Netrin-1 and clusterin: Innovative potential diagnostic biomarkers for early renal damage in ß-thalassemia major children.

BACKGROUND: Children with ß-thalassemia major (ß-TM) suffer from tubular dysfunction even before the onset of any renal impairment symptoms and/or clinical signs. Therefore, identifying innovative biomarkers allowing early renal damage detection has focused attention. AIM: This study aims to preliminary assess Netrin-1(NTN-1) and clusterin (CLU) in ß-TM children and explore their possible roles as surrogate noninvasive biomarkers of renal tubular dysfunction. SUBJECTS AND METHODS: In this study, 40 ß-TM children and 30 healthy children were enrolled. Routine serum and urinary biochemical variables were determined. Urinary NTN-1 and CLU levels were measured using ELISA and their mRNA expression in PBMCs were assayed using real-time PCR. Serum TNF-α, MDA levels and GST activity were measured. RESULTS: Urinary NTN-1 and CLU concentrations and mRNA relative expression levels in PBMCs were significantly increased in ß-TM children relative to controls. Oxidative stress and inflammatory markers revealed significant elevation in ß-TM children compared to controls. The change in these parameters correlated significantly with other renal parameters. ROC curves analysis showed that urinary NTN-1 and CLU levels are of promising diagnostic performance. CONCLUSION: Our results suggest that NTN-1 and CLU are qualified as new noninvasive biomarker panels for early detection of renal injury in ß-TM children. Moreover, urinary NTN-1 is recommended as a precise one during the clinical practices.

Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Neuronal clusterin expression is associated with cognitive protection in amyotrophic lateral sclerosis.

AIMS: Clusterin is a topologically dynamic chaperone protein with the ability to participate in both intra- and extacellular proteostasis. Clusterin has been shown to be upregulated in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and has been shown to protect against TDP-43 protein misfolding in animal and cell models. Previous studies have demonstrated an association between the pathological burden of TDP-43 misfolding and cognitive deficits in ALS, demonstrating high specificity, but correspondingly low sensitivity owing to a subset of individuals with no evidence of cognitive deficits despite a high burden of TDP-43 pathology, called mismatch cases. METHODS: Hypothesizing that differences in the ability to cope with protein misfolding in these cases may be due to differences in expression of protective mechanisms such as clusterin expression, we assessed the spatial expression of clusterin and another chaperone protein, HspB8, in post mortem brain tissue of mismatch cases. We employed a modified in situ hybridization technique called BaseScope, with single cell, single transcript resolution. RESULTS: Mismatch cases demonstrated differential spatial expression of clusterin, with a predominantly neuronal pattern, compared to cases with cognitive manifestations of their TDP-43 pathology who demonstrated a predominantly glial distribution of expression. CONCLUSIONS: Our data suggest that, in individuals with TDP-43 pathology, predominantly neuronal expression of clusterin in extra-motor brain regions may indicate a cell protective mechanism delaying clinical manifestations such as cognitive dysfunction.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Pseudoexfoliation and Alzheimer's associated CLU risk variant, rs2279590, lies within an enhancer element and regulates CLU, EPHX2 and PTK2B gene expression.

Genetic variants at PTK2B-CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer's disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele 'A' constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele 'G'. By binding to allele 'A', HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes.

Identification of plexin A4 as a novel clusterin receptor links two Alzheimer's disease risk genes.

