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W2036506653 abstract "Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA-expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3–5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re-established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40–80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation. Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA-expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3–5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re-established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40–80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation. heat shock protein 70 antisense phosphate-buffered saline constitutive heat shock protein 70 sodium nitroprusside 3-(4,5-dimethylthizolyl-2)-2,5-diphenyltetrazolium bromide malondialdehyde high pressure liquid chromatography (Z)-1-(2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate hydrogen peroxide nitric oxide superoxide anion Heat shock proteins (hsps)1 are a large group of evolutionary strongly conserved proteins with multiple tasks in trafficking, chaperoning, and stabilizing biomolecules such as mRNAs and proteins with enzymatic and structural functions (1.Günther E. Walter L. Experientia (Basel). 1994; 50: 987-1001Crossref PubMed Scopus (107) Google Scholar, 2.Liang P. MacRae T.H. J. Cell Sci. 1997; 110: 1431-1440Crossref PubMed Google Scholar). With these functions the hsps essentially contribute to the protection of vital cell components against injuries induced under conditions of physical (3.Li G.C. Li L. Liu Y.-K. Mak J.Y. Chen L. Lee W.M.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1681-1685Crossref PubMed Scopus (333) Google Scholar) or metabolic stress (4.Marber M.S. Mestril R. Chi S.-H. Sayen M.R. Yellon D.M. Dillmann W.H. J. Clin. Invest. 1995; 95: 1446-1456Crossref PubMed Scopus (765) Google Scholar) and by a variety of cytotoxic inflammatory mediators like cytokines (5.Jäättelä M. Wissing D. Bauer P.A. EMBO J. 1992; 11: 231-236Crossref Scopus (355) Google Scholar) and radicals (6.Bellmann K. Wenz A. Radons J. Burkart V. Kleemann R. Kolb H. J. Clin. Invest. 1995; 95: 2840-2845Crossref PubMed Scopus (146) Google Scholar, 7.Polla B.S. Kantengwa S. Francois D. Salvioli S. Franceschi C. Marsac C. Cossarizza A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6458-6463Crossref PubMed Scopus (391) Google Scholar). Among the hsps a special protective potential is attributed to the heat shock protein 70 (hsp70), which obviously plays a crucial role in the cellular defense against radical-induced injury. In insulin-producing pancreatic beta cells of rats up-regulation of hsp70 is associated with an improved resistance against NO, reactive oxygen species and the beta cell toxin streptozocin (6.Bellmann K. Wenz A. Radons J. Burkart V. Kleemann R. Kolb H. J. Clin. Invest. 1995; 95: 2840-2845Crossref PubMed Scopus (146) Google Scholar). Further evidence for the importance of hsp70 in beta cell defense comes from studies in which the selective overexpression of the hsp70 protein resulted in an improved resistance of rat insulinoma cells against NO (8.Bellmann K. Jäättelä M. Wissing D. Burkart V. Kolb H. FEBS Lett. 1996; 391: 185-188Crossref PubMed Scopus (163) Google Scholar), which has been identified as an important mediator of beta cell destruction in experimental systems of type 1 diabetes (9.Kolb H. Kolb-Bachofen V. Immunol. Today. 1992; 13: 157-160Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 10.Kolb H. Kolb-Bachofen V. Diabetologia. 1992; 35: 796-797PubMed Google Scholar). However, recent findings indicate strong species-specific differences in the sensitivity of beta cells toward NO-induced toxicity. Whereas human beta cells are largely resistant, beta cells from rodents exhibit an increased susceptibility toward the damaging effects of NO (11.Eizirik D.L. Sandler S. Welsh N. Cetkovic-Cvrlje M. Niemann A. Geller D.A. Pipeleers D.G. Bendtzen K. Hellerström C. J. Clin. Invest. 