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PubMed1998

Clusterin protects against oxidative stress in vitro through aggregative and nonaggregative properties.

Perturbations of cell interactions, an early event in acute renal injury, have important pathophysiologic consequences. We hypothesized that promotion of cell interactions protects cells from injury. To test this hypothesis, a single cell suspension of LLC-PK1 cells (porcine proximal tubular cell line) treated with albumin (control) was compared to cells aggregated with fibrinogen or purified human clusterin (aggregation graded 0 to 4). Following aggregation, the cells were injured with 1.5 mM hydrogen peroxide (H2O2) for three hours. Cell aggregation induced by clusterin but not fibrinogen protected against oxidant injury by H2O2. Complete abrogation of cytotoxicity occurred at a clusterin concentration of 2.5 micrograms/ml, which resulted in an aggregation score of 1. In the absence of aggregation, clusterin at concentrations of 20 and 50 micrograms/ml, but not lower doses, partially protected against injury induced by H2O2. Cell aggregation induced by both clusterin and fibrinogen partially protected against endogenously generated oxidant stress induced by incubating LLC-PK1 cells with aminotriazole and 1-chloro-2,4-dinitrobenzene (CDNB). In conclusion, clusterin protects against models of oxidant stress in vitro, whether generated by exogenously administered hydrogen peroxide, or from endogenously produced peroxide, and such protective effects can accrue from aggregative and nonaggregative properties of clusterin.

Amitrole↗
PubMed1997

Regulation of clusterin gene expression by transforming growth factor beta.

Transforming growth factor beta (TGFbeta) induces the expression of a wide variety of genes in many cell types. Our previous studies have shown that TGFbeta stimulates both clusterin mRNA and protein levels, and induces its accumulation in the nucleus of CCL64 cells. To further investigate the molecular mechanism of clusterin mRNA induction by TGFbeta, we created a 1.3-kilobase rat clusterin promoter/luciferase reporter construct. We demonstrate that TGFbeta enhances luciferase activity 2.5-6-fold in transient transfection assays of epithelial, endothelial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5'-TGAGTCA) in the minimal promoter region is necessary for initiating transactivation by TGFbeta. A single T to G base mutation in the AP-1 site (5'-TGAGGCA) abolishes TGFbeta-induced clusterin promoter transactivation. In transcription factor decoy experiments, 23-mer oligonucleotides of wild type AP-1 reduce TGFbeta induction of clusterin mRNA levels and promoter transactivation, while an oligonucleotide containing the mutated AP-1 site has no effect. Two specific protein kinase C inhibitors, GF109203X and calphostin C, block TGFbeta-induced clusterin mRNA levels and promoter transactivation. Together these results indicate that TGFbeta regulates clusterin gene expression through an AP-1 site and its cognate transcription factor AP-1, and requires the involvement of protein kinase C.

Animals↗
PubMed2006

Differential expression of serum clusterin isoforms in colorectal cancer.

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

Biomarkers, Tumor↗
PubMed1996

Expression and localization of clusterin mRNA in the small and large intestine of the irradiated rat: its relationship with apoptosis.

Apoptotic elimination of intestinal cells following irradiation has been studied in the small and large intestine of the rat, and correlated with the level of expression and localization of clusterin mRNA. Clusterin was moderately expressed in normal intestine where only small levels of apoptosis were found. After irradiation, however, there was a temporal correlation between an increased apoptotic index and increased clusterin expression. Localization of clusterin mRNA by in situ hybridization identified extensive labelling in the lower part (Paneth cell region) of small intestinal crypt, whereas epithelial cells in the large intestine were diffusely labelled. Clusterin expression was not localized over apoptotic cells and its role may be as a cell protection factor for surviving cells, as had been suggested by others. Clusterin may also be involved in remodelling of the intestinal crypt after radiation damage, a process that includes altering cell-to-cell contact, apoptosis, and sloughing of the dead cells from the intestinal villi. Our results do show a close temporal link between apoptosis and clusterin expression, and, as such, expression of the gene may be a useful indicator of presence of apoptosis in the irradiated intestine.

Animals↗
PubMed2005

A phase I pharmacokinetic and pharmacodynamic study of OGX-011, a 2'-methoxyethyl antisense oligonucleotide to clusterin, in patients with localized prostate cancer.

