Pro-survival Activity Research Articles

Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis

AbstractAbstract 204Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures:No relevant conflicts of interest to declare.

Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo

AbstractAccumulation of leukemic cells in patients with chronic lymphocytic leukemia (CLL) is due to prolonged cell survival rather than increased proliferation. Survival of CLL cells depends on microenvironmental factors. Even though long-lived in vivo, CLL cells rapidly die by spontaneous apoptosis in vitro unless cocultured with stromal cells or their conditioned medium. In the present study, we show that survival of CLL cells is maintained in high cell density cultures, where the main prosurvival activity is delivered by monocytes. Cytokine array and enzyme-linked immunosorbent assay studies revealed increased expression of soluble CD14 by monocytes in the presence of CLL cells. The addition of recombinant soluble CD14 to primary CLL cells resulted in significantly increased cell survival rates, which were associated with higher activity nuclear factor κB. Quantification of serum levels of soluble CD14 revealed abnormally high levels of this protein in CLL patients, indicating a potential role of soluble CD14 in vivo. In summary, the presented data show that monocytes help in the survival of CLL cells by secreting soluble CD14, which induces nuclear factor κB activation in these cells, and that CLL cells actively shape their microenvironment by inducing CD14 secretion in accessory monocytes.

Macrophage-induced preadipocyte survival depends on signaling through Akt, ERK1/2, and reactive oxygen species

Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction. We propose that these effects are dependent on an as yet unrecognized role for CD31-mediated homophilic interactions between T cells and antigen-presenting cells (APCs) during priming. We show that loss of CD31 interactions leads to enhanced primary clonal expansion, increased killing capacity, and diminished regulatory functions by T cells. Immunomodulation by CD31 signals correlates with a partial inhibition of proximal T-cell receptor (TCR) signaling, specifically Zap-70 phosphorylation. However, CD31-deficient mice do not develop autoimmunity due to increased T-cell death following activation, and we show that CD31 triggering induces Erk-mediated prosurvival activity in T cells either in conjunction with TCR signaling or autonomously. We conclude that CD31 functions as a nonredundant comodulator of T-cell responses, which specializes in sizing the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death.

Paeonol Oxime Inhibits bFGF-Induced Angiogenesis and Reduces VEGF Levels in Fibrosarcoma Cells

BackgroundWe previously reported the anti-angiogenic activity of paeonol isolated from Moutan Cortex. In the present study, we investigated the negative effect of paeonol oxime (PO, a paeonol derivative) on basic fibroblast growth factor (bFGF)-mediated angiogenesis in human umbilical vein endothelial cells (HUVECs) (including tumor angiogenesis) and pro-survival activity in HT-1080 fibrosarcoma cell line.Methodology/Principal FindingsWe showed that PO (IC50 = 17.3 µg/ml) significantly inhibited bFGF-induced cell proliferation, which was achieved with higher concentrations of paeonol (IC50 over 200 µg). The treatment with PO blocked bFGF-stimulated migration and in vitro capillary differentiation (tube formation) in a dose-dependent manner. Furthermore, PO was able to disrupt neovascularization in vivo. Interestingly, PO (25 µg/ml) decreased the cell viability of HT-1080 fibrosarcoma cells but not that of HUVECs. The treatment with PO at 12.5 µg/ml reduced the levels of phosphorylated AKT and VEGF expression (intracellular and extracelluar) in HT-1080 cells. Consistently, immunefluorescence imaging analysis revealed that PO treatment attenuated AKT phosphorylation in HT-1080 cells.Conclusions/SignificanceTaken together, these results suggest that PO inhibits bFGF-induced angiogenesis in HUVECs and decreased the levels of PI3K, phospho-AKT and VEGF in HT-1080 cells.

