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PubMed1992

Clusterin binds by a multivalent mechanism to the Fc and Fab regions of IgG.

Clusterin was purified from human serum by IgG and monoclonal antibody affinity chromatography. SDS-PAGE and immunoblotting revealed no major differences between clusterin prepared in these two ways. An ELISA method for measuring the binding of clusterin to immunoglobulins was developed. Clusterin purified by IgG affinity chromatography bound to pooled human IgG with a similar affinity (S0.5 5.9 +/- 0.4 micrograms/ml) as clusterin purified by monoclonal antibody chromatography (S0.5 6.1 +/- 0.2 micrograms/ml). The apparent affinity of clusterin for IgG immobilised on ELISA plates increased with increasing concentrations of IgG in the coating solution. Aggregated IgG in solution was a more potent inhibitor of the binding of clusterin to immobilised IgG than was monomer IgG. Clusterin bound to all of the isotypes of human IgG, and to human IgA and IgM, with apparent affinities in the order IgG3 > IgG4 > IgM > IgG1 > IgG2, IgA. Clusterin bound to both the Fab and Fc fragments of human IgG. The clusterin binding site(s) on the Fc do not overlap with those for protein A and Clq.

Antibodies, Monoclonal↗
PubMed1996

Induction of clusterin in the immature brain following a hypoxic-ischemic injury.

A unilateral hypoxic-ischemic (HI) insult in the 21 day old rat has been used to assess the role of clusterin in nerve cell death. Both clusterin mRNA and protein levels were measured at various time points after moderate (15 min) and severe (60 min) HI insult using in situ hybridisation and immunocytochemistry respectively. The severe HI insult lead primarily to necrotic neuronal death and showed very little if any clusterin mRNA and protein induction on the ligated side of the brain. However, following the moderate HI insult there was a dramatic time-dependent accumulation of clusterin protein in neurons of the CA1-CA2 pyramidal cell layers in the hippocampus and cortical layers 3-5, regions undergoing delayed neuronal death. Clusterin mRNA expression, in contrast to neuronal protein accumulation, appeared to be glial in origin (probably astrocytes) with increases in mRNA in and around the hippocampal fissure and only a weak signal over the CA1-CA2 pyramidal cell layer. These results support the hypothesis that the clusterin protein is synthesised in the astrocytes, secreted and then taken up by dying neurons. Clusterin immunoreactivity and in situ DNA end-labelling performed on the same sections revealed that clusterin was accumulating in neurons destined to die by programmed cell death. However the relative time-courses of DNA fragmentation and clusterin immunoreactivity suggest that clusterin production was a result of the selective delayed neuronal death rather than being involved in the biochemical cascade of events that cause it.

Animals↗
PubMed1994

Selective expression of clusterin (SGP-2) and complement C1qB and C4 during responses to neurotoxins in vivo and in vitro.

This study concerns expression of the genes encoding three multifunctional proteins: clusterin and two complement cascade components, C1q and C4. Previous work from this and other laboratories has established that clusterin, Clq and C4 messenger RNAs are elevated during Alzheimer's disease, and in response to deafferenting and excitotoxic brain lesion. This study addresses hippocampal clusterin, ClqB and C4 expression in response to neurotoxins that caused selective neuron death. Kainate, which preferentially kills hippocampal CA3 pyramidal neurons but not dentate gyrus granule neurons induced clusterin immunoreactivity in CA1 and CA3 pyramidal neurons and adjacent astrocytes, but not in dentate gyrus granule neurons. In contrast, colchicine, which preferentially kills the dentate gyrus granule neurons, induced clusterin immunoreactivity in the local neuropil as punctate deposits, but not in the surviving or degenerating dentate gyrus granule neurons. Clusterin messenger RNA was increased in astrocytes. ClqB and C4 messenger RNAs increased within 48 h after kainate injections, particularly in the CA3 pyramidal layer, less in the dentate gyrus-CA4, and less in CA1. Clq immunoreactivity was detected in CA1 pyramidal neurons and also as small punctate deposits in the CA1 region at eight and 14 days after kainate. The increase of both clusterin and ClqB messenger RNAs after kainate injections was blocked by barbiturates that prevented seizures and neurodegeneration. In primary hippocampal neuronal cultures treated with glutamate, a subpopulation of cultured neurons that survived glutamate toxicity also had parallel elevations of clusterin and ClqB messenger RNA. In conclusion, cytotoxins that target selective hippocampal neurons increase the expression of both clusterin and ClqB in vivo and in vitro. These results show that elevations of clusterin messenger RNA or protein can be dissociated from each other and from cell death. These increased messenger RNAs were associated with immunoreactive deposits that differed by cell type and intra- versus extracellular locations. These results suggest that the complement system is involved in brain responses to injury.