BACKGROUND: Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA and has been reported to inhibit clusterin expression and enhance drug efficacy in xenograft models. The primary objective of this clinical study was to determine a biologically effective dose of OGX-011 that would inhibit clusterin expression in human cancer. METHODS: Subjects (n = 25) with localized prostate cancer with high-risk features who were candidates for prostatectomy were treated with OGX-011 by 2-hour intravenous infusion on days 1, 3, and 5 and then weekly from days 8-29 combined with androgen blockade starting on day 1; prostatectomy was performed on days 30-36. Six different doses were tested, from 40 to 640 mg. OGX-011 plasma and prostate tissue concentrations were measured by an enzyme-linked immunosorbent assay method, and the pharmacokinetics of OGX-011 were determined from these data. Prostate cancer tissue, lymph nodes, and serial samples of peripheral blood mononuclear cells were assessed for clusterin expression using quantitative real-time polymerase chain reaction and immunohistochemistry. All statistical tests were two-sided. RESULTS: Only grade 1 and 2 toxicities were observed. The plasma half-life of OGX-011 was approximately 2-3 hours, and the area under the concentration versus time curve and CMAX (peak plasma concentration) increased proportionally with dose (Ptrend < .001). OGX-011 in prostate tissue increased with dose (Ptrend < .001). Dose-dependent decreases in prostate cancer and lymph node clusterin expression were observed by polymerase chain reaction of greater than 90% (Ptrend = .008 and Ptrend < .001, respectively) and by immunohistochemistry (Ptrend < .001 and Ptrend = .01, respectively). CONCLUSIONS: OGX-011 is well tolerated and reduces clusterin expression in primary prostate tumors. The optimal biologic dose for OGX-011 at the schedule used is 640 mg.

Aged↗
PubMed1987

Biosynthesis and secretion of clusterin by ram rete testis cell-enriched preparations in culture.

Rabbit polyclonal antibodies, directed specifically against clusterin purified from ram rete testis fluid, were employed in an investigation of the biosynthesis of clusterin by cultures of rete testis epithelial cells and by Sertoli cells prepared from testes of adult rams. Cells in serum-free medium were incubated in the presence of either [35S]methionine, [3H]leucine, or [3H]glucosamine, and radiolabeled proteins secreted were immunoprecipitated. The pellet was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the gels were then fluorographed. In other experiments, protein bands were transferred to nitrocellulose and visualized immunochemically. Under non-reducing conditions, a single band was detected, having a molecular weight of 80,000. Under reducing conditions, doublet bands were detected, having approximate molecular weights of 40,000 (major band) and 37,000 (minor band). These properties were indistinguishable from those obtained with authentic samples of pure clusterin subjected to gel electrophoresis and Western immunoblot procedures. Amounts of clusterin synthesized by rete testis cells in culture, quantitatively determined with a sandwich enzyme-linked immunosorbent assay procedure, were approximately 4 micrograms/micrograms cell DNA/48 h. Immunocytochemical localization investigations, using monoclonal antibodies against clusterin, revealed the presence of clusterin in the perinuclear of juxtanuclear regions, in both rete testis epithelial cells and Sertoli cells in culture. The possible functions of clusterin produced by rete testis epithelial cells and by Sertoli cells are discussed.

Animals↗
PubMed2004

Clusterin expression distinguishes follicular dendritic cell tumors from other dendritic cell neoplasms: report of a novel follicular dendritic cell marker and clinicopathologic data on 12 additional follicular dendritic cell tumors and 6 additional interdigitating dendritic cell tumors.

While tumors of dendritic cell lineage may have overlapping histomorphologic features, most but not all cases can be classified using an immunohistochemical panel, including CD21, CD23, CD35, CD1a, and S-100. Based on observations that clusterin is expressed in benign follicular dendritic cells, clusterin expression in 32 dendritic cell tumors was evaluated. Diffuse strong staining for clusterin was seen in 12 of 12 follicular dendritic cell tumors. Two of these cases were negative for traditional markers (CD21, CD23, CD35); they were classified based on characteristic ultrastructural features. Three of 6 interdigitating dendritic cell tumors were negative for clusterin and 3 showed focal weak positivity. Clusterin staining in Langerhans cell histiocytosis ranged from negative (6 of 14) to weak/moderate (8 of 14). Follicular dendritic cell tumors behaved as benign tumors or low-grade sarcomas. Interdigitating dendritic cell tumors demonstrated a widely variable behavior, ranging from benign to rapidly fatal disease. Based on this initial study, strong clusterin staining supports a diagnosis of follicular dendritic cell tumor. Thus, staining for clusterin is useful in classification of dendritic cell tumors, particularly when the more common markers of follicular dendritic cells are not expressed.

