Angelo F. Borghetti

Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis.

To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30-60% when osmolality was increased from 0.35 to 0.53 osmol/kg of water by the addition of NaCl, KCl, CH(3)CO(2)Na or CH(3)CO(2)K. In contrast, equivalent additions of the compatible osmolytes betaine or myo -inositol caused a 40-50% increase in the rate of translation, whereas amino acids (50-135 mM) that are transported via system A had no significant effect. Addition of 75 mM KCl caused a dramatic fall in the amount of the 43 S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150 mM betaine. The formation of a non-enzymic initiation complex between rabbit [(3)H]Phe-tRNA, poly(U) and the 80 S ribosomes was unaffected by the addition of 75 mM NaCl or KCl, but translation of the complex decreased by 70%. Density-gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150 mM betaine had no effect, whereas addition of 75 mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80 S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo -inositol.

Cell susceptibility to apoptosis by glutamine deprivation and rescue: survival and apoptotic death in cultured lymphoma-leukemia cell lines.

Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS‐supplemented RPMI 1640 medium containing 2 mM L‐glutamine CEM, Namalwa, HL‐60 and U937 cells, seeded at initial densities of 0.2 to 1 × 106 cells/ml, ceased growing within 3–5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy‐source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine‐free or glucose‐free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12–24 h incubation in glutamine‐depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3–0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death.

Adaptive regulation of amino acid transport across the cell membrane in avian and mammalian tissues

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in a variety of mesenchymal and epithelial cells and tissues of avian and mammalian origin. Changes in transport activity as a function of time under various in vitro conditions (amino acid dependence, active and inhibited protein synthesis) have been evaluated by measurements of initial entry rates with representative amino acids. Results and conclusions based on the adopted experimental approach include the following. (1) An adaptive control mechanism for the transport of neutral amino acids corresponding to the typical substrates of the A mediation is operative in (a) mesenchymal cells (fibroblasts, chondroblasts, osteoblasts and myoblasts) from embryonic tissues of avian (chick embryo) origin and (b) mesenchymal cells from immature rat uterus (fibroblasts and smooth muscle cells) and other mammalian tissues (cardiac cells from newborn mouse and rat heart). (2) Adaptive regulation is restricted to a discrete subgroup of amino acids (L-proline, glycine and the analogue alpha-aminoisobutyric acid) in rat peritoneal macrophages and thymic lymphocytes. (3) Adaptive regulation is absent in erythroid cells (human erythrocytes, rabbit erythrocytes and reticulocytes, avian erythrocytes) which lack the A mediation and are incapable of active gene transcription. (4) Adaptive regulation is absent in the epithelial kidney cortex tissue and possibly absent in the epithelial component of liver tissue from adult rats; it is fully operative in the chick embryo crystalline lens, i.e. an epithelial preparation of embryonic origin. (5) These observations indicate that adaptive control mechanisms of amino acid transport across the cell membrane are quite common among tissues and species and emphasize their broad biological significance in eukaryotes.

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

Osmoregulation of amino acid transport activity in cultured fibroblasts

The effect of exposure of chick embryo cells to increasing concentrations of Na+ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na+. Changes were measurable as early as 1 h after altering Na+ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na+ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with L-proline and L-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na+. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na+ treatment occurred through a mechanism affecting Vmax rather than Km.

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.

Effectiveness of very low doses of immunotherapy in advanced renal cell cancer.

Twenty-one nephrectomized patients with metastatic renal cell cancer were treated with recombinant interleukin 2 (rlL-2) and interferon alpha (rIFN alpha). rIL-2 was administered s.c. at a dose of 1 x 10(6) IU m(-2) every 12 h on days 1 and 2, followed by 0.5 x 10(6) IU twice daily on days 3-5; rIFN alpha-2 was given i.m. as 1.8 x 10(6) IU m(-2) on days 3 and 5 of each week for 4 consecutive weeks. The cycle was regularly repeated at 4-month intervals and continued ad libitum in patients showing some response and in patients with progressing disease. Of 20 patients evaluable for treatment response, one (5%) had a complete response and three (15%) showed partial response. Three patients (15%) achieved stable disease and 13 (65%) were evaluated as having progressive disease. The estimated actuarial 44-month survival rate was 44%. Toxicity was limited to WHO grades 1 and 2 only.

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.

Amino acid transport in pig lymphocytes enhanced activity of transport system asc following mitogenic stimulation

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of 14C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27-32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na+-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na+-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity (Km = approximately 4 mM) and a high affinity (Km = approximately 0.2 mM) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity (V) of the high affinity component without any substantial change in the apparent affinity constant (Km).

Adaptive cellular response to osmotic stress in pig articular chondrocytes

The authors studied the effects of a wide range of medium osmolarities (from 0.28 osM (physiological osmolarity of plasma and synovial fluid) to 0.58 osM) by altering Na+ concentration in high density cultures of pig articular chondrocytes in order to analyze the behaviour of some functional and structural parameters during cell adaptation to these imposed changes in the ionic environment. Biochemical and morphological results indicated that, even if isolated from the tissue matrix and cultured in vitro, chondrocytes maintained active osmoregulation systems which are present in living conditions. They showed a similar biochemical and morphological behavior when cultured at 0.28 osM and 0.38 osM but they were able, with regard to protein synthesis, aminoacid transport and proliferation rates, to respond quickly and to adapt to 0.48 osM medium as well. On the contrary, the treatment at the highest osmolarity (0.58 osM) early altered these biochemical parameters and was detrimental or even gave rise to lethal damage during long-term treatment. Furthermore, while chondrocytes cultured in 0.28-0.38 osM medium maintained phenotypic characteristics in culture, the higher osmolarities (0.48-0.58 osM) caused morphological changes in cell populations resulting in loss of phenotypic cell stability as demonstrated by their taking on a fibroblast-like shape as well as a lack of ability to assembly matrix proteoglycans.