Induction Of Bcl-xL Research Articles

Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering CD1d trafficking through the endocytic pathway.

NKT cells are a unique subset of T cells that recognize glycolipid Ags presented in the context of CD1d molecules. NKT cells mount strong antitumor responses and are a major focus in developing effective cancer immunotherapy. It is known that CD1d molecules are constantly internalized from the cell surface, recycled through the endocytic compartments, and re-expressed on the cell surface. However, little is known about the regulation of CD1d-mediated Ag processing and presentation in B cell lymphoma. Prosurvival factors of the Bcl-2 family, such as Bcl-xL, are often upregulated in B cell lymphomas and are intimately linked to sphingolipid metabolism, as well as the endocytic compartments. We hypothesized that Bcl-xL can regulate CD1d-mediated Ag presentation to NKT cells. We found that overexpression or induction of Bcl-xL led to increased Ag presentation to NKT cells. Conversely, the inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Furthermore, knockdown of Bcl-xL resulted in the loss of CD1d trafficking to lysosome-associated membrane protein 1(+) compartments. Rab7, a late endosomal protein, was upregulated and CD1d molecules accumulated in the Rab7(+) late endosomal compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated Ag processing and presentation to NKT cells by altering the late endosomal compartment and changing the intracellular localization of CD1d.

Bcl-xL Protein Protects from C/EBP Homologous Protein (CHOP)-dependent Apoptosis during Plasma Cell Differentiation

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate.

Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells (APP3P.114)

Abstract Natural killer T (NKT) cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. Once activated, NKT cells can mount strong anti-tumor responses. However, little is known about the regulation of CD1d-mediated antigen processing and presentation to NKT cells, particularly in the context of B cell lymphoma. Pro-survival factors of the Bcl-2 family, such as Bcl-xL are often upregulated in B cell lymphomas, and are associated with changes in the endocytic pathway, which is paramount for CD1d-mediated antigen presentation. We hypothesized that Bcl-xL regulates this process, and found that over-expression or induction of Bcl-xL led to increased antigen presentation to NKT cells. Conversely, inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Knockdown of Bcl-xL also resulted in the loss of CD1d trafficking to LAMP1+ compartments. Furthermore, Rab7, a late endosomal marker was upregulated following Bcl-xL knockdown, and CD1d molecules accumulated in the Rab7+ compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering the intracellular localization of CD1d. Thus, we have identified a new pathway involved in antigen presentation that can impact current therapies targeting the Bcl-2 family, as well as emerging NKT cell based cancer immunotherapeutic strategies.

Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes.

Erythropoiesis: from molecular pathways to system properties.

Erythropoiesis is regulated through a long-range negative feedback loop, whereby tissue hypoxia stimulates erythropoietin (Epo) secretion, which promotes an increase in erythropoietic rate. However, this long-range feedback loop, by itself, cannot account for the observed system properties of erythropoiesis, namely, a wide dynamic range, stability in the face of random perturbations, and a rapid stress response. Here, we show that three Epo-regulated erythroblast survival pathways each give rise to distinct system properties. The induction of Bcl-xL by signal transducer and activator of transcription 5 (Stat5) is responsive to the rate of change in Epo levels, rather than to its absolute level, and is therefore maximally but transiently activated in acute stress. By contrast, Epo-mediated suppression of the pro-survival Fas and Bim pathways is proportional to the levels of stress/Epo and persists throughout chronic stress. Together, these elements operate in a manner reminiscent of a "proportional-integral-derivative (PID)" feedback controller frequently found in engineering applications. A short-range negative autoregulatory loop within the early erythroblast compartment, operated by Fas/FasL, filters out random noise and controls a reserve pool of early erythroblasts that is poised to accelerate the response to acute stress. Both these properties have previously been identified as inherent to negative regulatory motifs. Finally, we show that signal transduction by Stat5 combines binary and graded modalities, thereby increasing signaling fidelity over the wide dynamic range of Epo found in health and disease.