Adolescent↗
PubMed2006

Clusterin enhances proliferation of primary astrocytes through extracellular signal-regulated kinase activation.

Clusterin, a secretory glycoprotein, has been shown to be up-regulated in the reactive astrocytes in response to brain injury and neurodegenerative diseases, but its function has not been clearly elucidated. In this study, we investigate whether clusterin has growth-stimulatory activity in astrocytes. Suppression of clusterin with antisense oligonucleotide induced growth arrest, whereas transient overexpression of clusterin by cDNA transfection or exogenous treatment with purified clusterin promoted proliferation of the primary astrocytes in culture. This clusterin-stimulated proliferation was abrogated by PD98059, an inhibitor of mitogen-activated protein kinase kinase. These results suggest that clusterin might play an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway.

Animals↗
PubMed2001

Introduction of clusterin gene into human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy through inhibition of apoptosis both in vitro and in vivo.

Recent studies have revealed the powerful antiapoptotic activity of clusterin in various malignant tumors; however, the significance of clusterin expression in the acquisition of a resistant phenotype against several kinds of treatment in human renal cell carcinoma (RCC) has not been well characterized. We, therefore, transfected the clusterin cDNA into RCC ACHN cells, that scarcely express clusterin protein, to examine whether overexpression of clusterin inhibits chemotherapy-induced apoptosis both in vitro and in vivo. Although no significant differences were observed in the in vitro growth rates between clusterin-transfected ACHN (ACHN/CL) and the vector only-transfected cell line (ACHN/Co), ACHN/CL exhibited high resistance to cisplatin treatment compared with ACHN/Co, with a greater than 5-fold higher IC(50) through the inhibition of apoptotic cell death, which was demonstrated by DNA fragmentation analysis and western blotting of PARP protein. Moreover, intravenous administration of cisplatin into athymic nude mice bearing ACHN/CL tumors resulted in 2- to 3-times faster tumor growth compared with ACHN/Co tumors. These findings suggest that clusterin overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis in human RCC cells.

Animals↗
PubMed2005

Adult motor neurons show increased susceptibility to axotomy-induced death in mice lacking clusterin.

Clusterin is a highly conserved, amphiphatic glycoprotein present in most tissues. It has been shown to be involved in the regulation of lipid transportation, clearance of cellular debris from the extracellular space and intracellular signal transduction. Clusterin is markedly up-regulated after neural injury but the functional significance of this response is unclear. Here, we show that clusterin up-regulation is substantially greater in hypoglossal motor neurons after hypoglossal nerve avulsion compared with nerve transection. Quantitative analyses of motor neuron numbers after the same lesions in clusterin(-/-) and clusterin(+/+) mice showed significantly larger numbers of surviving motor neurons in clusterin(+/+) mice. These results suggest that clusterin has a neuroprotective role after axotomy.

Animals↗
PubMed1996

Serum clusterin and vitronectin in alcoholic cirrhosis.

Clusterin and vitronectin are multifunctional regulatory proteins which both serve as complement lysis inhibitors. Previous data have strongly suggested that serum vitronectin is mainly produced in the liver, whereas the biosynthetic origin for serum clusterin has not been determined. In the present study we aimed to determine the role of the liver in producing these proteins and to evaluate the proteins as possible markers of liver failure. We therefore quantified clusterin and vitronectin in serum from patients suffering from alcoholic liver cirrhosis (n = 83), and in serum-free culture supernatants from the hepatoma cell line HepG2. The median clusterin concentration was 0.20 g/l in cirrhosis and 0.37 g/l in the controls, whereas corresponding vitronectin values were 0.19 and 0.26 g/l, respectively. The concentration of both proteins showed significant correlation (p < 0.0001) with disease severity and with established plasma markers of hepatic synthetic function, such as albumin and prothrombin complex. The clusterin level, but not the vitronectin level, correlated with survival (p = 0.005). The rates of synthesis of clusterin, vitronectin and C3 from HepG2 cells were 0.02, 0.21 and 1.9 micrograms/10(6) cells/24 h, respectively. From the present data we conclude that clusterin (as vitronectin and C3) is mainly produced in the liver and may be a useful marker in the evaluation of severity of liver disease and prognosis of patients with alcoholic cirrhosis.