1994; 93: 1968-1974Crossref PubMed Scopus (295) Google Scholar). The parallel finding of a considerably increased spontaneous expression of hsp70 in human beta cells compared with rodent beta cells (12.Eizirik D.L. Horm. Metabol. Res. 1996; 28: 302-305Crossref PubMed Scopus (33) Google Scholar) led to the suggestion that hsp70 contributes to the strong natural resistance of human beta cells against NO-induced damage (13.Eizirik D.L. Pipeleers D.G. Zhidong L. Welsh N. Hellerström C. Andersson A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9253-9256Crossref PubMed Scopus (261) Google Scholar). To prove this issue we established a human beta cell line in which the expression of hsp70 is selectively suppressed by transfection with a plasmid designed for the constitutive expression of antisense-hsp70 mRNA. Our experiments show that the selective inhibition of hsp70 expression results in an increased susceptibility of human beta cells toward NO-induced toxicity. The preservation of the respiratory activity in NO-exposed islet cells identified the mitochondria as the primary targets of hsp70-mediated protection. The study was performed with cells of the rat insulinoma line RINm5F (14.Chick W. Warren S. Chute R.N. Like A.A. Lauris V. Kitchen K.C. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 628-632Crossref PubMed Scopus (238) Google Scholar), the mouse beta cell line MIN-6 (kindly provided by Drs. J. Miyazaki, Y. Oka, H. Ishihara, Tokyo, Japan) (15.Miyazaki J.-I. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K.-I. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1033) Google Scholar), and the human beta cell line CM, which was originally isolated from tumor cells present in the ascitic fluid of a patient suffering from insulinoma (16.Gueli N. Toto A. Palmieri G. Carmenini G. Delfino A. Ferrini U. J. Exp. Clin. Cancer Res. 1987; 4: 281-285Google Scholar). The cells were cultivated (37 °C, 5% CO2) in RPMI 1640 (Life Technologies, Inc.) supplemented with 125 mg/liter ampicillin, 75 mg/liter penicillin, 50 mg/liter streptomycin (Serva, GmbH, Heidelberg, Germany), 2 mmol/liter l-glutamine, 10 ml/liter 100x nonessential amino acids (Life Technologies, Inc.), 3.4 g/liter NaHCO3, 2.38 g/liter HEPES (pH 7.3, Serva), and 5% fetal calf serum (Life Technologies, Inc.) (culture medium). Cell cultures were regularly tested for the absence of mycoplasma contamination. The antisense (as) hsp70pcDNA3 plasmid was constructed by insertion of a 500-base pair fragment of the human inducible hsp70cDNA in antisense orientation (974–475) at the multiple cloning site downstream the cytomegalovirus promoter in the pcDNA3 vector (17.Jäättelä M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (614) Google Scholar). The eukaryotic expression vector pZEM-neo was used as control. This vector, with a size (6.3 kilobases) comparable to that of pcDNA3, also contains the neomycin resistance gene but lacks any fragment interfering with hsp70 transcripts. Transfection of the CM cells was performed by electroporation as described previously (8.Bellmann K. Jäättelä M. Wissing D. Burkart V. Kolb H. FEBS Lett. 1996; 391: 185-188Crossref PubMed Scopus (163) Google Scholar). CM cells (2 × 107) were resuspended in 800 μl of phosphate-buffered saline (PBS) containing 40 μg of plasmid DNA and exposed to a double pulse (pulse 1: 330 V/cm, ∞ Ω, 1500 microfarads; pulse 2: 100 V/cm, 192 Ω, 900 microfarads) in a Cellject electroporation device (Eurogentec, Sart Tilman, Belgium). After electroporation the cells were seeded in cloning plates (Greiner, Solingen, Germany) in the presence of 1600 μg/ml G418 (Geneticin, Roche Molecular Biochemicals). From the pool of the surviving G418-resistant cells single cell-derived clones were selected and expanded to a cell number sufficient for analysis by Western blot and for the in vitroexperiments. Proteins from lysates of CM cells cultivated under standard conditions or exposed to heat shock (42.5 °C, 60 min) (6.Bellmann K. Wenz A. Radons J. Burkart V. Kleemann R. Kolb H. J. Clin. Invest. 