Targeting Microenvironment-Mediated NFκb Activation With MLN4924, An Inhibitor Of The Nedd8-Activating Enzyme, In Chronic Lymphocytic Leukemia B Cells

Gene expression profiling identified lymph nodes as the site of cell activation and proliferation with upregulation of NFκB. B-cell receptor (BCR) and CD40 signaling enhances NFκB pathway activity in CLL cells resident in the microenvironment, leading to their survival, proliferation and chemoresistance. The NFκB pathway has been successfully targeted in CLL in vitro, yet lack of clinical advances with those agents necessitates development of novel approaches. Ubiquitination of IκBα may partially account for constitutive activation of NFκB in this setting. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme and thereby prevents neddylation of Cullin-RING ubiquitin ligases, resulting in a decrease of their activity and stabilization of their protein substrates. This study reports that MLN4924 abrogates pro-survival microenvironment stimuli in CLL cells in vitro.Primary B cells from 50 patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NFκB pathway activity, Bcl-2 family members and cell cycle was assessed by flow cytometry, western blotting, PCR and immunocytochemistry. MLN4924 was provided by Millennium Pharmaceuticals.Treatment with MLN4924 led to reduced neddylation of Cullin-RING ubiquitin ligases, accumulation of phospho-IκBa and induced modest apoptosis in CLL cells cultured off stroma (12.8±1.7% after a 24 hour incubation with 1 μM MLN4924 compared to untreated control). Incubation with the CD40L-expressing stroma resulted in activation of the canonical and non-canonical NFκB pathways in the CLL cells, induction of Mcl-1 and Bcl-xL, leading to protection from spontaneous apoptosis. This was accompanied by repression of Bim, a pro-apoptotic BH3-only protein of the Bcl-2 family. CD40L-expressing stroma rescued CLL cell from drug-induced apoptosis, including bendamustine, fludarabine, chlorambucil and CAL-101. By contrast, CD40L-expressing stroma failed to elicit resistance to MLN4924. Moreover, CLL cells cultured on stroma were more sensitive to Nedd8-activating enzyme inhibition compared to cells off stroma (1 μM MLN4924 induced apoptosis in 55.3±3.4% cells cultured on stroma). MLN4924 promoted apoptosis regardless of the common adverse prognostic factors (IGHV mutational status, cytogenetic markers, or ZAP-70 expression). In the presence of MLN4924, CLL cells demonstrated a significant decrease in nuclear translocation of the NFκB subunits p65 and p52 thus marking de-activation of both canonical and non-canonical pathways. Using gene microarray analysis we determined that receptor signaling and expression target pathways involving NFκB were most significantly associated with genes downregulated in CLL cells by MLN4924 (p<0.0001). We noted reduced transcription of several groups of NFκB target genes, including anti-apoptotic Bcl-2 family members and genes involved in cell cycle progression (p<0.01). Furthermore, treatment with MLN4924 resulted in a significant downregulation of a number of important cytokine ligands and receptors expressed by the CLL cells, including CCL5, CCL22, CXCR7, CXCR5 and CD40. Concomitantly, MLN4924 promoted induction of Bim and Noxa in the CLL cells which preceded apoptosis as evidenced by PARP cleavage. siRNA-mediated knockdown of either Bim or Noxa resulted in partial protection from MLN4924-mediated apoptosis irrespective of expression of the pro-survival Bcl-2 family members (Bcl-2, Bcl-xL or Mcl-1). This indicates that MLN4924 leads to rebalancing of Bcl-2 family members towards the pro-apoptotic BH3-only proteins in CLL. Furthermore, MLN4924 reversed stroma-mediated protection from fludarabine and the alkylators.In summary, we were able to abrogate the protective effect of the CLL microenvironment in vitro using MLN4924, a Nedd8-activating enzyme inhibitor. MLN4924 reduced NFκB activity in CLL cells, led to re-expression of the pro-apoptotic BH3-only proteins and interfered the chemokine network thus carrying a potential to disrupt cell homing. These results suggest a rationale for clinical investigation of MLN4924 as a novel tool to target microenvironment in CLL. Disclosures:No relevant conflicts of interest to declare.