Altered Retinoid Uptake and Action Contributes to Cell Survival in Endometriosis

Retinoic acid (RA) controls multiple biological processes via exerting opposing effects on cell survival. Retinol uptake into cells is controlled by stimulated by RA 6 (STRA6). RA is then produced from retinol in the cytosol. Partitioning of RA between the nuclear receptors RA receptor α and peroxisome-proliferator-activated receptor β/δ is regulated by cytosol-to-nuclear shuttling proteins cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5), which induce apoptosis or enhance survival, respectively. The roles of these mechanisms in endometrium or endometriosis remain unknown. The aim was to determine the regulation of retinoid uptake and RA action in primary stromal cells from endometrium (n = 10) or endometriosis (n = 10). Progesterone receptor was necessary for high STRA6 and CRABP2 expression in endometrial stromal cells. STRA6, which was responsible for labeled retinoid uptake, was strikingly lower in endometriotic cells compared to endometrial cells. CRABP2 knockdown in endometrial cells increased survival, and FABP5 knockdown in endometriotic cells decreased survival without altering the expression of downstream nuclear retinoic acid receptor α and peroxisome-proliferator-activated receptor β/δ. In endometrial stromal cells, progesterone receptor up-regulates expression of STRA6 and CRABP2, which control retinol uptake and growth-suppressor actions of RA. In endometriotic stromal cells, decreased expression of these genes leads to decreased retinol uptake and dominant FABP5-mediated prosurvival activity.

Prosurvival activity for airway neutrophils in severe asthma

BackgroundAirway neutrophilia is a recognised feature of chronic severe asthma, but the mechanisms that underlie this phenomenon are unknown. Evidence for factors present in airway secretions that prolong neutrophil survival...

Transgenic expression of human wild‐type amyloid precursor protein decreases neurogenesis in the adult hippocampus

Besides its role in Alzheimer's disease, the amyloid precursor protein (APP) is implicated in several physiological functions in neuronal tissue such as cell survival, neurite outgrowth, synaptic formation, and neuronal plasticity. The present study analyzed effects of human wild-type APP (hAPP) overexpression on adult hippocampal neurogenesis in transgenic mice. Mice were housed under either standard or enriched conditions, the latter to boost neurogenetic activity. Different aspects of neurogenesis including proliferation, survival, and differentiation were assessed by employing the BrdU-incorporation method and, in parallel, immunohistochemistry for the neuronal and glial markers NeuN and S100b, respectively. Overexpression of hAPP caused a significant decrease in cell proliferation under standard housing conditions. The relative increase in the proliferation rate following housing in enriched environment was not different to that observed in wild-type mice. Overexpression of hAPP, on the other hand, promoted the survival of newly generated cells, but just under conditions of standard housing. Findings further suggest that overexpression of hAPP suppresses the phenotypic shift toward neuronal differentiation under conditions of enriched environment. In summary, the results reveal a dual effect of APP on adult hippocampal neurogenesis, comprising antiproliferative and prosurvival activities.

Aven blocks DNA damage-induced apoptosis by stabilising Bcl-xL

Induction of apoptosis by DNA-damaging agents involves the activation of mitochondrial apoptotic pathway. Aven has been identified as an antiapoptotic protein and has been shown to activate ATM in response to DNA damage. In this study, we demonstrated that enforced expression of Aven blocks UV-irradiation-, SN-38- or cisplatin-induced apoptosis upstream of mitochondria by stabilising Bcl-xL protein levels in breast cancer cells. Aven silencing by RNA interference markedly enhanced apoptotic response following treatment with DNA-damaging agents. Aven is complexed with Bcl-xL in untreated breast cancer cells and treatment with DNA-damaging agents led to decreased Aven/Bcl-xL interaction. Importantly, Bcl-xL was necessary for the prosurvival activity of Aven and depletion of Bcl-xL abrogated Aven-mediated protection against DNA damage-induced apoptosis. Analysis of breast cancer tissue microarrays revealed decreased Aven nuclear expression in breast cancer tissues compared with non-neoplastic breast tissues. In particular, we detected reduced nuclear expression of Aven in infiltrating ductal carcinoma and papillary carcinoma breast cancer subtypes compared with non-neoplastic breast tissues and infiltrating lobular breast cancer tissues. Our results suggest that Aven is an important mediator in DNA damage-induced apoptotic signalling in breast cancer cells and its nuclear expression is altered in breast cancer tissues, which may contribute to genomic instability in breast cancer tumours.

Casein Kinase II: An attractive target for anti-cancer drug design

Casein Kinase II (CK2) is a ubiquitous serine/threonine kinase that is highly conserved in eukaryotic cells. CK2 has been shown to impact cell growth and proliferation, as numerous growth-related proteins are substrates of CK2. More importantly, experimental evidence linking increased expression and activity of CK2 to human cancers underscores the relevance of CK2 biology to cellular transformation and carcinogenesis. Due to the critical regulatory role CK2 plays in cell fate determination in cancer cells, there is a tremendous interest in the development of CK2-specific therapies. Supporting this, recent reports have demonstrated that genetic manipulation of CK2 expression as well as pharmacological inhibition of its enzymatic activity sensitizes cancers to apoptotic stimuli. Here we provide a succinct account of the biology of CK2, its cellular substrates, its pro-survival and pro-proliferation activity, and highlight evidence for its involvement in human cancer.

