Silvia Desenzani

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.

Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats

Proteasome activity is known to decrease with aging in ad libitum (AL) fed rats. Severe caloric restriction (CR) significantly extends the maximum life-span of rats, and counteracts the age-associated decrease in liver proteasome activities. Since few investigations have explored whether lower CR diets might positively counteract the age associated decrease in proteasome activity, we then investigated the effects of a mild CR regimen on animal life-span, proteasome content and function. In addition, we addressed the question whether both CR regimens might also affect the expression of Hsc70 protein, a constitutive chaperone reported to share a role in the function of proteasome complex and in the repair of proteotoxic damage, and whose level decreased during aging. In contrast to severe CR, mild CR had a poor effect on life-span; however, it better counteracted the decrease of proteasome activities. Both regimens, however, maintain Hsc70 in liver of old rats at level comparable to that of young rats. Interestingly, the effects of aging and CRs on liver proteasome enzyme activities did not appear to be associated with parallel changes in the amount of proteasome proteins suggesting that the quality (molecular activity of the enzymes) rather than the quantity are likely to be modified with age. In conclusion, the results presented in this work show that a mild CR can have beneficial effects on liver function of aging rats because is adequate to counteract the decrease of proteasome function and Hsc70 chaperone level.

Increased Levels of Inducible HSP70 in Cells Exposed to Electromagnetic Fields

Abstract Alfieri, R. R., Bonelli, M. A., Pedrazzi, G., Desenzani, S., Ghillani, M., Fumarola, C., Ghibelli, L., Borghetti, A. F. and Petronini, P. G. Increased Levels of Inducible HSP70 in Cells Exposed to Electromagnetic Fields. Radiat. Res. 165, 95–104 (2006). Because reports in the literature on the effects of electromagnetic fields (EMFs) on expression of the 70-kDa heat-shock protein (HSP70) are somewhat contradictory, we studied the influence of low-frequency EMFs on the accumulation of inducible HSP70 in several cell models. Some of the cell types tested showed increased levels of HSP70 protein when exposed for 24 h to 50 Hz, 680 μT EMFs. In endothelial cells, EMFs alone induced only a poor and transient activation of the heat-shock transcription factor 1 (HSF1); however, neither the level of HSP70 mRNA nor the synthesis of HSP70 appeared to be altered significantly. Accordingly, transfection experiments involving HSP70 promoter showed that gene transcription was not affected. We also noted a marked reduction in proteasome activities in cell extracts exposed to EMFs. Interestingly, the heat-shock-induced levels of HSP70 mRNA and protein were increased by a concomitant weak stressor like EMFs. Taken together, our results indicate that in EMF-exposed endothelial cells, HSP70 gene transcription and translation are unaffected; however, EMFs alone promoted accumulation of the inducible HSP70 protein, probably by increasing its stability, and it enhanced accumulation and translation of the heat-induced HSP70 mRNA when applied in concert with heat shock.

Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells.

Cholangiocarcinoma is the second most common primary hepatic neoplasia and the only curative therapy is surgical resection or liver transplantation. Biphosphonates (BPs) are an emerging class of drugs widely used to treat bone diseases and also appear to possess direct antitumor activity. In two human cholangiocarcinoma cell lines (TFK-1 and EGI-1) we investigated, for the first time, the activity of zoledronic acid by determining proliferation, cell cycle analysis and apoptosis. The results obtained indicate that zoledronic acid induces cell-narrowing and growth inhibition, both reversed by 25 microM GGOH, and significantly affects the colony-forming ability of these cells. The inhibition by zoledronic acid of Rap1A prenylation was reversed in cell co-treated with GGOH. At 10-50 microM zoledronic acid exerted an S-phase cell cycle arrest which was confirmed by changes in the level of cyclins and of regulators p27(KIP1) and pRb. Interestingly, the expression level of cyclin A (putative S-phase marker) shows a dose-dependent increment in contrast to the decrement of cyclin D1 (putative G1 phase marker). However, neither hypodiploid cells nor cleaved PARP or caspase-3 was detected. The lack of TP53 or loss of its function, the large constitutive expressions of anti-apoptotic proteins Bcl-xL and HSP27 together with the low level of the pro-apoptotic Bax are the likely factors which protect cells from apoptosis. In conclusion, our study indicates that zoledronic acid induces S-phase arrest and cell-narrowing, both reversed by GGOH and, by changing the delicate balance between pro- and anti-apoptotic proteins, allows survival of cholangiocarcinoma cells.