Bcl-xL protects from UPR-associated apoptosis during plasma cell differentiation. (P1457)

Abstract Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. Plasma cell differentiation requires activation of the unfolded protein response, a set of signaling pathways in which prolonged activation leads to apoptosis in other cell types. Plasma cells activate this pathway during differentiation suggesting compensatory survival signals are activated. Consistent with this hypothesis, we found that LPS-driven differentiation protects Bcl1 cells against tunicamycin-activated ER stress-induced death. LPS plus cytokine-induced differentiation was associated with an increase in Bcl-xL expression accompanied by a decrease in Mcl-1 or Bcl-2 in Bcl1 cells and primary B cells respectively. Moreover, differentiation-induced protection against ER stress was shown to be Bcl-xL-dependent as tunicamycin-induced death was restored by the addition of the Bcl-xL/Bcl-2 inhibitor, ABT-737. Importantly, we also showed that during differentiation, Bcl1 cells and primary B cells are Bcl-xL-dependent as ABT-737 resulted in significant apoptosis in the absence of any additional death signal. Finally, we found that differentiation was accompanied by a change in Bim binding from primarily Mcl-1 and/or Bcl-2 to Bcl-xL in differentiating cells. These data demonstrate that Bcl-xL induction protects from death signals activated during plasma cell differentiation.

Mutually exclusive regulation of T cell survival by IL-7R and antigen receptor-induced signals

Two major processes govern T cell proliferation and survival: interleukin-7-mediated homeostasis and antigen-induced selection. How cells transit between the two states is unknown. Here we show that T cell receptor ligation actively inhibits homeostatic survival signals while initiating a new, dominant survival programme. This switch is mediated by a change in the expression of pro- and anti-apoptosis proteins through the downregulation of Bcl-2 and the induction of Bim, A1 and Bcl-xL. Calcineurin inhibitors prevent the initiation of the new survival programme, while permitting the dominant repression of Bcl-2. Thus, in the presence of these drugs the response to antigen receptor ligation is cell death. Our results identify a molecular switch that can serve as an attractive target for inducing antigen-specific tolerance in treating autoimmune disease patients and transplant recipients.

Anti-Apoptotic Effect of Human Telomerase Reverse Transcriptase on Endothelial Cells under Oxidative Stress, Independent of Telomere Elongation and Telomerase Activity

Increasing age and replicative senescence with critical shortening of telomeres is associated with endothelial dysfunction followed by atherosclerosis. Although human telomerase reverse transcriptase (hTERT) is suggested to play an important role in endothelial cells, the precise effect of hTERT is still uncertain. In this study we investigated telomerase- independent hTERT function against oxidative stress using a primary culture of endothelial cells in which telomerase enzymatic activity was inhibited by addition of 3’-azido-3’-deoxythymidine (AZT). Overexpression of hTERT attenuated H2O2-induced endothelial apoptosis, assessed by cell viability assay and flow cytometry, accompanied by significant reduction of caspase-3 activity. Of importance, it was suggested that this effect was independent of telomerase enzymatic activity because hTERT expression did not alter telomere length under addition of AZT. Also, suppression of human telomerase-associated protein 1 (TEP-1) by introduction of TEP-1-specific small interfering RNA (siRNA) did not affect the anti-apoptotic effect of hTERT, suggesting that hTERT does not cooperate with TEP-1, another component of telomerase. Moreover, the anti-apoptotic effect of hTERT was through attenuation of H2O2-induced upregulation of p53, followed by reduction of Bax and induction of Bcl-xL. Also, overexpression of hTERT resulted in activation of the transcription factor, NFκB, which is an anti-apoptotic factor. Altogether, the present study demonstrated the anti-apoptotic effect of not telomerase but hTERT itself on cultured endothelial cells under oxidative stress, independent of telomere elongation, followed by acquired replicative ability. Also, this function of hTERT did not require another protein component of telomerase. Moreover, the anti-apoptotic effect of hTERT on endothelial cells was mediated by two pathways; reduction of p53/Bax and activation of NFκB. Keywords: Apoptosis, atherosclerosis, AZT, Bax, Bcl-xL, caspase-3, endothelial cell, hTERT, NFκB, oxidative stress, p53, telomerase, telomere length, TEP-1.

Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation

Abstract 3288Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures:No relevant conflicts of interest to declare.