Animals↗
PubMed2004

Enhanced chemosensitivity of bladder cancer cells to cisplatin by suppression of clusterin in vitro.

We examined the functional role of clusterin in chemotherapy-induced apoptosis and tested whether anti-sense transfection targeted against clusterin enhances the chemosensitivity in human bladder cancer cells in vitro. Clusterin mRNA and protein expression of 253J cells, a human bladder carcinoma cell line, after treatment with cisplatin were measured by RT-PCR and Western blot analysis. Clusterin expression and cell growth were compared between 253J cells transfected with constructed a clusterin anti-sense plasmid vector (pCR-CLU-AS) and controls. Tumor cell viability was measured with MTT assay after cisplatin treatment. DNA fragmentation and CPP32 assay were performed. Clusterin expression was increased after treatment with cisplatin and highest at 8 h in 253J cells. Clusterin anti-sense transfectants were highly sensitive to apoptotic cell death induced by cisplatin compared with parental 253J cells or control transfectants. Collectively, our results showed that expression of clusterin was increased in the acute phase of cell death caused by cisplatin and that suppressing the expression of clusterin enhanced the susceptibility of apoptosis caused by cisplatin in human bladder cancer cells. These results suggest that lowering the expression of clusterin might increase the sensitivity of bladder cancer cells to chemotherapeutic agents.

Antineoplastic Agents↗
PubMed2006

Expression of clusterin in prostate cancer correlates with Gleason score but not with prognosis in patients undergoing radical prostatectomy without neoadjuvant hormonal therapy.

OBJECTIVES: To determine whether the expression level of clusterin in prostate cancer could be used as a prognostic predictor in patients who have undergone radical prostatectomy (RP). METHODS: This study included 172 consecutive patients undergoing RP for clinically organ-confined prostate cancer without neoadjuvant hormonal therapy. Immunohistochemical staining was performed in RP specimens obtained from these patients to evaluate the expression level of clusterin protein. The cell proliferative activities and apoptotic features in these specimens were investigated using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, respectively. RESULTS: Varying levels of clusterin expression were noted in 169 of 172 prostate cancer specimens; 32 of the 172 normal prostatic tissue specimens did not exhibit any clusterin staining. Clusterin expression in prostate cancer tissue was significantly related to the Gleason score, but not to the other parameters, including age, serum prostate-specific antigen level, pathologic stage, perineural invasion, tumor volume, and lymph node metastasis. In addition, cell proliferative activity in the prostate cancer specimens was significantly associated with clusterin expression; however, no correlation was found between the apoptotic index and clusterin expression. In this series, 34 (19.8%) of 172 patients developed biochemical recurrence. No significant difference was found in biochemical/recurrence-free survival between patients with strong clusterin expression and those with weak expression. CONCLUSIONS: Despite its detection in most prostate cancer tissue, clusterin expression failed to show a significant association with prognosis in patients undergoing RP without neoadjuvant hormonal therapy. This suggests a limited role for clusterin in the progression of clinically organ-confined prostate cancer in the absence of proapoptotic stimuli.

Aged↗
PubMed1998

Deleterious actions of chronic ethanol treatment on the glycosylation of rat brain clusterin.