Abstract 1394: Pro-apoptotic and anti-tumorigenic effects of a naturally occurring proteasome inhibitor on hormone-refractory prostate cancer

Abstract A major mode by which cancer cells evade death is by an acquired ability to suppress apoptosis. In hormone refractory prostate cancer, the overexpression of pro-survival/anti-apoptotic proteins results in resistance to chemo- and radio-therapy. Hence, developing co-therapies that sensitize cells to standard treatment regimens by overcoming this apoptotic block could improve treatment outcomes in advanced prostate cancer. The naturally occurring proteasome inhibitor celastrol has been previously reported to inhibit DNA damage processing and synergize with radiotherapy to kill PC-3 cells. To further explore the mechanism by which celastrol might potentiate cell death, several lines of questioning were explored. First, the open question regarding whether or not celastrol inhibits DNA damage repair, not simply processing, was tested in the comet assay. The tail moments measured in cells treated with celastrol, irradiated and allowed to recover were significantly greater than those in untreated cells, indicating that DNA damage repair was inhibited. In other experiments, cells treated with celastrol displayed an increased expression of the pro-apoptotic, BH3 only protein Noxa. This correlated with an increase in apoptotic cell death which was further augmented by ionizing radiation treatment. The pro-survival protein NFkB is tightly controlled by IkB-alpha binding. Proteasome inhibition can suppress the pro-survival activity of NFkB by decreasing the turnover of IkB-alpha. Indeed, IkB-alpha was found to accumulate in celastrol treated cells. Further, NFkB target gene activation was markedly reduced. To test the feasibility of celastrol treatment in animals, the PC-3 xenograft model in the athymic nude mouse was employed. When celastrol was combined with radiotherapy, tumor volume decreased, apoptosis increased, angiogenesis decreased, and NFkB target gene mRNA levels were found to be lower. Taken together, these data show that celastrol sensitized PC-3 cells to radiation both in vitro and in vivo by impairing DNA damage repair and augmenting apoptosis through suppressing NFkB activation and increasing the protein levels of Noxa. Celastrol may hold promise as a co-therapy with radiation for the treatment of hormone-refractory prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1394.

DYRK1A and DYRK3 Promote Cell Survival through Phosphorylation and Activation of SIRT1

DYRK1A (the dual specificity tyrosine phosphorylation-regulated kinase 1A) plays an important role in body growth and brain physiology. Overexpression of this kinase has been associated with the development of Down syndrome in both human and animal models, whereas single copy loss-of-function of DYRK1A leads to increased apoptosis and decreased brain size. Although more than a dozen of DYRK1A targets have been identified, the molecular basis of its involvement in neuronal development remains unclear. Here we show that DYRK1A and another pro-survival member of the DYRK family, DYRK3, promote cell survival through phosphorylation and activation of SIRT1, an NAD(+)-dependent protein deacetylase that is essential in a variety of physiological processes including stress response and energy metabolism. DYRK1A and DYRK3 directly phosphorylate SIRT1 at Thr(522), promoting deacetylation of p53. A SIRT1 phosphorylation mimetic (SIRT1 T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (SIRT1 T522V) fails to mediate DYRK-induced deacetylation of p53 and cell survival. We show that knockdown of endogenous DYRK1A and DYRK3 leads to hypophosphorylation of SIRT1, sensitizing cells to DNA damage-induced cell death. We also provide evidence that phosphorylation of Thr(522) activates SIRT1 by promoting product release, thereby increasing its enzymatic turnover. Taken together, our findings provide a novel mechanism by which two anti-apoptotic DYRK members promote cell survival through direct modification of SIRT1. These findings may have important implications in understanding the molecular mechanisms of tumorigenesis, Down syndrome, and aging.