BCL-Xl Contributes to Decreased Cellular Respiration During Erythroid Differentiation

AbstractAbstract 3201The BCL2 family of proteins are well known for their ability to both positively and negatively regulate mitochondrial mechanisms of apoptosis. The anti-apoptotic members of this family can decrease mitochondrial outer membrane permeability and cytochrome c release. By stabilizing the cell against apoptosis, these proteins allow cell survival even in states of low energy. However, despite this intimate link between the BCL2 family proteins and mitochondria, their direct effect on metabolism is less clearly understood. It is generally expected that metabolic changes induced by the BCL2 family of proteins will further impact cell survival as murine hepatoma and cancer cells overexpressing Bcl-xL are sensitive to Bcl-xL inhibition1 but BCL-xL is also known to be essential for erythroid differentiation and more recently was linked specifically to heme synthesis2. We therefore set out to investigate whether there was a connection between BCL-xL induced changes in cellular respiration and erythroid differentiation.Murine erythroleukemia (MEL) cells were differentiated by exposure to 2% DMSO for 5 days and then real time oxygen consumption was measured on the Seahorse extracellular flux analyzer (XFA). DMSO induced differentiation yielded a 4-fold decrease in oxygen consumption. Western blot analysis revealed that BCL-xL was induced during differentiation. We then generated cell lines in which BCL-xL was knocked down with small hairpin RNA (shRNA). As differentiation has previously been reported to be fatal in MEL cells without BCL-xL activity, both parental cells and BCL-xL knockdowns were infected with a vector over expressing BCL2. Differentiation over 5 days with 2% DMSO was performed on these new cell lines. Erythroid differentiation was confirmed using Benzidine staining. While the control cell line showed high rates of Benzidine staining after exposure to DMSO, the BCL-xL knockdown cell line consistently showed <5% benzidine positivity. Western blot analysis confirmed the absence of BCL-xL induction by DMSO exposure in the knockdown cell line. Using the Seahorse XFA the control cell line was shown to have significant decrease in oxygen consumption when exposed to DMSO, while DMSO exposed BCL-xL knockdown cells showed less than half this drop in oxygen consumption. However, both control and BCL-xL knockdowns have limited respiratory reserve as the response to CCCP, an uncoupler of electron transport, is diminished after DMSO exposure as compared to their undifferentiated counterparts.Our results suggest that erythroid differentiation is associated with a significant decrease in cellular respiration. Although, not the only contributor to the decreased dependence on oxidative phosphorylation of cells undergoing erythroid differentiation, BCL-xL expression is clearly a necessary factor. Our data is able to connect BCL-xL expression to both erythroid differentiation and this distinct metabolic phenotype. As BCL-xL's role in erythroid differentiation has previously been reported to be associated with heme synthesis, future work will focus on identifying oxidative metabolic pathways associated with BCL-xL expression and heme synthesis. Disclosures:No relevant conflicts of interest to declare.

A role for Bcl-xL in CD1d-mediated Natural Killer T cell responses to B cells (46.39)

Abstract Lymphomas are a heterogeneous group of malignancies, primarily divided into Hodgkin's and non-Hodgkin's lymphomas (NHL). Unlike other cancers, the incidence of NHL is steadily rising. Natural Killer T (NKT) cells are innate-like lymphocytes that recognize glycolipid antigens in the context of CD1d, an MHC class I-like molecule. They have strong anti-tumor activity and respond by massive cytokine production and/or cytotoxicity. It is known that pro-survival factors are upregulated in B cell lymphomas, but their role in antigen processing and presentation remains unknown. The effect of Bcl-xL on CD1d-mediated antigen presentation was examined utilizing the well-characterized WEHI-231 B cell lymphoma cell line. Bcl-xL expression was modulated via genetic, pharmacologic and biological approaches. We found a concomminant increase in CD1d-mediated NKT cell responses following the induction of Bcl-xL; however, cell surface expression of CD1d molecules remained unchanged. Induction of Bcl-xL in primary B cells led to increased CD1d-mediated activation of NKT cells in response to exogenous antigens, resulting in increased Th1 not Th2 responses. Taken together, these data suggest that aberrant expression of Bcl-xL may act as a sentry system to alert the CD1d/NKT system to neoplastic transformation.