1995; 95: 2840-2845Crossref PubMed Scopus (146) Google Scholar) were separated on 10% SDS- polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were incubated for 1 h with 1:1000 dilutions of a mouse monoclonal antibody directed against the inducible form of the human hsp70 (BIOMOL, Hamburg, Germany), a rat monoclonal antibody raised against the constitutive form of Chinese hamster heat shock protein 70 (hsc 70, BIOMOL) or a mouse monoclonal antibody specific for the human mitochondrial heat shock protein 70 (GRP 75, BIOMOL). The detection was performed with sheep peroxidase-labeled anti-mouse or anti-rat antibodies (1:10,000, Amersham Pharmacia Biotech) using the ECL detection system (Amersham Pharmacia Biotech) (8.Bellmann K. Jäättelä M. Wissing D. Burkart V. Kolb H. FEBS Lett. 1996; 391: 185-188Crossref PubMed Scopus (163) Google Scholar, 18.Bellmann K. Hui L. Radons J. Burkart V. Kolb H. Diabetes. 1997; 46: 232-236Crossref PubMed Google Scholar). For flow cytometric analysis single cell suspensions of ashsp70-transfected cells and CM cells were fixed in methanol/acetone (30 min, 4 °C). The cells were washed with PBS and incubated for 18 h (4 °C) with a 1:150 dilution of the mouse anti-human hsp70 antibody (see Western blot analysis). After washing in PBS with 2% fetal calf serum the cells were incubated with a rat fluorescein isothiocyanate-conjugated anti-mouse antibody (1:60, Sigma) for 1 h. The cell suspension was washed again and resuspended in PBS with 2% fetal calf serum for flow cytometric analysis in a FACScan (Becton-Dickinson, San José, CA). Cells were seeded at a concentration of 1 × 105/200 μ l/well of 96-well culture plates (Falcon/Becton Dickinson, Franklin Lakes, NJ) or laminin- (Sigma) coated chamber slides (Nunc, Naperville, IL) and incubated for 1 day (37 °C, 5%CO2). (Z)-1-(2-(2-Aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium-1,2-diolate) (ALEXIS, Grünberg, Germany) and sodium nitroprusside (NP, Sigma) served as NO-generating agents. NP was added in the presence of rhodanese and thiosulfate to scavenge cyanide ions eventually released during the decomposition process (19.Kallmann B. Burkart V. Kröncke K.-D. Kolb-Bachofen V. Kolb H. Life Sciences. 1992; 51: 671-678Crossref PubMed Scopus (112) Google Scholar). Xanthine oxidase (EC 1.1.3.22, grade III from buttermilk, specific activity 1.2 units/mg protein, Sigma) and its substrate hypoxanthine (Sigma) were used to expose the cells to superoxide anion (O⨪2) and hydrogen peroxide (H2O2) (20.Burkart V. Koike T. Brenner H.-H. Kolb H. Diabetologia. 1992; 35: 1028-1034Crossref PubMed Scopus (68) Google Scholar). The proportion of dead cells was determined by the trypan blue exclusion assay (19.Kallmann B. Burkart V. Kröncke K.-D. Kolb-Bachofen V. Kolb H. Life Sciences. 1992; 51: 671-678Crossref PubMed Scopus (112) Google Scholar) in 96-well flat bottom plates (Falcon/Becton-Dickinson). After incubation under various conditions, 150 μl of the culture supernatant were removed from each well and 15 μl of a trypan blue solution (0.4% in PBS) were added, and the cells were incubated for another 15 min (37 °C, 5% CO2). Then 200 cells were counted in adjacent microscopic fields in each well, and the percentage of dead cells from the total cell number was calculated. To investigate the reactivity of CM cells toward glucose stimulation the cells were cultivated in medium containing 16.7 mm glucose. After 0, 4, and 24 h the cells were disrupted by ultrasound treatment, and insulin was extracted by incubation of the cell lysates in acidified ethanol (75% ethanol, 1.5% HCl (12 m), 23.5% H2O) for 18 h at 4 °C. The insulin contents of the ethanol extracts were determined by a microparticle enzyme immunoassay insulin kit (IMX, ABBOTT Diagnostics, Wiesbaden, Germany). The insulin concentrations were quantified by the use of standard preparations of human insulin included in the IMX kit. The metabolic activity of the cells was assessed by their capacity to convert 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma) into its formazan product. Recent studies demonstrated that this reaction reflects metabolic processes that mainly depend on the activity of mitochondrial redox processes, such as the activity of succinate dehydrogenase (21.Janjic D. Wollheim C.B. Diabetologia. 