The p75 neurotrophin receptor, semaphorins, and sympathetic traffic in the heart

the sympathetic and parasympathetic branches of the autonomic nervous system control cardiac function through the coordinated activity generated by neurons within the intracardiac autonomic ganglia and central nervous system (3). Activation of the sympathetic efferent projections to the heart increases heart rate, modifies the pattern and conduction velocity of the impulse through the heart, and augments both atrial and ventricular contractility while hastening myocardial relaxation. In the vasculature, the sympathetic nervous system (SNS) reduces venous capacitance and constricts resistance vessels, increasing blood pressure. Conversely, the parasympathetic nervous system (PSNS) decreases heart rate and cardiac impulse conduction. More recent evidence has suggested that the PSNS may also affect ventricular function, indirectly by countering β-adrenergic action or directly via inhibition of L-type Ca2+ channels (34). A transient imbalance between these two branches can lead to increased heart rate and blood pressure when cardiac output is increased. When subnormal cardiac PSNS activity concomitant with sympathetic hyperactivation persists, such as after myocardial infarction, this disequilibrium may increase the risk for cardiac adverse events, including arrhythmias, hypertension, cardiac hypertrophy, and, ultimately, heart failure. This condition is called “autonomic neural remodeling” and can greatly contribute to the induction of ventricular fibrillation, thus increasing the patient's susceptibility to sudden cardiac death (41, 47). It is plausible that neurotrophic factors, target organ-secreted proteins essential for the development and differentiation of neurons, are involved in the remarkable neuroplasticity exhibited by the SNS in the adult life, including autonomic remodeling after infarction and/or in the onset and progression of congestive heart failure (17, 30, 32).

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Human Herpesvirus 8-Encoded Cytokines

Human herpesvirus (HHV)-8, also called Kaposi's sarcoma-associated herpesvirus, was discovered in 1994 and was rapidly sequenced, revealing several unique and surprising features of its genetic makeup. Among these discoveries was the identification of the first viral homolog of IL-6 and three CC/beta-chemokine ligands (viral CCL-1, -2 and -3), not previously found in gamma-herpesviruses. Viral IL-6 was immediately recognized as a potential contributor to HHV-8 pathogenesis, specifically endothelial-derived Kaposi's sarcoma and the B-cell malignancy multicentric Castleman's disease with which IL-6, a proangiogenic and B-cell growth factor, had previously been implicated. The roles of the viral chemokines were speculated to involve immune evasion; however, like viral IL-6, the viral chemokines have the potential to contribute to pathogenesis through their shared angiogenic activities, known to be important for Kaposi's sarcoma and HHV-8-associated primary effusion lymphoma, and also via direct prosurvival activities. This article will discuss the molecular properties, activities and functions of viral IL-6 and the viral CCLs, proteins that could provide appropriate targets for antiviral and therapeutic strategies.

The prosurvival activity of ascites against TRAIL is associated with a shorter disease-free interval in patients with ovarian cancer.

BackgroundThe production of ascites is a common complication of ovarian cancer. Ascites constitute a unique tumor microenvironment that may affect disease progression. In this context, we recently showed that ovarian cancer ascites may protect tumor cells from TRAIL-induced apoptosis. In this study, we sought to determine whether the prosurvival effect of ascites affects disease-free intervals.MethodsPeritoneal fluids were obtained from 54 women undergoing intra-abdominal surgery for suspected ovarian cancer (44 cancers and 10 benign diseases). The ability of peritoneal fluids to protect from TRAIL was assessed in the ovarian cancer cell line CaOV3, and IC50 were determined. The anti-apoptotic activity of 6 ascites against cisplatin, paclitaxel, doxorubicin, etoposide and vinorelbine was also assessed in CaOV3 cells, and the prosurvival activity of two ascites was assessed in 9 primary ovarian cancer cultures.ResultsAmong the 54 peritoneal fluids tested, inhibition of TRAIL cytotoxicity was variable. Fluids originating from ovarian cancer were generally more protective than fluids from non-malignant diseases. Most of the 44 ovarian cancer ascites increased TRAIL IC50 and this inhibitory effect did not correlate strongly with the protein concentration in these ascites or the levels of serum CA125, a tumor antigen which is used in the clinic as a marker of tumor burden. The effect of ascites on cisplatin- and paclitaxel-induced cell death was assessed with 4 ascites having inhibitory effect on TRAIL-induced cell death and 2 that do not. The four ascites with prosurvival activity against TRAIL had some inhibitory on cisplatin and/or paclitaxel. Two ovarian cancer ascites, OVC346 and OVC509, also inhibited TRAIL cytotoxicity in 9 primary cultures of ovarian tumor and induced Akt activation in three of these primary cultures. Among a cohort of 35 patients with ascites, a threshold of TRAIL IC50 with ascites/IC50 without ascites > 2 was associated with shorter disease-free interval.ConclusionsThe prosurvival activity of ascites against TRAIL is associated with shorter disease-free interval, which may be explained, at least in part, by ascites-induced cisplatin/paclitaxel resistance. Our findings suggest that ascites may contain prosurvival factors that protect against TRAIL and chemotherapy and consequently affect disease progression.