Adult↗
PubMed1984

Isoelectric forms of clusterin isolated from ram rete testis fluid and from secretions of primary cultures of ram and rat Sertoli-cell-enriched preparations.

Clusterin, a cell aggregating factor isolated from ram rete testis fluid (RTF), is shown to contain 14.7% hexoses, 13.6% glucosamine, and 7.9% sialic acid. The isoelectric point (pI) of the predominant electrophoretic form of clusterin from ram RTF is 3.7. After treatment with neuraminidase, the pI values become more basic, with the majority of the material being eluted from a chromatofocusing column at pH values between 4.9 and 5.1. Intact clusterin binds quantitatively to wheat germ agglutinin - Sepharose 6 MB, but after treatment with neuraminidase only 49% specifically binds. Clusterin isolated from proteins secreted by primary cultures of ram Sertoli-cell-enriched preparations was shown to have properties similar to those of intact clusterin isolated from ram RTF. In contrast, clusterin isolated from proteins secreted by primary cultures of rat Sertoli- or granulosa-cell-enriched preparations has isoelectric forms which more closely resemble those of neuraminidase-treated ram clusterin.

Animals↗
PubMed2002

Clusterin protects renal tubular epithelial cells from gentamicin-mediated cytotoxicity.

Clusterin is a heterodimeric secreted glycoprotein that is upregulated after acute renal injury. In aminoglycoside nephrotoxicity, clusterin is induced in the tubular epithelium and increased levels are found in the urine. In this study, we developed an in vitro model of gentamicin-induced cytotoxicity in renal proximal tubule cells and tested whether clusterin protected these cells from injury. LLC-PK(1) cells were incubated with varying concentrations of gentamicin in serum-free media, and cytotoxicity was quantified by lactate dehydrogenase release and confirmed by vital dye exclusion. A dose-dependent increase in cytotoxicity occurred with gentamicin concentrations up to 27 mg/ml. Clusterin decreased cytotoxicity in a dose- and time-dependent manner at 6, 12, and 24 h, whereas albumin, used as a control protein, had no effect. In contrast to the aminoglycoside model, when cells were injured by depletion of ATP, clusterin had only a minimally protective effect. LLC-PK(1) cells did not express megalin, a receptor that can mediate the uptake of both clusterin and aminoglycosides into proximal tubule cells. Uptake of gentamicin into LLC-PK(1) cells was observed despite the absence of megalin. In conclusion, clusterin specifically protects against gentamicin-induced renal tubular cell injury by a megalin-independent mechanism.

Adenosine Triphosphate↗
PubMed2006

Expression of the antiapoptotic proteins clusterin and bcl-2 in laryngeal squamous cell carcinomas.

Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02-0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors.

Apoptosis↗
PubMed2006

Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke.

RATIONALE: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. OBJECTIVES: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. METHODS: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. CONCLUSIONS: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.

Clusterin↗
PubMed1996

Multiple signal transduction pathways regulate clusterin (gp 80) gene expression in MDCK cells.

Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell-cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Clusterin is expressed in the anterior and intermediate lobes, but not in the posterior pituitary of sheep.

We have examined the expression of the ovine clusterin gene in the sheep pituitary gland, with the aim of determining its site of synthesis in this tissue. Northern blotting analysis of extracted polyadenylated RNA, using a (32)P-labelled rat clusterin cDNA probe, detected the greatest amounts of clusterin mRNA in the anterior part of dissected pituitary glands. In situ hybridisation studies showed clusterin mRNA in anterior and intermediate pituitary cells, with lower amounts in vascular endothelium and posterior pituicytes. Clusterin protein, detected by immunohistochemistry, was observed in some single secretory cells, within the capillary lumen and in cells around capillaries in the anterior and intermediate lobes, but no immunoreactivity was observed in posterior pituitary tissue. The pattern of clusterin expression in anterior and intermediate pituitary cells suggests possible roles for the protein in secretory cell turnover and/or hormone secretion or lipid uptake. Clusterin does not appear to be involved in ovine posterior pituitary hormone neurosecretion.

Animals↗
PubMed2004

Alterations in expression, proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma.

AIM: To investigate biogenesis and intracellular localizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa. METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular localizations of clusterin were carried out in both tissues and cell lines of ESCC. RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform, were the major alterations in cancer cells of esophagus. Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia. CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

Carcinoma, Squamous Cell↗