Glucocorticoid resistance in thymocytes from mice expressing a T cell receptor transgene

A majority of thymocytes undergo apoptosis during differentiation due to lack of survival signals provided by T cell receptor (TCR) activation. As glucocorticoids (GC) have been suggested to be involved in this process, we have investigated the GC sensitivity in thymocytes from mice expressing a transgenic selecting TCR. We now report that immature CD4(+)CD8(+) double-positive thymocytes from these mice are comparatively more resistant to corticosterone-induced apoptosis. This is associated with reduced glucocorticoid receptor (GR) expression, increased levels of membrane CD28, increased NF-kappaB DNA binding activity, and increased binding to the CD28 response element in the interleukin-2 gene promoter. Analysis of NF-kappaB/Rel proteins from nuclear extracts demonstrated altered levels of some of these proteins. Our results suggest that TCR recognition of self major histocompatibility antigens generates intracellular signals which alter the thymocyte GC sensitivity and thereby protect them against apoptosis induced by endogenous GC.     

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase–1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi–1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.     

Calcium homeostasis in the thymocyte apoptosis II. Accumulation of calcium in mitochondria and endoplasmic reticulum

The activity of energy-dependent Ca2+-accumulation systems in rat thymocytes mitochondria and endoplasmic reticulum (ER) in control and at the early stage of X-irradiation or H2O2-induced apoptosis were determined in experiments using the model of digitonin-permeabilized cells with addition of thapsigargin and ruthenium red. The mitochondrial Ca2+-transporting system proved to be more sensitive to both apoptotic stimuli. The stationary level of Ca2+, accumulated in mitochondria and initial rate of Ca2+ accumulation in ER were reduced 15 min after H2O2 treatment. The parameters of mitochondrial Ca2+-accumulation system in irradiated cells were decreased 30 min after irradiation. Cyclosporin A almost completely inhibited DNA fragmentation in irradiated and partly--in peroxide-treated cells. The mitochondrial calcium homeostasis imbalance is suggested to be an early event in thymocytes apoptosis initiation.     

Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor. Although cisplatin is the primary chemotherapy used in osteosarcoma treatment, the cisplatin resistance remains a big challenge for improving overall survival. The store-operated calcium (Ca²⁺) entry (SOCE) and its major mediator Stim1 have been shown to be implicated in a number of pathological processes typical for cancer. In this study, we showed that Stim1 expression was significantly increased in chemo-resistant osteosarcoma tissues compared with chemo-sensitivity tissues. Patients with Sitm1 expression exhibited poorer overall survival than Stim1-negative patients. Moreover, un-regulation of Stim1 expression and SOCE were also observed in cisplatin-resistant MG63/CDDP cells compared with their parental cells. Cisplatin treatment obviously reduced Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, cisplatin resulted in a more pronounced increase of endoplasmic reticulum (ER) stress in MG63 cells than in their resistant variants, which was evidenced by the activation of molecular markers of ER stress, GRP78, CHOP and ATF4. Knockdown of Stim1 using siRNA remarkably enhanced cisplatin-induced apoptosis and ER stress in MG63/CDDP cells, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of Stim1 markedly reversed apoptosis and ER stress following cisplatin treatment. Taken together, these results demonstrate that Stim1 as well as Ca²⁺ entry contributes cisplatin resistance via inhibition of ER stress-mediated apoptosis, and provide important clues to the mechanisms involved in cisplatin resistance for osteosarcoma treatment. Stim1 represents as a target of cisplatin and blockade of Stim1-mediated Ca²⁺ entry may be a useful strategy to improve the efficacy of cisplatin to treat osteosarcoma.     

Regulation of Bim by TCR signals in CD4/CD8 double-positive thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3epsilon injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim(EL) is rapidly phosphorylated in thymocytes in response to CD3epsilon cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x(L) bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x(L) in immature thymocytes.     

Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry.

Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiation-induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A0 regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A0 region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C

Glucocorticoid hormones and Ca2+ ionophores stimulate a suicide process in immature thymocytes, known as apoptosis or programmed cell death, that involves extensive DNA fragmentation. We have recently shown that a sustained increase in cytosolic Ca2+ concentration stimulates DNA fragmentation and cell killing in glucocorticoid- or ionophore-treated thymocytes. However, a sustained increase in the cytosolic Ca2+ level also mediates lymphocyte proliferation, suggesting that apoptosis is blocked in proliferating thymocytes. In this study we report that phorbol esters, which selectively stimulate protein kinase C (PKC), blocked DNA fragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone. The T cell mitogen, concanavalin A, which stimulates thymocytes by a mechanism that involves PKC activation, caused concentration-dependent increases in the cytosolic Ca2+ level that did not result in DNA fragmentation, but incubation with concanavalin A and the PKC inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) resulted in both DNA fragmentation and cell death. Phorbol ester directly inhibited Ca2+-dependent DNA fragmentation in isolated thymocyte nuclei. Our results strongly suggest that PKC activation blocks thymocyte apoptosis by preventing Ca2+-stimulated endonuclease activation.     

Liver poly(ADP-ribose)polymerase is resistant to cleavage by caspases

In hepatocytes the DNA repair enzyme poly(ADP-ribose)polymerase (PARP) is not proteolytically cleaved during apoptosis. The reason for this was investigated using a cell-free system that consisted of isolated nuclei from hepatocytes or thymocytes and cytosolic extracts from hepatocytes or thymocytes undergoing apoptosis. It was found that liver PARP is resistant to proteolytic cleavage by the caspases present in the cytosolic extracts. Furthermore, liver PARP was not cleaved by recombinant human caspase-3. It is concluded that PARP proteolysis cannot be used as a marker for hepatocyte apoptosis.     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

Roles of CHOP/GADD153 in endoplasmic reticulum stress

Endoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.     

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.     

Murine retrovirus-induced depletion of T cells is mediated through activation-induced death by apoptosis.

ts1, a mutant of Moloney murine leukemia virus, causes neurologic disorders and acute immunodeficiency associated with the destruction of thymocytes and helper T cells. In this study, we examined whether apoptosis was involved in ts1-induced killings of T cells. Neonatal mice were inoculated with ts1, and 20 to 23 days postinoculation, when cytopathic effects on T cells normally appear, thymocytes and splenic lymphocytes were isolated and examined. Our results showed that several features of apoptosis were present in ts1-infected thymocytes and splenic lymphocytes. Apoptotic fragmented DNA, condensation of the chromatin, and enhanced cell death after stimulation with mitogens which was preventable with protein synthesis inhibitors, all of which are common features of apoptotic cell death, were observed in ts1-infected cells. Several other viruses, including human immunodeficiency virus, have been shown to cause apoptotic death of T cells. Here we show for the first time that a murine retrovirus which also induces immunodeficiency can cause apoptotic T-cell death. Future studies with this murine retrovirus may provide important results to help us better understand the mechanisms of retrovirus-induced apoptosis of T cells.