Interleukin-22 Ameliorates Cerulein-Induced Pancreatitis in Mice by In-hibiting the Autophagic Pathway (44.15)

Abstract Pancreatitis occurs when digestive enzymes are activated in the pancreas. Severe pancreatitis has a 10-30% mortality rate. No specific treatments for pancreatitis exist now. Here, we discovered that interleukin-22 (IL-22) may have therapeutic potential in treating acute and chronic pancreatitis. Wild-type and IL-22 knockout mice were equally susceptible to ceru-lein-induced acute and chronic pancreatitis, whereas liver-specific IL-22 transgenic mice were completely resistant to cerulein-induced elevation of serum digestive enzymes, pancreatic necrosis and apoptosis, and inflammatory cell infiltration. Treatment of wild-type mice with recombinant IL-22 or adenovirus IL-22 markedly attenuated the severity of cerulein-induced acute and chronic pancreatitis. Mechanistically, we show that the protective effect of IL-22 on pancreatitis was mediated via the induction of Bcl-2 and Bcl-XL, which bind to Beclin-1 and subsequently inhibit autophagosome formation to ameliorate pancreatitis. In conclusion, IL-22 ameliorates cerulein-induced pancreatitis by inhibiting the autophagic pathway. IL-22 could be a promising therapeutic drug to treat pancreatitis.

Myb overexpression overrides androgen depletion-induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

AbstractSurvival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-xL is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-xL pathway “resets,” allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using “knock-in” mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-xL response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-xL induction provides a “stop-gap” in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-xL may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.

Relative role of hepatocyte growth factor in mediating epithelial cell-protective effects in lung fibrosis – a role of Bcl-xL induction

Einleitung: Hepatocyte growth factor (HGF) is a cytokine with pleiotropic functions during wound healing and repair. Anti-fibrotic effects have been shown in animal models of lung fibrosis and may be mediated via multiple direct and indirect mechanisms, including promoting endothelial and epithelial cell survival and proliferation and reduction of myofibroblast accumulation. In liver and kidney epithelial cells the increased expression of Bcl-xL protein has been suggested as an important anti-apoptotic mechanism, but it remains to be elucidated in the lung.

Molecular investigation of the effects of lindane in rat hepatocytes: Microarray and mechanistic studies

Although many studies of lindane toxicity have been carried out, we still know little about the underlying molecular mechanisms. We used a microarray specifically designed for studies of the hepatotoxic effects of xenobiotics to evaluate the effects of lindane on specific gene expression in primary cultured rat hepatocytes. These genes were assigned to detoxication processes (CYP3A4, Gsta2, CYP4A1), cell signalling pathways and apoptosis (Eif2b3, Eif2b4, PKC). In this study, we demonstrate that lindane up-regulates PKC by increasing oxidative stress. TEMPO (a well known free radical scavenger) and Ro 31-8220 (an inhibitor of classical PKCs) prevented the inhibition of spontaneous and intrinsic apoptosis pathway (characterised by Bcl-xL induction, Bax down-regulation, caspases inhibition) and the induction of necrosis by lindane in rat hepatocytes. Thus, these findings indicate that several dependent key signalling pathways, including detoxification, apoptosis, PKC activity and redox status maintenance, contribute to lindane-induced toxicity in primary cultured rat hepatocytes. This may account more clearly for the acute and chronic effects of lindane in vivo, with the induction of cell death and tumour promotion, respectively.

Activation of Akt/GSK3β and Akt/Bcl-2 signaling pathways in nickel-transformed BEAS-2B cells

The Akt signaling pathway has been implicated in a wide range of cellular functions involving cell survival and proliferation, angiogenesis, metabolism and cell migration. Accumulating evidence suggests that Akt perturbations play an important role in human malignancy. Here, we investigated Akt perturbation in nickel-transformed cells. Chronic treatment of human bronchial epithelial BEAS-2B cells with low doses of nickel chloride resulted in cell transformation demonstrated by anchorage-independent (AI) growth, increased cell growth and alterations of cell growth pattern. Western blot assays show that phosphorylation of Akt at Ser473, but not that of p38, JNK and ERK, was increased in nickel-transformed cells compared with controls. Inhibition of Akt or PI3K by pharmacological or biochemical interference suppressed nickel AI growth and cell growth of transformed cells. Activation of Akt led to inhibition of GSK3β by phosphorylation at Ser9 in nickel-transformed cells. In addition, two major anti-apoptotic proteins of the Bcl family, Bcl-2 and Bcl-XL, were increased in nickel-transformed cells. By employing the small interfering RNA technique (siRNA), our results showed that siRNA Akt attenuated the expression of Bcl-2 and Bcl-XL in nickel-transformed cells, indicating that induction of Bcl-2 and Bcl-XL was likely mediated through Akt. ROS generation was decreased in nickel-transformed cells compared with controls. Moreover, down-regulation of retinoblastoma protein (Rb) was observed in nickel-transformed cells. Taken together, these findings demonstrate that activation of Akt, followed by GSK3β inhibition and Bcl-2, Bcl-XL up-regulation and decrease of ROS generation, along with a synergistic effect of Rb down-regulation may cause apoptosis resistance, contributing to the overall mechanism of nickel carcinogenesis.