1992; 35: 482-485Crossref PubMed Scopus (143) Google Scholar, 22.Segu V. Li G. Metz S.A. Metabolism. 1998; 47: 824-830Abstract Full Text PDF PubMed Scopus (27) Google Scholar). After termination of the various treatments 25 μl of the MTT stock solution (5 mg/ml) were added to each well (100 μl) of the cultivated cells. After 4–5 h of incubation (37 °C, 5% CO2) the supernatant was removed and the formazan crystals were dissolved in 100 μl of isopropanol. The OD was measured at 550 nm with 650 nm as the reference wavelength in an Emax precision 96-well microplate reader (Molecular Devices Corporation, Graefeling, Germany) (20.Burkart V. Koike T. Brenner H.-H. Kolb H. Diabetologia. 1992; 35: 1028-1034Crossref PubMed Scopus (68) Google Scholar). For the detection of apoptotic alterations of cell nuclei the cells were cultivated on laminin-coated chamber slides. After exposure to NO (48 h, 37 °C, 5% CO2) 10 μl of a solution of acridin orange (3 mm, Serva) was added to each well. The supernatant was removed, and the cells were observed under the fluorescence microscope (526 nm). The percentage of cells containing two or more apoptotic bodies was calculated from the total cell number. At least 200 cells/sample were counted in triplicates. DNA fragmentation analysis was performed by the use of the InViSorb Apoptosis Detection Kit (InViTek, Berlin, Germany). Cells (2 × 105/200 μl) were seeded in the wells of 96-well flat bottom microtiter plates and exposed to NO. After 72 h the cells were lysed, and the released DNA was bound to a mineral carrier material. The fixed DNA was washed over a column (12,000 × g, 2 min) and eluted by incubation in elution buffer (2 min), followed by centrifugation (12,000 × g, 1 min). The DNA fragments were separated by electrophoresis on an agarose gel (2%, Metaphor, FMC Bioproducts, Rockland, ME) containing ethidium bromide and visualized by UV exposure. DNA isolated from apoptotic murine thymocytes included in the InViSorb kit was used as a positive control for a ladder-like DNA fragmentation pattern. Lipid peroxidation was assessed by measurement of malondialdehyde (MDA). The cells (2 × 106 in 10 ml) were seeded in Petri dishes and incubated for 24 h in the presence of NP (0.6 mm). To induce lipid peroxidation in the cells FeCl3 (0.1 mm) and ascorbate (0.25 mm) were used as a positive control. Then the cells were scraped from the Petri dishes in the presence of butylhydroxytoluol (Sigma), disrupted by sonification and frozen at −20 °C until use. To 1 ml of the cell lysate 0.35 ml of 20% trichloroacetic acid (Sigma) and 0.5 ml of 1.4% (w/v) thiobarbituric acid (Merck) were added. After heating (15 min, 95 °C) the samples were centrifuged (5 min, 250 × g), and 2 ml of the supernatant were mixed with 2 ml of 1-butanol. The resulting thiobarbituric acid-MDA complex was separated on reversed-phase HPLC and quantitated with a fluorescence detector (excitation 515 nm, emission 553 nm) (23.Lepage G. Munoz G. Champagne J. Roy C.C. Anal. Biochem. 