Clusterin is a N-glycosylated sialoglycoprotein present in rat brain cells. Clusterin, which elicits aggregation in a wide variety of cells, has been suggested to play an important role in synaptic remodeling through its cell adhesion property or lipid transport capacity in the brain. Sialic acid residues in clusterin may be responsible for its structural conformation, stability and functional ability. Maturation of clusterin is governed by the relative actions of sialyltransferases and sialidases that are present in brain microsomes, golgi bodies, cytosol and plasma membranes. We have earlier reported that chronic ethanol treatment in rats has a damaging effect on the hepatic glycosylation machinery. Others have reported increased hydrolysis of brain sialoconjugates in rats following chronic ethanol administration. Specificity of the effects of chronic ethanol treatment in the brain in relation to the glycosylation process, is still obscure. Therefore, in this investigation, we have studied the specific effects of chronic ethanol treatment on the glycosylation of rat brain clusterin and the causes that may lead to any possible defects in the glycosylation process. We have determined the effects of chronic ethanol treatment on (i) the incorporation of labeled leucine and N-acetylmannosamine into immunoprecipitable clusterin in whole brain homogenate, microsomes, golgi, cytosol, plasma membrane and synaptosomes, (ii) enzymatic activities of sialyltransferases in golgi and synaptosomes, and sialidase in brain cytosol and plasma membranes, and (iii) de novo synthetic rate of rat brain cytosolic sialidase. Our results showed that chronic ethanol treatment in rats resulted in (1) a decreased sialation index of brain clusterin by 47. 2% (p<0.001), 56.7% (p<0.05), 51.7% (p<0.05), 64.8% (p<0.001), and 54.5% (p<0.05), respectively, in whole brain homogenate, golgi, cytosol, plasma membranes, and synaptosomes; (2) a 46.1% (p<0.05) and 12.5% (p<0.05) decreased activities of brain sialyltransferases, respectively, in the golgi and the synaptosomal fractions; (3) a 70. 1% (p<0.05) and 42.6% (p<0.05) increased activities of sialidases, respectively, in the cytosol and plasma membrane fractions; and (4) a 22.2%-64.3% (p<0.001) increased incorporation of labeled leucine into brain cytosolic sialidase. Our findings have clearly established that long-term ethanol treatment in rats leads to a marked impairment in the glycosylation of rat brain clusterin as a result of altered activities of brain sialation and desialation enzymes. In particular, the specific increase noted in brain sialidase activity was due to concomitant increases in its synthetic rate. These defects in the glycosylation of brain clusterin may lead to changes in the molecular conformation of clusterin, and thus, may result in its structural instability and/or functional impairment.

Alcoholism↗
PubMed2002

Over expression of clusterin is an independent prognostic factor for nonpapillary renal cell carcinoma.

PURPOSE: Recent studies have revealed the powerful anti-apoptotic activity of clusterin in several types of malignant tumors. However, the significance of clusterin expression in human renal cell carcinoma has not been well characterized. We determined whether the expression level of clusterin in nonpapillary renal cell carcinoma may be used as predictor of tumor progression and prognosis. MATERIALS AND METHODS: Total RNA was extracted from 93 nonpapillary renal cell carcinomas obtained at surgery and expression levels of clusterin messenger (m)RNA in these specimens were measured by Northern blot analysis. Findings were analyzed with respect to several clinicopathological factors. RESULTS: There were 48 cases (52%) with strong clusterin mRNA expression and 45 (48%) in which it was weak or nonexistent. The expression level of clusterin mRNA did not significantly correlate with patient gender, age, tumor grade or size, or histological cell type, whereas pathological stage and the incidence of tumor recurrence were closely associated with clusterin expression (p <0.005 and <0.01, respectively). The recurrence-free and overall survival rates in patients with strong clusterin expression were significantly lower than in those with weak or no expression (p <0.05). Furthermore, multivariate analyses revealed that strong expression of clusterin was an independent predictor of tumor recurrence and overall survival (p <0.05). CONCLUSIONS: These findings suggest that the over expression of clusterin may be a useful prognostic parameter in patients with nonpapillary renal cell carcinoma after radical nephrectomy.