Different activation forms of MMP-2 oppositely affect the fate of endothelial cells

Detachment of endothelial cells (ECs) from the extracellular matrix (ECM) is required not only for angiogenesis, but also for EC apoptosis. Matrix metalloproteinase (MMP)-2 plays a major role in the degradation of the ECM, supporting an essential role for this enzyme in both survival (angiogenesis) and death of ECs. Our aim was to study these seemingly paradoxical effects of MMP-2. We rationalized that inhibiting apoptosis would drive MMP-2 toward a prosurvival activity, clarifying the mechanisms involved. By employing specific inhibitors to two major apoptotic pathways in ECs, caspases and p38 MAPK (p38), we demonstrated that they differently affected EC behavior as well as MMP-2 expression. The p38 pathway appears to enhance MMP-2 synthesis, its partial ("intermediate") and its full activation, probably via membrane type (MT)1-MMP, while caspases enhance MMP-2 synthesis and full activation but reduce MT1-MMP and MMP-2 intermediate form. Evaluation of the reciprocal influences of MMP-2 on ECs showed that the intermediate form supported survival and migration, and the fully active form led to cell death. In addition, a pro- and intermediate form-rich environment, even in the presence of the fully active form, exerted protective effects. Thus the seemingly conflicting effects of MMP-2 on EC survival may be explained by the ratio between the MMP-2 activation forms. A regulatory loop between active MMP-2 and p38 but not between MMP-2 and caspases was also observed, suggesting that MMP-2 is downstream to caspases where it serves as an "exterminator" molecule. Altogether, modification of caspase and p38 pathways, via changes of local MMP-2, affect survival and angiogenic steps in ECs.

Genetic dissection of the miR-17∼92 cluster of microRNAs in Myc-induced B-cell lymphomas

The miR-17 approximately 92 cluster is frequently amplified or overexpressed in human cancers and has emerged as the prototypical oncogenic polycistron microRNA (miRNA). miR-17 approximately 92 is a direct transcriptional target of c-Myc, and experiments in a mouse model of B-cell lymphomas have shown cooperation between these two oncogenes. However, both the molecular mechanism underlying this cooperation and the individual miRNAs that are responsible for it are unknown. By using a conditional knockout allele of miR-17 approximately 92, we show here that sustained expression of endogenous miR-17 approximately 92 is required to suppress apoptosis in Myc-driven B-cell lymphomas. Furthermore, we show that among the six miRNAs that are encoded by miR-17 approximately 92, miR-19a and miR-19b are absolutely required and largely sufficient to recapitulate the oncogenic properties of the entire cluster. Finally, by combining computational target prediction, gene expression profiling, and an in vitro screening strategy, we identify a subset of miR-19 targets that mediate its prosurvival activity.

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema.

Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.

Nicotine, through upregulating pro‐survival signaling, cooperates with NNK to promote transformation

Cigarette smoking is a mixture of thousands of compounds, many of which are carcinogens, such as NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Nicotine, as an addictive substance in cigarette, has been shown to promote growth of non-neuronal cells. It is unclear how nicotine cooperates with tobacco-related carcinogens during tumorigenesis. Here, by concurrent treatment of nicotine and NNK, we investigate the effect of the cooperation of these two compounds on cell growth and apoptosis in various different lung epithelial (RLE) or cancer (LKR) cells. We demonstrated that short-term nicotine exposure moderately activated mitogenic signaling pathways (such as PKC, ERK, and Akt) and a mediocre protection against cisplatin-mediated apoptosis. In contrast, NNK strongly stimulated mitogenic signaling and rendered the cells a high resistance to cisplatin. The pre-ligation of nAChR by nicotine interfered with NNK-mediated mitogenic signaling and resistance to cisplatin, the magnitude of which was similar as that exposed to nicotine alone. Interestingly, a week after the exposure to nicotine or nicotine plus NNK, Bcl-2 expression was augmented, accompanied with the increased resistance to cisplatin-induced apoptosis. In comparison, long-term NNK treatment provided very little protection of the cells from cisplatin. We also showed that the combination treatment promoted more cells to grow in an anchorage-independent fashion than NNK exposure alone. Thus, the data suggest that through occupying nAChR, nicotine appears to modulate NNK-mediated signaling and persistently sustain pro-survival activities to promote transformation.