1991; 197: 277-283Crossref PubMed Scopus (160) Google Scholar). Statistical differences were calculated using the Student's t test with a significance level ofp < 0.05. The study was performed to investigate the role of hsp70 in the natural resistance of human beta cells against damage induced by reactive radicals. For the experiments, cells of the human insulinoma line CM were used, which was maintained under tissue culture conditions since its isolation from an insulinoma patient. To confirm the beta cell characteristics of this cell line, the insulin contents of the cells was determined before and after stimulation by an increased glucose level in the culture medium. As shown in TableI the CM cells contained measurable amounts of insulin (8.5 microunits/106 cells) and were able to release the stored hormone in a time-dependent manner in response to stimulation with an elevated glucose concentration.Table IEffect of glucose stimulation on insulin contents of CM cellsDuration of stimulationInsulin contentshmicrounits/10 6 cells08.5 ± 2.780.4 ± 0.2240.05 ± 0.04CM cells were incubated at 16.7 mm glucose, and after 0, 8, and 24 h of stimulation their insulin content was determined by measuring the amount of immunoreactive insulin as described under “Experimental Procedures.” Data show mean ± S.D. from three determinations. Open table in a new tab CM cells were incubated at 16.7 mm glucose, and after 0, 8, and 24 h of stimulation their insulin content was determined by measuring the amount of immunoreactive insulin as described under “Experimental Procedures.” Data show mean ± S.D. from three determinations. To compare the sensitivity of human and rodent beta cells to radical-induced injury, CM cells and cells of the rat line RINm5F and of the mouse line MIN-6 were exposed to NO and reactive oxygen species generating agents for 18 h. Both agents dose dependently damaged cell functions as determined by the MTT test. The human insulinoma cell line showed a significantly improved metabolic activity in the presence of the NO donor NP (Fig. 1 A) as well as after exposure to the O⨪2/H2O2 generating system hypoxanthine/xanthine oxidase (Fig. 1 B) compared with the insulinoma cells from mouse and rat (p < 0.05 andp < 0.005, respectively). Next we analyzed for spontaneous expression of hsp70 in untreated cells. Western blot analysis revealed spontaneous expression of hsp70 in human CM cells, whereas the rodent cells contained almost undetectable amounts of the protein (inset in Fig. 1 B). Both rodent cell lines expressed hsp70 in response to heat stress (not shown). To investigate the role of hsp70 in the enhanced natural resistance of the human insulinoma cells against NO radicals, the expression of hsp70 protein was suppressed in CM cells by transfection with a plasmid expressing an antisense hsp70 mRNA. The efficiency and the selectivity of the antisense strategy was proven by Western blot analysis. Transfection of the CM cells with the ashsp70 construct resulted in a 15-fold reduction of hsp70 expression in cells cultivated under standard conditions (37 °C, 5% CO2), whereas cells transfected with the control plasmid pZEM showed an hsp70 protein expression comparable to the level in the untransfected controls (TableII). Expression of the antisense construct did not influence the morphology and growth rate of the cells. Exposure of the cells to heat shock (42.5 °C, 60 min) 4 h prior to protein extraction resulted in a significant increase of the hsp70 signal in all three cell lines tested. In the control lines (CM and pZEM-transfected CM) the hsp70 signal increased about 1.7-fold. Interestingly, heat shock induced a more than 30-fold increase of the hsp70 signal also in ashsp70-transfected cells thereby reaching a level of expression comparable to the control cells (Table II). The deficient expression of hsp70 in the antisense line was also confirmed by cytofluorometry. As shown in Fig. 2, there was a reduction of the fluorescence activity in ashsp70-transfected cells yielding a single peak, which indicated homogeneously reduced hsp70 expression in antisense plasmid-transfected cells.Table IIhsp70 protein expression in untransfected, ashsp70-transfected, and pZEM-transfected CM cellsCM cells untransfectedCM cells ashsp70 transfectedCM cells pZEM transfectedUntreated354.9 ± 20.220.4 ± 10.2ap < 0.01 compared to the signal of untransfected CM cells or the pZEM-transfected cells.319.8 ± 