Adult↗
PubMed2002

Overexpression of clusterin in transitional cell carcinoma of the bladder is related to disease progression and recurrence.

OBJECTIVES: To determine whether the expression level of clusterin in transitional cell carcinoma (TCC) of the bladder could be used as a predictor of tumor recurrence and prognosis. METHODS: Total RNA samples were extracted from 89 specimens of TCC of the bladder, and the expression level of clusterin mRNA in these specimens was measured by Northern blot analysis. The results were evaluated with respect to several clinicopathologic factors. RESULTS: The mean level of clusterin mRNA expression in invasive TCC of the bladder was fourfold and fivefold higher than that in superficial TCC and normal urothelial tissue, respectively. The expression level of clusterin mRNA showed no significant correlation with sex, age, tumor size, or multiplicity, and the pathologic stage and tumor grade showed close associations with clusterin expression. The overall survival rate of patients with strong clusterin expression was significantly lower than that of patients with weak expression. Among the 43 patients with invasive TCC who underwent complete resection, the recurrence-free survival rate of patients with strong clusterin expression was significantly lower than that of patients with weak expression. Moreover, multivariate analyses indicated that among these 43 patients, strong expression of clusterin was an independent predictor of tumor recurrence. CONCLUSIONS. These findings indicate that clusterin mRNA is strongly expressed in invasive TCC of the bladder compared with the expression in superficial TCC and that strong clusterin expression could be used as a novel predictor of prognosis of patients with TCC of the bladder.

Adult↗
PubMed2001

Clusterin, a binding protein with a molten globule-like region.

Clusterin is a heterodimeric glycoprotein found in many tissues of the body and is the most abundant protein secreted by cultured rat Sertoli cells. The function of clusterin is unknown, but it has been associated with cellular injury, lipid transport, apoptosis, and it may be involved in the clearance of cellular debris caused by cell injury or death. Consistent with this last idea, clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate (ANS). Given this variety of ligands, clusterin must have specific structural features that provide the protein with its promiscuous binding activity. Using sequence analyses, we show that clusterin likely contains three long regions of natively disordered or molten globule-like structures containing putative amphipathic alpha-helices. These disordered regions were highly sensitive to trypsin digestion, indicating a flexible nature. The effects of denaturation on the fluorescence of the clusterin-ANS complex were compared between proteins with structured binding pockets and molten globular forms of proteins. Clusterin bound ANS in a manner that was very similar to that of molten globular proteins. Furthermore, we found that, when bound to ANS, at least one cleavage site within the protease-sensitive disordered regions of clusterin was protected from trypsin digestion. In addition, we show that clusterin can function as a biological detergent that can solubilize bacteriorhodopsin. We propose that natively disordered regions with amphipathic helices form a dynamic, molten globule-like binding site and provide clusterin the ability to bind to a variety of molecules.

Amino Acid Sequence↗
PubMed1998

Characterization of the heparin-binding properties of human clusterin.

Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation. Both gave dose-dependent increases in ELISA signal with increasing concentrations of biotinylated heparin, with the latter giving signals an order of magnitude greater than the former. There was a dose-dependent increase in the ELISA signal from bound biotinylated heparin with increasing concentrations of plate-bound clusterin. The apparent affinity constant for binding of biotinylated heparin to plate-bound clusterin at pH 6.0 was estimated as 0.06 +/- 0.02 microM. Unlabeled heparin blocked the binding of biotinylated heparin to clusterin over a concentration range similar to that of the binding of biotinylated heparin to plate-bound clusterin. The binding of biotinylated heparin to clusterin was independent of the presence or absence of Ca2+. The binding of biotinylated heparin to plate-bound clusterin increased with decreasing pH over the range 5.5-8.0 and was characterized by an apparent pKa of 6.9. Clusterin in human serum bound to heparin-Sepharose at pH 6.0 but not at pH 7.4. Dot-blot experiments showed that one of the polypeptide chains of clusterin which had been reduced and alkylated under denaturing conditions bound to heparin-Sepharose. This chain was identified as the alpha chain from its N-terminal amino acid sequence.