Although abundant genetic and biochemical evidence strongly links Clusterin (CLU) to Alzheimer disease (AD) pathogenesis, the receptor for CLU within the adult brain is currently unknown. Using unbiased approaches, we identified Plexin A4 (PLXNA4) as a novel, high-affinity receptor for CLU in the adult brain. PLXNA4 protein expression was high in brain with much lower levels in peripheral organs. CLU protein levels were significantly elevated in the cerebrospinal fluid (CSF) of Plxna4-/- mice and, in humans, CSF levels of CLU were also associated with PLXNA4 genotype. Human AD brains had significantly increased the levels of CLU protein but decreased levels of PLXNA4 by ∼50%. To determine whether PLXNA4 levels influenced cognition, we analyzed the behaviour of Plxna4+/+, Plxna4+/-, and Plxna4-/- mice. In comparison to WT controls, both Plxna4+/- and Plxna4-/- mice were hyperactive in the open field assay while Plxna4-/- mice displayed a hyper-exploratory (low-anxiety phenotype) in the elevated plus maze. Importantly, both Plxna4+/- and Plxna4-/- mice displayed prominent deficits in learning and memory in the contextual fear-conditioning paradigm. Thus, even a 50% reduction in the level of PLXNA4 is sufficient to cause memory impairments, raising the possibility that memory problems seen in AD patients could be due to reductions in the level of PLXNA4. Both CLU and PLXNA4 have been genetically associated with AD risk and our data thus provide a direct relationship between two AD risk genes. Our data suggest that increasing the levels of PLXNA4 or targeting CLU-PLXNA4 interactions may have therapeutic value in AD.

Clusterin inhibition mediates sensitivity to chemotherapy and radiotherapy in human cancer.

Since its discovery in 1983, the protein clusterin (CLU) has been isolated from almost all human tissues and fluids and linked to the development of different physiopathological processes, including carcinogenesis and tumor progression. During the last few years, several studies have shown the cytoprotective role of secretory CLU in tumor cells, inhibiting their apoptosis and enhancing their resistance to conventional treatments including hormone depletion, chemotherapy, and radiotherapy. In an effort to determine the therapeutic potential that the inhibition of this protein could have on the development of new strategies for cancer treatment, numerous studies have been carried out in this field, with results, in most cases, satisfactory but sometimes contradictory. In this document, we summarize for the first time the current knowledge of the effects that CLU inhibition has on sensitizing tumor cells to conventional cancer treatments and discuss its importance in the development of new strategies against cancer.

Expression of clusterin in colorectal carcinoma in relation to clinicopathological criteria.

BACKGROUND/AIM: Colorectal carcinoma (CRC) carries a high incidence of morbidity and mortality. Prognosis is related to nodal metastasis and stage. Clusterin is a widely distributed glycoprotein with not yet fully understood functions. Clusterin may be overexpressed in some tumours or under expressed in other tumours. The aim behind this study is to examine the relation of clusterin cytoplasmic immunostaining to tumour characteristics, disease relapse, and survival in CRC. MATERIALS AND METHODS: Paraffin blocks of 133 CRCs were retrieved from the Department of Pathology, King Abdulaziz University, Jeddah, Saudi Arabia. Immunostaining was done using antibody to clusterin. Staining expression in 10% of malignant cells was used as a cut-off to determine low immunostaining and high immunostaining. Statistical tests were used to evaluate the association of clusterin immunostaining with clinicopathological parameters. RESULTS: Immunohistochemical results showed clusterin low immunostaining in CRC and nodal metastases. No association was found between clusterin immunostaining and tumour grade, age, tumour invasiveness, distant metastases, vascular invasion, nodal metastases, relapse, and survival. CONCLUSION: Our study showed low clusterin immunostaining in CRC with lack of association with prognostic indicators in CRC. These results raise the controversy of understanding the role of clusterin in CRC. Further molecular studies are required to explore more about possible mechanisms of clusterin association with tumorigenicity, apoptosis, tumour growth progression, local and vascular invasion, and metastasis of CRC.

Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5' end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Clusterin expression level correlates with increased oxidative stress in asthmatics.