23.5Heat shock treated570.9 ± 27.1bp < 0.01 versus the signal of the corresponding samples without heat shock treatment.682.1 ± 18.7bp < 0.01 versus the signal of the corresponding samples without heat shock treatment.571.1 ± 24.7bp < 0.01 versus the signal of the corresponding samples without heat shock treatment.Untransfected, ashsp70-transfected, and pZEM-transfected CM cells remained untreated or were exposed to heat shock (42.5 °C, 60 min). Lysates were prepared from untreated cells or after heat shock treatment plus a 4 h recovery period. The signals on the films resulting from the Western blot analysis were scanned in a densitometer. The data show densitometrical units (mean ± S.D.) from scans of three experiments.a p < 0.01 compared to the signal of untransfected CM cells or the pZEM-transfected cells.b p < 0.01 versus the signal of the corresponding samples without heat shock treatment. Open table in a new tab Untransfected, ashsp70-transfected, and pZEM-transfected CM cells remained untreated or were exposed to heat shock (42.5 °C, 60 min). Lysates were prepared from untreated cells or after heat shock treatment plus a 4 h recovery period. The signals on the films resulting from the Western blot analysis were scanned in a densitometer. The data show densitometrical units (mean ± S.D.) from scans of three experiments. Because the constitutively expressed hsc70 and the mitochondrial GRP75 have a high amino acid sequence homology with hsp70, we investigated whether the expression of these proteins was impaired in cells transfected with ashsp70. As shown in Fig.3 (lane 1) the CM cells spontaneously express hsp70, hsc70, and GRP75. Transfection with the ashsp70 construct strongly reduced the expression of hsp70, whereas the signal strengths of hsc70 and GRP75 remained unchanged (lane 2). CM cells transfected with the control plasmid pZEM expressed the same levels of hsp70, hsc70, and GRP75 as the wild type CM cells (lane 3). After heat shock treatment a strong increase mainly of the hsp70 protein expression was observed in all three cell lines (lanes 4 -6). To investigate whether the suppression of spontaneous hsp70 protein expression by the antisense plasmid would increase the sensitivity of the CM cells toward NO radical-induced injury, the cell lines were exposed to the NO donors NP (Fig.4 A) and DETA/NO (Fig.4 B). As an end point of necrotic cell death, the irreversible loss of membrane integrity was determined by the inability of the cells to exclude trypan blue. Within the first 24 h of NO exposure the rates of cell death slightly increased up to 5–10% (Fig.4, A and B). No significant difference in sensitivity could be observed between the ashsp70-transfected cells and the cells transfected with the control plasmid. Prolongation of the exposure time resulted in a steady increase of the death rate in the control cells up to a maximum of 10% after 48 h. In contrast, ashsp70-transfected cells showed a strongly accelerated death rate reaching 50% in the NP- (0.6 mm) exposed cells and more than 30% in the DETA/NO- (0.2 mm) exposed cells (p < 0.05 compared with the specific lysis of the pZEM-transfected cells after 48 h). These findings clearly show an increased susceptibility toward NO-induced necrosis in cells with suppressed spontaneous hsp expression. To further prove the role of hsp70 we tested the hypothesis that the (re-)induction of hsp70 protein in ashsp70-transfected CM cells by heat shock exposure (Fig. 3) will re-establish the resistance of the cells toward NO-induced damage. In fact, heat shock treatment resulted in a significant reduction of DETA-NO-induced lysis of ashsp70-transfected cells from 38.4 to 12.0% for 0.1 mm DETA-NO and from 55.6 to 17.3% for 0.2 mm DETA-NO (p < 0.01, Fig. 5). Heat shock did not improve the resistance of untransfected and pZEM-transfected CM cells that constitutively express hsp70. In parallel samples the effect of hsp70 expression on the NO-induced apoptotic