Biotin↗
PubMed1995

Clusterin expression and apoptosis in tissue remodeling associated with renal regeneration.

To analyze the role of clusterin in renal diseases involving a regenerative process, we have used a novel rodent model to compare temporal and spatial expression of clusterin mRNA. Thus, renal artery stenosis was used to induce unilateral non-infarctive renal atrophy. After several weeks, when cellular pathology of atrophic kidneys involved minimal apoptosis or inflammatory response and mitosis was at normal levels, regeneration of atrophic kidneys was stimulated by removal of the contralateral healthy kidneys. The regrowth response was very rapid and involved renal hyperplasia rather than hypertrophy. Regenerating kidneys were studied 0, 4, 8, 24 hours and 2, 3, 5, 7, and 14 days after contralateral nephrectomy. Several parameters were compared: level and localization of clusterin mRNA; cell proliferation; cell dedifferentiation and redifferentiation and apoptosis. During the acute regenerative phase (first 24 hr) clusterin expression was markedly increased, decreasing to untraceable levels by five days of regeneration. Clusterin mRNA was localized in dilated or collapsed atrophic tubules that had lost identifying surface structures of normal tubular epithelium (termed dedifferentiated). Clusterin was also localized in the periphery of some blood vessel walls. Cell proliferation peaked at three to five days of regeneration, and was also localized in dedifferentiated tubules. Despite the regenerative stimulus, an unexpected result was a transient but marked increase in apoptotic cell death in atrophic tubules in the first 24 hours of regeneration. Our results provide evidence of a temporal association between increased clusterin expression and apoptosis, but in situ localization showed clusterin mRNA over apparently viable, as well as apoptotic, cells in the epithelium of tubules showing clusterin expression. Clusterin mRNA was rarely identified over epithelial cells in foci of non-atrophic (non-dedifferentiated) nephrons that responded to the regenerative stimulus by cellular hypertrophy. The dramatic response after initiation of regeneration, especially the initiation of apoptosis in the tubular epithelium, may have applications for the study of genetic changes leading to renal oncogenesis.

Animals↗
PubMed2003

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model.

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.

Apoptosis↗
PubMed1996

Clusterin (Apo J) protects against in vitro amyloid-beta (1-40) neurotoxicity.

Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta. Given this interaction with A beta, the goal of this study was to determine whether clusterin could modulate A beta neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against A beta (1-40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A beta (1-40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-beta plaque pathology.

Amyloid beta-Peptides↗
PubMed1993

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Amino Acid Sequence↗
PubMed1994

Immunohistological localization of clusterin in the male genital tract in humans and marmosets.

Clusterin is a multifunctional protein, first described in the reproductive tracts of the rat and the ram. It is produced by several cell types and exists in at least two differentially glycosylated forms. The aim of this study was to extend knowledge of clusterin expression in the primate (human and marmoset) male reproductive tracts by means of clusterin-specific immunohistochemical techniques. In both normal and abnormal testicular tissue, clusterin was found in association with Sertoli cells, lumenal sperm, proacrosomal Golgi complexes, residual bodies, and degenerating germ cells. The major differences observed between the two groups were attributable primarily to morphological differences rather than to clusterin expression specifically. There was no correlation between testicular clusterin content and the cause and severity of spermatogenic disorders. Within normal epididymides, regional differences in clusterin staining similar to those reported in the rat were observed. The seminal vesicles contained large amounts of positive clusterin staining, whereas normal human prostate was completely negative. Low levels of clusterin expression were observed in the marmoset prostate. This study suggests that clusterin is an important and widespread product in the human and marmoset reproductive tracts and is likely to have a role in spermatogenesis.

Animals↗
PubMed2001

Expression of clusterin in pancreatic acinar cell injuries in vivo and in vitro.