BACKGROUND: Oxidative stress is thought to play a role in the pathogenesis of asthma. Clusterin is a sensitive cellular biosensor of oxidative stress and has antioxidant properties. The function and expression of clusterin in patients with asthma have not been fully investigated. OBJECTIVE: To investigate whether the expression of clusterin in patients with asthma is regulated by increased oxidative burden and whether clusterin expression could be used to assess the response to inhaled corticosteroids. METHODS: Clusterin levels in serum, induced sputum, and peripheral blood mononuclear cells of patients with asthma were measured by enzyme-linked immunosorbent assay and western blotting and compared with pulmonary function and levels of expression of hyperoxidized peroxiredoxins. Serum concentrations of clusterin in treatment-naive patients were compared before and after inhaled corticosteroid use. RESULTS: Serum clusterin concentration was significantly elevated in patients with severe asthma and was inversely correlated with pulmonary function. The expression of hyperoxidized peroxiredoxins was greatly increased in peripheral blood mononuclear cells of patients with asthma and was strongly correlated with clusterin expression. Serum clusterin concentrations in treatment-naive patients with asthma were decreased significantly after initial treatment with inhaled corticosteroids. CONCLUSION: Clusterin may be a biomarker of asthma severity and the burden of oxidative stress in patients with asthma. Moreover, clusterin may be useful for the prompt assessment of airway inflammation.

Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis.

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

Changes in mRNA and protein expression in the renal cortex of male and female F344 rats treated with bromate.

Bromate (BrO3(-)), a by-product of ozonation of drinking water, induces nephrotoxicity in male rats at much lower doses than in female rats. This difference appears to be related to the development of α-2u-globulin nephropathy in males. To determine sex-dependent changes in mRNA and protein expression in the renal cortex attributable to α-2u-globulin nephropathy, we performed microarray and immunohistochemical analyses in proximal renal tubules of male and female F344 rats treated with KBrO3 for 28 days. Particular attention was paid to molecular biomarkers of renal tubular injury. Microarray analysis of male and female rats treated with BrO3(-) at low doses (125 mg/L KBrO3) displayed marked sex-dependent changes in renal gene expression. The greatest differences were seen in genes encoding for cellular differentiation, apoptosis, ion transport, and cell proliferation. Differences by sex were especially prominent for the cell cycle checkpoint gene p21, the renal injury protein Kim-1, and the kidney injury and cancer biomarker protein osteopontin. Dose-related nephrotoxicity, assessed by hematoxylin and eosin staining, was greater in males compared to female rats, as was cellular proliferation, assessed by bromodeoxyuridine staining. The fraction of proximal renal cells with elevated 8-oxodeoxyguanosine (8-OH-dG) was only increased at the high dose and did not differ by sex. Dose-dependent increases in the expression of osteopontin were detected immunohistochemically only in male rats and were localized in proximal tubule cells. Similarly, BrO3(-) treatment increased clusterin and Kim-1 staining in the proximal tubules; however, staining for these proteins did not differ appreciably between males and females. These data demonstrate both qualitative and quantitative differences in the response of male versus female kidneys to BrO3(-)-treatment. These sex-dependent effects likely contribute to renal carcinogenesis of BrO3(-) in the male rat.

Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats.

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

Age-changes in gene expression in primary mixed glia cultures from young vs. old rat cerebral cortex are modified by interactions with neurons.

Astrocytic GFAP expression increases during normal aging in many brain regions and in primary astrocyte cultures derived from aging rodent brains. As shown below, we unexpectedly found that the age-related increase of GFAP expression was suppressed in mixed glia (astrocytes+microglia). However, the age-related increase of GFAP was observed when E18 neurons were co-cultured with mixed glia. Thus, the presence of microglia can suppress the age-related increase of GFAP, in primary cultures of astrocytes. To more broadly characterize how aging and co-culture with neurons alters glial gene expression, we profiled gene expression in mixed glia from young (3 mo) and old (24 mo) male rat cerebral cortex by Affymetrix microarray (Rat230 2.0). The majority of age changes were independent of the presence of neurons. Overall, the expression of twofold more genes increased with age than decreased with age. The minority of age changes that were either suppressed or revealed by the presence of neurons may be useful to analyze glial-neuron interaction during aging. Some in vitro changes are shared with those of aging rat hippocampus in studies from the Landfield group (Rowe et al., 2007; Kadish et al., 2009).

Clinical significance of clusterin expression in pancreatic adenocarcinoma.