pathway of cell death was examined by analyzing nuclear chromatin condensation and DNA fragmentation. As shown in Fig. 6exposure to NO resulted in an increased proportion of cells showing apoptotic alterations. Acridin orange staining revealed condensation of nuclear chromatin and formation of apoptotic bodies in a significantly higher percentage of ashsp70-transfected CM cells (24.0 ± 0.5%) (Fig. 6 A) when compared with identically treated cells transfected with the control plasmid pZEM (13.8 ± 2.2%) (p < 0.05). In the untreated samples, about 4% of the cells formed apoptotic bodies. To investigate the mode of DNA degradation, DNA was isolated from ashsp70-transfected CM cells and controls after 72 h of NO exposure. After separation of the DNA by electrophoresis a ladder-like fragmentation pattern was clearly visible in ashsp70-transfected cells (lane 3), whereas only faint signals of DNA degradation were detectable in untransfected CM cells (lane 1) and in CM cells transfected with the control plasmid pZEM (lane 4) (Fig. 6 B). These observations indicate that suppression of spontaneous hsp70 expression increased the susceptibility of the CM cells toward NO-induced apoptosis. Exposure to NO radicals may lead to cell injury via the formation of toxic compounds from cellular components. It has been suggested that lipid peroxides mediate radical toxicity in islet cells (24.Rabinovitch A. Suarez W.L. Thomas P.D. Strynadka K. Simpson I. Diabetologia. 1992; 35: 409-413Crossref PubMed Scopus (123) Google Scholar, 25.Rabinovitch A. Suarez-Pinzon W.L. Strynadka K. Lakey J.R. Rajotte R.V. J. Clin. Endocrinol. Metab. 1996; 81: 3197-3202PubMed Google Scholar). Therefore, it was analyzed whether hsp70-mediated protection from NO toxicity affects lipid peroxidation. As a measure of lipid peroxidation, we determined MDA. CM cells transfected with the ashsp70 plasmid or the pZEM control plasmid were incubated in the presence of the NO donor NP or the potent reactive oxygen species generating system FeCl3 and ascorbate. As expected, HPLC analysis of the cell lysates revealed a strong accumulation of MDA (about 8800 nm/106 cells) in cells exposed to FeCl3/ascorbate for 24 h (Fig.7). In contrast, NP-exposed cells showed only a slight, but significant (p < 0.05) increase to 0.86 nm MDA/106 cells compared with untreated cells (0.07 nm MDA/106 cells). However, the analysis of the ashsp70-transfected cells and the pZEM transfected CM cells did not reveal any difference in the degree of lipid peroxidation in response to either NO or reactive oxygen species (Fig. 7). Because the mitochondrial respiratory system and energy metabolism in general were found to be highly susceptible to NO radicals, it was determined whether hsp70 might exert its protective action at this level. CM cells transfected with the ashsp70 construct and with the control plasmid pZEM were incubated for 24 h in the presence of increasing doses of NP or DETA/NO, and after different time intervals the residual metabolic activity was assessed by the capacity of the cells to reduce MTT into its formazan product (Fig.8). In CM cells transfected with pZEM, the metabolic activity was decreased by 40% after exposure to NP, and no significant changes were noted after DETA/NO exposure. In contrast, the ashsp70-transfected CM cells showed a significantly decreased capacity to convert MTT after 24 h of exposure to either NP or DETA/NO (p < 0.01). In an additional sample CM cells were used, which derived from the pool of ashsp70-transfected cells after selection for G418 resistance but before selection of clones. After NO exposure (0.6 mm NP) these cells showed a significant reduction of mitochondrial activity to a level comparable to th" @default.
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W2036506653 title "Natural Resistance of Human Beta Cells toward Nitric Oxide Is Mediated by Heat Shock Protein 70" @default.
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