Transfection of NS0 myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis

Mouse myeloma NS0 cells widely used in hybridoma technology lack the expression of a major stress protein Hsp70 which is the principal component of the basic cellular defense mechanism. These cells rapidly undergo apoptosis at the late-stationary phase of batch culture following nutrient exhaustion. Since Hsp70 was recently demonstrated to protect cells against numerous apoptotic stimuli, the aim of the present study was to examine the protective potential of the protein expression in engineered myeloma NS0 cells and in resulting hybridomas. Myeloma cells were transfected with the hsp70 gene under beta-actin gene promoter. To imitate harmful conditions that hybridoma or myeloma cells often experience when cultivated in large scale for an antibody production, NS0(wt) and NS0(hsp70) cell cultures were maintained without changing the medium for a few days, and the expression of apoptotic markers has been studied. It was found that long-term cultivation induced apoptosis in original cells manifested by typical nuclei fragmentation, DNA ladders and activation of caspase-3. In contrast, in transfected cells under the same conditions the outcome of apoptosis was postponed for 24 hours. Most relevant was that the fusion of transfected myeloma cells with immune splenocytes resulted in twofold hybridomas output compared with wild-type fusion partner. Almost half of the hybridomas continued to be hsp70-positive and maintained higher robustness in culture. The level of monoclonal antibodies production by hybridoma cells obtained with the use of NS0(wt) and NS0(hsp70) was similar, however, the secreted product was better preserved in culture supernatants of Hsp70-positive cells. It is concluded that transfection of mouse myeloma cells with the hsp70 gene can be a novel means to increase hybridoma yield and reduce the sensitivity of myeloma and hybridoma cells to culture conditions insults accompanying monoclonal antibody production.     

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone hsp70

The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.     

Hsp90 Is Involved in Apoptosis of Candida albicans by Regulating the Calcineurin-Caspase Apoptotic Pathway

Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway.     

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.     

Glucoronic acid is a novel inducer of heat shock response

The elevated expression of 70 kDa heat shock protein (Hsp70) induces resistance to stress-induced apoptosis. We have screened a variety of natural products for their ability to enhance Hsp70 expression as anti-apoptotic agent. We found that glucuronic acid (GA) induced the synthesis of Hsp70 and that cells pretreated with GA were significantly tolerant to stress including heat shock and hydrogen peroxide. We also found that GA induces the production of reactive oxygen species (ROS), a process inhibited by NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) and antioxidant N-acetylcysteine (NAC). GA-induced ROS production was also inhibited in RacN17 cell line overexpressing a dominant negative mutant of Rac1. Furthermore, GA treatment induces MAPKs activation (SAPK/JNK and p38) and Hsp70 expression in ROS dependent manner, suggesting that GA turns on the signaling pathway by generation of ROS through Rac1. We analyzed the profiles of newly synthesized proteins by GA with 2-dimensional gel electrophoresis and MALDI-TOF MS and found that two families of proteins were expressed by GA. One was similar to the protein family synthesized by heat shock (Hsp70, Hsp73, Hsp65, Hsp90, vimentin, tubulin, Ras homolog); and the other was a family of protein specific to GA (calreticulin, annexin III, thioredoxin peroxidase). These results suggest that GA-induced stress responses are mediated by ROS generation and are similar, in part, to heat shock-induced responses and GA can be possibly adopted for the protecting agent from cell death.     

Hsp70 Architecture: The Formation of Novel Polymeric Structures of Hsp70.1 and Hsc70 after Proteotoxic Stress

Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.     

Heat-shock protein 70 antagonizes apoptosis-inducing factor

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.     

Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis. This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2 arrest and apoptosis of target T cells. A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest. Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress. In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells. Overexpression of Hsp70 reduced the Vpr-dependent G2 arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1. Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1. Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished. Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells. Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr.     

Engineering responsiveness to cell culture stresses: growth arrest and DNA damage gene 153 (GADD153) and the unfolded protein response (UPR) in NS0 myeloma cells

The successful movement of a newly synthesized protein through the endoplasmic reticulum (ER) and associated membranous compartments is dependent on appropriate recognition by complex processing systems. Failure to perceive appropriately processed or modified intermediates in the pathway can initiate a series of cellular signaling events (ER stress or unfolded protein response, UPR) that can lead to cell apoptosis and loss of biomass in culture processes. We have shown that expression of growth arrest and DNA damage gene 153 (GADD153) is associated with recognition of damaged or mis-processed proteins within the secretory processes of CHO and NS0 myeloma cells. To directly characterize the roles of GADD153 in UPR-directed apoptosis, we have generated stable clones of NS0 myeloma cells with elevated (constitutive and inducible) and deleted GADD153 expression. Although GADD153 is a robust indicator of the onset of ER stress or the UPR, GADD153 expression alone is not sufficient to provoke NS0 myeloma apoptosis and it is not required for apoptosis to occur.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.     

Encapsulated Hsp70 decreases endotoxin-induced production of ROS and TNFα in human phagocytes

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.     

Salicylic acid-mediated potentiation of Hsp70 induction correlates with reduced apoptosis in tobacco protoplasts.

BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.     

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1–3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.     

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.     

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

Hsp105α and Hsp105β are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105α is expressed constitutively in the cytoplasm of cells, while Hsp105β, an alternatively spliced form of Hsp105α, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105β but also Hsp105α accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105α and Hsp105β with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105α and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105α and Hsp105β on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105α and Hsp105β suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.