Clusterin is a secretory glycoprotein that is highly induced in several tissues in response to injury. The pathophysiologic significance of clusterin in the pancreas remains largely unknown. The aim of this work was to examine whether clusterin is expressed in spontaneous chronic pancreatitis in the WBN/Kob rat and to investigate the relationship between clusterin and apoptosis in pancreatic acinar AR4-2J cells. In the in vivo study, 4-week-old male WBN/Kob rats developed chronic pancreatitis at 12 weeks. Clusterin mRNA was expressed after 12 weeks and then decreased. Immunohistochemistry showed clusterin expression in the acinar cells. In the in vitro study, clusterin mRNA and protein were strongly induced in AR4-2J cells treated either with arginine, menadione, tumor necrosis factor-alpha or transforming growth factor-beta1. In the time course study with arginine or menadione, clusterin mRNA was expressed after 4 hours and peaked at 8 and 24 hours, whereas DNA fragmentation peaked at 72 hours. Our results show that clusterin is overexpressed in the pancreas at the onset of chronic pancreatitis in vivo and in cultured acinar cells in response to various stimuli in vitro, suggesting that clusterin is a defense mechanism of the exocrine pancreas.

Animals↗
PubMed2007

Ethylenediaminetetraacetic acid affects subcellular expression of clusterin protein in human colon adenocarcinoma COLO 205 cell line.

The aim of our study was to determine the expression of various isoforms of clusterin and to evaluate how etoposide or calcium chelators [ethylenediaminetetraacetic acid and (2-aminoethoxyethane)-N,N,N',N'-tetraacetic acid] affect the subcellular expressions of the 50-kDa isoform of clusterin protein in colon adenocarcinoma COLO 205 cells. We then determined how the cytoplasmic vs. nuclear expression of the 50-kDa isoform of clusterin correlates with the viability of COLO 205 cells. To identify the clusterin isoforms, and its nuclear and cytoplasmic expression in COLO 205 cells, Western bloting was used. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay. Etoposide decreased the viability of COLO 205 cells with a concomitant increase in the 50-kDa clusterin concentration in the cell nucleus. Chelation of the extracellular calcium ions by (2-aminoethoxyethane)-N,N,N',N'-tetraacetic acid did not modulate the subcellular distribution of clusterin. The use of ethylenediaminetetraacetic acid, which reduces the intracellular and extracellular calcium levels, stimulated nuclear expression of clusterin protein and was accompanied by extensive cell death. Intracellular calcium determines cytoplasmic expression and antiapoptotic activity of the intracellular protein clusterin. The depletion of intracellular calcium leads to increased nuclear expression of the 50-kDa clusterin protein, which is accompanied by cell death. We concluded that there is at least one cell death-promoting pathway in COLO 205 cells that is dependent on intracellular calcium and nuclear localization of 50-kDa clusterin.

Adenocarcinoma↗
PubMed1996

Clusterin, a putative complement regulator, binds to the cell surface of Staphylococcus aureus clinical isolates.

The ability of Staphylococcus aureus Cowan I strain and a number of S. aureus clinical isolates to bind to the human blood glycoprotein clusterin was investigated. Binding of clusterin to these strains was tested by both enzyme-linked immunosorbent assay and flow cytometry. All of the S. aureus strains examined appeared to bind clusterin to some extent, while nonpathogenic control strains Bacillus subtilis BR151 and Escherichia coli JM109 did not. Three S. aureus isolates were selected for more detailed study; binding of labeled clusterin was saturable, inhibited in the presence of excess unlabeled clusterin, and prevented by pretreatment of bacteria with proteases. From the saturation binding studies, estimates of the affinity constants for the binding of clusterin to the bacteria ranged from 31 to 57 nM. Addition of clusterin to S. aureus cultures was also found to result in aggregation of the bacterial cells; aggregation was not detected when clusterin was added to B. subtilis BR151 or E. coli JM109 cultures. These results suggest that at least some S. aureus strains possess specific proteinaceous receptors for clusterin. Such receptors may be an important new bacterial virulence determinant for S. aureus, as clusterin has been proposed to have a role in the regulation of complement activity.

Clusterin↗