 rat prostatic adenocarcinoma (the CP strain) were monitored for 1 month during growth in control and hormonally manipulated male Fischer X Copenhagen rats. The tumor was implanted into control rats and into rats 1 month following orchiectomy. Twenty-nine days following tumor implantation, 1 group of unoperated rats was orchiectomized while the rats implanted subsequent to orchiectomy were repleted with pharmacological doses of testosterone. At 2 and 4 weeks following treatment, half the original number of rats from each group were sacrificed and the growth rate (doubling time), per cent of aneuploid cells and androgen receptor levels (total, cytoplasmic and nuclear) were determined for each tumor. Orchiectomy increased tumor doubling time, while testosterone repletion decreased it, demonstrating the hormonal dependence of this tumor strain. Orchiectomy also decreased the levels of aneuploid cells in the tumor; however, repletion of testosterone to rats orchiectomized prior to implantation did not restore the aneuploid cell number to control levels. A sensitive indicator of the hormonal status of the tumor was the per cent of androgen receptors in the nucleus. Tumors grown in rats orchiectomized after implantation had the lowest percentage of androgen receptor in the nucleus while orchiectomized rats repleted with testosterone had the highest percentage. Comparison of the levels of androgen receptors in the tumors from the various groups (androgen receptor per gram of tissue) unexpectedly revealed that tumors grown in the orchiectomized rats had slightly higher total receptor levels than did control tumors, while the tumors of orchiectomized rats repleted with testosterone had lower amounts than did the control tumors. In contrast to these findings, the prostates of orchiectomized rats replenished with testosterone had higher levels of total androgen receptor than did the prostates of control rats.
Adenocarcinoma↗
Androgen receptor assays in advanced prostatic cancer.
Most technical obstacles in performing biochemical androgen receptor assays on malignant prostatic tissue have been surmounted. The available data, while suggesting that nuclear androgen receptor content in malignant tissues may be a useful criterion in this regard, remain meager and contradictory. The authors suggest that the clinical nature of prostatic cancer, including the tendency for metastases to occur in osseous and deep lymphatic regions and the heterogeneous intermingling of benign and malignant elements in the primary tumor, may preclude the success of biochemical assays for androgen receptors to predict hormonal dependency of prostatic tumors.
Binding Sites↗
Steroid receptor-nuclear matrix interactions. The role of DNA.
The interaction of sex steroid hormone receptors with the nuclear matrix (NM) of target and non-target tissue was investigated using a simple in vitro binding assay. Steroid receptors can recognize acceptor sites on the NM of target cells; androgen receptor binds with the highest apparent affinity to rat prostate NM; similarly estrogen receptor binds with the highest apparent affinity to uterine NM. Furthermore, the steroid receptor-NM interaction depends upon the hormonal status of the animal. The binding of androgen receptor to rat prostate NM was drastically reduced upon hormone withdrawal (castration) and fully recovered upon hormonal stimulation. When NM were prepared by an alternate method (DNase I digestion prior to high salt extraction) known to digest "active" chromatin, no preferential receptor binding to target tissue NM was observed. Although the NM fraction contains less than 1% of the total nuclear DNA, the matrix-associated DNA sequences seem to be, at least in part, responsible for specific receptor recognition. DNA extracted from the prostate NM was shown to be a potent competitor for androgen receptor binding as measured by DNA-cellulose competition experiments. Moreover, this DNA recognition also depends upon the hormonal status of the animal. These studies are consistent with the notion that hormonal manipulation induces changes in the NM-associated DNA sequences of steroid hormone target tissue.
Animals↗