Role of antiapoptotic STAT3 and BCL-2 signaling in promoting lung tumor cells to survive against combined MET-/EGFR-targeted inhibitors.

10644 Background: Epidermal growth factor receptor (EGFR) inhibitors in lung cancer are largely limited by the development of resistant disease that ultimately limits their clinical utility. Combined targeted therapy strategies using both MET and EGFR small molecular tyrosine kinase inhibitors (TKIs) are under preclinical and clinical investigation to enhance the therapeutic efficacy and to circumvent acquired EGFR TKI resistance in lung cancer. These combined therapeutic approaches are likely to have unique mechanisms of resistance. Methods: We investigated the effects of combined MET/EGFR TKI treatment on signal transducer and activator of transcription 3 (STAT3) and B-cell lymphoma protein 2 (BCL-2) family members in lung cancer cell lines and xenografts models that are resistant to EGFR inhibitors. Results: Activated STAT3 and BCL-2/BCL-XL induction were seen in lung tumor cells that survived targeted inhibition by MET kinase inhibitors (SU11274 and PHA665752), either alone or in combination with EGFR inhibitors. Using in vivo xenograft model with luciferase-expressing H1975 cells for bioluminescence imaging (BLI), we show that while the H1975 xenograft exhibited excellent imaging tumor response when treated with combined SU11274 (MET TKI) and erlotinib (EGFR TKI), activated P-STAT3[Y705] expression was evident in the residual microscopic tumor survivor cells within the treated xenograft. Interestingly, the resistant survivor tumor cells were located predominantly along the peripheral rind juxtaposing the host stroma. BCL-2 expression was also found induced in the tumor survivor cells during the course of targeted combined MET/EGFR inhibition in vivo. Conclusions: Results of our study support the role of activated STAT3, and its downstream transcriptional targets of the antiapoptotic BCL-2 family members as potential markers for tumor resistance against combined MET/EGFR targeted inhibitors in lung cancer. Further investigations to target the intrinsic apoptotic pathway with BCL-2 family inhibitors, which may overcome or prevent resistance against combined MET/EGFR TKIs, are warranted to impact on the long term outcome in lung cancer therapy. No significant financial relationships to disclose.

The catalytic PI3K isoforms p110γ and p110δ contribute to B cell development and maintenance, transformation, and proliferation

Class I PI3K-dependent signaling regulates cell proliferation, differentiation, and survival. Analysis of gene-deficient mice revealed specific roles for the hematopoietically expressed PI3K catalytic subunits, p110gamma and p110delta, in development and function of T and B lymphocytes. However, the functional redundancy between these two PI3K isoforms in the B cell lineage remains unclear. Here, we demonstrate that p110delta and p110gamma are expressed in B cells at early developmental stages. Normal B cell differentiation requires both isoforms, as p110gamma/p110delta double deficiency causes an increased percentage of CD43(hi)/B220(+)/CD19(-) cells as compared with single deficiency. Interestingly, initial transformation efficiency of B cell precursors was strongly reduced in double-deficient cells following transformation by p185 bcr-abl or v-abl oncogenes as compared with single-deficient cells. The requirement of p110gamma and p110delta in B cell development is underlined by reduced splenic B cell numbers of p110gamma/p110delta double-deficient mice and of lethally irradiated wild-type mice reconstituted with double-deficient BM. Moreover, the peripheral maintenance of p110gamma/p110delta double-deficient T and B cells was highly impaired following adoptive transfer of double-deficient splenocytes into wild-type mice. Functionally, LPS stimulation of splenocytes revealed proliferation defects resulting in decreased survival of p110gamma/p110delta double-deficient B cells, which correlated with impaired induction of D-type cyclins and Bcl-X(L). Surprisingly, this was not observed when purified B cells were analyzed, indicating a contribution of likely cell-extrinsic factor(s) to the impaired proliferation of double-deficient B cells. Thus, we provide novel evidence that p110gamma and p110delta have overlapping and cell-extrinsic roles in the development, peripheral maintenance, and function of B cells.