BACKGROUND: Clusterin is known to be expressed in many human neoplasms, and is believed to participate in the regeneration, migration, and anti-apoptosis of tumor cells. However, few reports have addressed the relationship between the manifestation of clusterin and clinicopathologic parameters in pancreas cancer patients. In the present study, the authors investigated the expression of clusterin and its clinical significance in pancreatic adenocarcinoma. METHODS: Immunohistochemical staining was performed for clusterin in tumor tissues obtained from patients who received pancreatic resection with radical intent, and the associations of clusterin expression with various clinicopathologic parameters were analyzed in addition to the relation between its expression and survival. RESULTS: Immunoreactivity for clusterin was observed in 17 of the 52 (33%) pancreatic adenocarcinomas examined. In addition, clusterin positivity was found to be associated with preoperative serum carcinoembryonic antigen level, perineural invasion, and, most strongly, lymph node metastasis. The survival analysis identified tumor differentiation and lymph node metastasis as the only significant prognostic factors. CONCLUSION: Although not an independent prognostic factor, clusterin immunoreactivity can be used in conjunction with lymph node metastasis to predict survival in cases of pancreatic adenocarcinoma.

Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel.

Understanding the molecular events that lead to paclitaxel (TX) resistance is necessary to identify effective means to prevent chemoresistance. Previously, results from our lab revealed that secretory clusterin (CLU) form positively mediates TX response in ovarian cancer cells. Thus, we had interest to study the role of another non-secreted form (intracellular clusterin (i-CLU)) in chemo-response. Here, we provide evidences that i-CLU form localizes mainly in the nucleus and differentially expressed in the TX-responsive KF cells, versus TX-resistant, KF-TX, ovarian cancer cells and negatively regulate cellular chemo-response. I-CLU was cloned, by deleting the secretion-leading signaling peptide from full-length CLU cDNA, and transiently over-expressed in OVK-18 cells. Forced expression of truncated i-CLU was mainly detectable in the nuclei and significantly reduced cellular growth, accumulating cells in G1 phase which finally died through apoptosis. Importantly, compromised expression of i-CLU under an inducible promoter was tolerated and did not induce apoptosis but sensitized ovarian cancer cells to TX. We then demonstrated that this sensitization mechanism was cell cycle independent and relied on i-CLU/Ku70 binding probably due to controlling the free amount of Ku70 available for DNA repair in the nucleus. Results from CLU immunohistochemistry in ovarian tumor tissues verified the retardation of nuclear CLU staining in the recurrent tumor even though their primary counterparts showed nuclear CLU staining. Thus, the controversial data on CLU function in chemo-response/resistance may be explained by a shift in the pattern of CLU expression and intracellular localization as well when tumor acquires chemoresistance.

Clusterin confers gemcitabine resistance in pancreatic cancer.

OBJECTIVE: To measure clusterin expression in pancreatic cancer tissues and cell lines and to evaluate whether clusterin confers resistance to gmcitabine in pancreatic cancer cells. METHODS: Immunohistochemistry for clusterin was performed on 50 primary pancreatic cancer tissues and 25 matched backgrounds, and clusterin expression in 5 pancreatic cancer cell lines was quantified by Western blot and PT-PCR. The correlation between clusterin expression level and gmcitabine IC50 in pancreatic cancer cell lines was evaluated. The effect of an antisense oligonucleotide (ASO) against clusterin (ASO-CLU) [corrected] on gmcitabine resistance was evaluated by MTT assays. Xenograft model was used to demonstrate tumor growth. RESULTS: Pancreatic cancer tissues expressed significantly higher levels of clusterin than did normal pancreatic tissues (P < 0.01). Clusterin expression levels were correlated with gmcitabine resistance in pancreatic cancer cell lines, and ASO-CLU [corrected] significantly decreased BxPc-3 cells resistance to gmcitabine (P < 0.01). In vivo systemic administration of AS clusterin and gmcitabine significantly decreased the s.c. BxPC-3 tumor volume compared with mismatch control ODN plus gmcitabine. CONCLUSION: Our finding that clusterin expression was significantly higher in pancreatic cancer than in normal pancreatic tissues suggests that clusterin may confer gmcitabine resistance in pancreatic cancer cells.

Serum and tissue profiling in bladder cancer combining protein and tissue arrays.

Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n = 12) and controls without the disease (n = 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n = 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosis.