Alessandro E. Caccamo

Compatible osmolytes modulate the response of porcine endothelial cells to hypertonicity and protect them from apoptosis

Porcine pulmonary arterial endothelial cells accumulated myo‐inositol and taurine, as well as betaine, during adaptation to hypertonic stress. The cells grew and maintained their normal morphology during culture in hypertonic (0.5 osmol (kg H2O)−1) medium that contained osmolytes such as betaine, myo‐inositol or taurine at concentrations close to reported physiological values. The cells did not grow well in hypertonic medium depleted of potential compatible osmolytes. After a few days, cell density decreased by about 50 % and many cells rounded up and detached from the plates, their nuclei showing clear apoptotic morphology. The caspase‐3 activity of the cells also increased dramatically under these conditions, but remained negligibly low when betaine and myo‐inositol were added to the medium. Addition of betaine and myo‐inositol to hypertonic medium depleted of compatible osmolytes increased the number of colonies remaining after 12 days of culture; with each solute at 30–100 μmol l−1 the number increased about sixfold. In the absence of compatible osmolytes, increased mRNA levels and corresponding activities of betaine/γ‐aminobutyric acid transporter (BGT1) and sodium/myo‐inositol transporter (SMIT) induced by hypertonicity remained high after 72 h incubation, whereas they were down regulated in the presence of betaine and myo‐inositol. Similarly, the down regulation of the amino acid System A transporter (ATA2) was markedly slowed in the absence of compatible osmolytes. We conclude that these compatible osmolytes at concentrations close to physiological values enable the endothelial cells to adapt to hypertonic stress, protecting them from apoptosis, and also modulate the adaptation process.

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.

BACKGROUND Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. METHODS We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription-polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. RESULTS Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). CONCLUSIONS We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.

Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin

Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G0/G1-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell-detachment-induced apoptosis, suggesting a possible major role for clusterin as an anti-proliferative gene in human prostate epithelial cells.

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

Nuclear Translocation of a Clusterin Isoform Is Associated with Induction of Anoikis in SV40‐Immortalized Human Prostate Epithelial Cells

Abstract: Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti‐apoptotic and pro‐apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over‐expression inhibited cell cycle progression of SV40‐immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum‐starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum‐starvation conditions, decreased growth rate, slow rounding‐up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment‐induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45–40 kDa isoforms. On the 8th day of serum‐free culturing, only the higher molecularweight protein‐band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45‐kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45‐kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis‐death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti‐oncogene in the prostate.

Clusterin is a short half‐life, poly‐ubiquitinated protein, which controls the fate of prostate cancer cells

The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra‐ and intracellular CLU forms regulate cell proliferation and apoptosis. Dis‐regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over‐expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC‐3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT‐qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half‐life is less than 2 h. CLU protein products were found poly‐ubiquitinated by co‐immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase‐dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor. J. Cell. Physiol. 219: 314–323, 2009.

Nuclear clusterin accumulation during heat shock response: Implications for cell survival and thermo-tolerance induction in immortalized and prostate cancer cells

Clusterin (CLU), whose role is still debated, is differentially regulated in several patho‐physiological processes and invariably induced during apoptosis. In heat shock response, CLU is considered a stress‐inducible, pro‐survival/cyto‐protective factor via an HSE element present in his promoter. In both human prostate PNT1A and PC‐3 epithelial cells we found that apoptotic stimuli induced nuclear localization of CLU (nCLU), and that overexpression of nCLU is pro‐apoptotic. We show here that CLU time‐course accumulation kinetic is different from that of HSP70 in these cells, thus other factor(s) might mediate HSF‐1 activation and CLU expression. Sub‐lethal heat shock inhibited the secretion of CLU (sCLU), leading to increased cytoplasm accumulation of CLU (cCLU) in association to cell survival. At difference, lethal heat stress caused massive accumulation of pro‐apoptotic nCLU in cells dying by caspase‐3‐dependent apoptosis. Double heat stress (sub‐lethal heat shock followed by recovery and lethal stress) induced HSP70 and thermo‐tolerance in PNT1A cells, but not in PC‐3 cells. In PNT1A cells, CLU secretion was inhibited and cCLU was accumulated, suggesting that cCLU might be pro‐survival, while in PC‐3 cells accumulation of nCLU was concomitant to caspase‐3 induction and PARP activation instead. Thus, CLU expression/sub‐cellular localization is strictly related to cell fate. In particular, nCLU and physiological levels of HSP70 affected cell survival in an antagonistic fashion. Prevalence of heat‐induced nCLU, not allowing PC‐3 cells to cope with heat shock, could be the rational explaining why malignant cells are more sensitive to heat when delivered by minimally invasive procedures for ablation of localized prostate cancer. J. Cell. Physiol. 207: 208–219, 2006.