Arnaldo Corti

Clusterin (SGP‐2, ApoJ) expression is downregulated in low‐ and high‐grade human prostate cancer

Clusterin is overexpressed during tissue and cell involution and downregulated in proliferating cells. Its role in cell survival, cell death and neoplastic transformation remains debated. We studied the expression and distribution of clusterin mRNA and protein in human prostate carcinoma (CaP) specimens of different degrees of malignancy. Fresh CaP specimens were obtained from 25 patients subjected to long‐term androgen ablation before surgery. Clusterin expression was studied by Northern and Western analysis, in situ hybridization and immunohistochemistry, in comparison with Gas1 and histone H3 mRNA (markers of cell quiescence and S phase of the cell cycle, respectively). Clusterin is downregulated in CaP in comparison with matched benign controls. In low‐grade CaP, clusterin colocalized with Gas1 to the stromal compartment, and in some glands, the basal lamina was heavily stained. In high‐grade CaP clusterin stained the remnants of stromal matrix while histone H3 localized to cancer cells, which were very rarely clusterin positive. High clusterin expression was found in the branches of a nerve infiltrated by tumor. The periglandular clusterin expression found in low‐grade CaP could result from induction of quiescence and/or apoptosis of prostatic fibroblasts lining those glands in which tumor invasion is at an initial stage, involving basal lamina. In advanced CaP, the staining of the remnants of the extracellular matrix suggests a role for clusterin in the process of dismantling the stromal organization caused by cancer progression.

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death.

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.

Clusterin (SGP-2) transient overexpression decreases proliferation rate of SV40-immortalized human prostate epithelial cells by slowing down cell cycle progression

Clusterin is a highly conserved, widely distributed glycoprotein whose biological significance is still debated. Involved in many biological processes and disease states, clusterin is induced by cell injury and tissue regression, but is repressed during cell proliferation. We have previously reported that clusterin mRNA induction is associated with epithelial cell atrophy in the rat prostate and both clusterin transcript and protein accumulated in quiescent normal human skin fibroblasts. Here we show that transient clusterin overexpression, in SV40-immortalized human prostate epithelial cells (PNT2), resulted in increased accumulation of cells in the G0/G1 phases of the cell cycle, accompanied by slowdown of cell cycle progression and decrease of DNA synthesis. The activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and the level of histone H3 mRNA (markers of cell proliferation) concomitantly decreased, while Gas1 mRNA (a marker of cell quiescence) accumulated. Thus it appears that clusterin, by opposing the effect of SV40 on the proliferation rate of PNT2 cells, acts as an anti-oncogene in the prostate, suggesting a role for this gene in controlling proliferation of normal and transformed prostate epithelial cells.

Paradoxical enhancement of S-adenosylmethionine decarboxylase in rat tissues following administration of the specific inhibitor methyl glyoxal bis(guanylhydrazone)

Abstract Treatment of rats with large but sublethal doses of methyl glyoxal bis(guanylhydrazone), a potent in vitro inhibitor of animal S-adenosylmethionine decarboxylases, causes marked increases in the enzyme activity of extracts of kidney, ventral prostate, and testis which had been extensively dialyzed to remove any remaining drug. One day after administration of the inhibitor to female rats, the renal S-adenosylmethionine decarboxylase activity was 12 times the normal level and remained greatly enhanced for a further 24 hr. As indicated by decline in decarboxylase activity following depression of protein biosynthesis by injection of cycloheximide, the apparent half-life of the kidney enzyme in normal female rats is roughly 2 hr; in contrast, the apparent half-life of the enzyme is elevated to a value of more than 20 hr in animals that were previously treated with methyl glyoxal bis(guanylhydrazone). The increased renal S-adenosylmethionine decarboxylase activity following administration of the specific enzyme inhibitor in vivo may thus be due, at least in part, to stabilization of the enzyme against intracellular inactivation as a result either of direct combination of the enzyme protein with the inhibitor, or with substance(s) in the tissue whose levels are influenced by treatment with methyl glyoxal bis(guanylhydrazone).

The gene for SP-40,40 human homolog of rat sulfated glycoprotein 2, rat clusterin, and rat testosterone-repressed prostate message 2, maps to chromosome 8

Sulfated glycoprotein 2 (SGP-2) is a rat glycoprotein that is particularly abundant in seminal fluid, where it is found associated with the acrosome and the tail of mature spermatozoa; for this reason it has been suggested that it has an important role in spermatogenesis. On the basis of nucleotide sequence homology, it has been proposed that the orthologous human gene is that coding for serum protein-40,40 (SP-40,40), a serum protein also called complement lysis inhibitor (CLI), SP-40,40 has been shown to act as a control mechanism of the complement cascade: in fact, it prevents the binding of a C5b-C7 complex to the membrane of the target cell and in this way inhibits complement-mediated cytolysis. SGP-2 and SP-40,40 seem then to be part of different biological systems. Furthermore it has been shown that another protein, testosterone-repressed prostate message 2 (TRPM-2), shares sequence homology with SGP-2 and SP-40,40. TRPM-2 is expressed at high levels and in a temporally precisely defined manner in dying cells, an observation that would suggest its involvement in the cascade of events leading to cell death. We have used a large panel of 24 mouse/human hybrid cell lines and a cDNA for SGP-2, which is also highly homologous to that for rat clusterin, to map the chromosomal location of the orthologous human gene. The mapping data and the Southern analysis presented in this paper, in addition to the data available from the literature, strongly suggest that in the human genome there is a single locus homologous to the probe used and that it codes for the protein which has been called, in different species, SP-40,40, SGP-2, clusterin, and TRPM-2. The chromosomal mapping of the locus for this multiname protein should facilitate its cloning and a better understanding of the apparently many biological functions of its product.

Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosyn‐thetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.

Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts

In human dermal fibroblasts, brought to quiescence (G0) by serum starvation, the S phase peaked 24 h and G2/M phases 36 h after serum re‐addition. Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h. Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater. S‐Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter. Spermidine/spermine N 1‐acetyltransferase mRNA and activity reached the highest values at 36–48 h. Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine. We discuss how this comprehensive study may help to understand the involvement of polyamines in the control of cell proliferation.

Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

Ca 2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR

Background Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP. Methodology/Principal Findings The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (80±5)% accuracy, (81±6)% sensitivity and (78±7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus ≥7, an important predictor of final outcome. Conclusions/Significance The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the “cancer field effect”, in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method posses the potential to enhance conventional diagnosis.

Studies on the relationship between cell proliferation and cell death: Opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in pha-stimulated human lymphocytes

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.