Roles of compatible osmolytes and heat shock protein 70 in the induction of tolerance to stresses in porcine endothelial cells

Studies of the responses of porcine pulmonary endothelial cells to acute hypertonic stress have been extended by examining the induction and underlying mechanisms of cell tolerance to both osmotic and heat stresses. Preliminary adaptation of these cells to 0.4osmol (kg H2O)−1 rendered them tolerant either to subsequent severe osmotic stress (0.7osmol (kg H2O)−1) or to subsequent severe heat shock (50 min at 49°C). In contrast, preliminary exposure of the cells to mild heat shock (44°C for 30 min) induced tolerance only to severe heat shock, not to hyperosmotic stress. Induction of tolerance to heat shock by either procedure correlated with the induced expression of heat shock protein 70 (HSP70). Induction of tolerance to hyperosmotic stress, on the other hand, was associated with the cellular accumulation of osmolytes, such as amino acids, betaine and myo‐inositol, and did not correlate with the induced expression of HSP70. It also required a reduction in the final change of osmotic pressure applied to the cells, such that maximum cell shrinkage would not be much more than 40%. In general, therefore, HSP70 and compatible osmolytes have distinct roles in cellular adaptation to these stresses.

Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts

At the end of their replicative potential in vitro, late passage WI-38 human diploid fibroblasts (HDF) have a low basal expression of heat shock protein 72 (HSP72) and an attenuated ability to induce it in response to heat shock. The transient exposure to the specific and reversible proteasome inhibitor MG132 during a mild heat shock induced late passage HDF to synthesize and accumulate high levels of HSP72. This HSP72 expression was long-lasting and appeared to result from both increased cytoplasmic levels and enhanced translation of HSP72 mRNA. The level of HuR, a stabilizing mRNA-binding protein, increased following the MG132 treatment. This result is consistent with the proposed role of HuR in assisting mRNA export to the cytoplasm and in antagonizing its degradation. Furthermore, the previous exposure of late passage HDF to a mild heat shock in the presence of MG132 protected these cells against the otherwise lethal effect of a subsequent severe heat shock. This acquisition of thermotolerance appeared to be correlated with the level of HSP72.

Hypertonic stress and amino acid deprivation both increase expression of mRNA for amino Acid transport system A.

The activity of amino acid transport system A ([Oxender and Christensen, 1963][1]) is regulated in a variety of different ways, the best studied being the increases of its activity caused by starving cells of amino acids or by exposing them to hypertonicity (for review see [McGivan and Pastor-

The BH3-mimetic ABT-737 targets the apoptotic machinery in cholangiocarcinoma cell lines resulting in synergistic interactions with zoledronic acid

PurposeIn TFK-1 and EGI-1 cholangiocarcinoma cell lines, zoledronic acid (ZOL) determines an S-phase block without apoptosis. Here, we investigated the occurrence of apoptosis stigmata when ZOL is associated to the BH3-mimetic ABT-737.MethodsIn EGI-1 and TFK-1 cholangiocarcinoma cell lines untreated or treated with ABT-737 alone or in combination with ZOL, the pro-survival protein’s pattern (BCL-2, BCL-XL, MCL-1, HSP72, HSP27) was investigated by biochemical criteria along with the occurrence of mitochondrial damage evaluated by cytofluorimetric analysis using a cationic dye.ResultsABT-737 induced growth inhibition and significantly affected the colony-forming ability of both EGI-1 and TFK-1 cells. However, activated PARP-1 or/and caspase-3 cleavage (apoptosis markers) were detected only at the highest ABT-737 concentrations used. Combined treatment showed synergistic effect by converting the predominant cytostatic effect of ZOL into a cytotoxic one as shown by striking increment of mitochondrial harmed cells along with PARP-1 activation and caspase-3 cleavage.ConclusionThe lack of apoptosis following ZOL treatment in these cholangiocarcinoma cell lines appears to be multifactorial and could be ascribed to the large constitutive expression of pro-survival proteins. The efficacy of ZOL treatment requires a concomitant unleashing of apoptosis using a selective BH3-mimetic as ABT-737. The rational targeting of specific components of the apoptotic pathway may appear a useful approach to improve the treatment of refractory or relapsed cholangiocarcinoma. Combined treatment could be further explored in in vivo tumor model of cholangiocarcinoma.