Bcl-xL mRNA Research Articles

Downregulation of STAT3 signaling induces apoptosis but also promotes anti-apoptotic gene expression in human pancreatic cancer cell lines

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of cytokine signaling pathways that regulates gene expression. In pancreatic cancer, constitutive activation of STAT3 contributes to oncogenesis by preventing apoptosis through upregulation of anti-apoptotic proteins. We have examined the inhibition of STAT3 as a potential therapeutic approach in pancreatic cancer. siRNA targeting STAT3 was used to evaluate the role of STAT3 in modulating the expression of Survivin/BIRC5 and BCL-xL in the pancreatic cancer cell lines PANC-1 and BxPC-3 and induction of apoptosis. Expression of STAT3, Survivin/BIRC5, and BCL-xL on mRNA and protein level was measured by real-time RT-PCR and Western blot analysis 24, 48, and 72 h after transfection. STAT3 downregulation resulted in a decrease of cell viability in both cell lines and induced apoptosis in BxPC-3 cells. Despite significant inhibition of STAT3, the expression of the anti-apoptotic genes Survivin/BIRC5 and BCL-xL were not subsequently downregulated. Even more, the cell line BxPC-3 shows a significant increase of Survivin/BIRC5 and BCL-xL mRNA after 48-72 h as a result of STAT3 downregulation. Inactivation of STAT3 in pancreatic cancer cell lines induces apoptosis but also may promote the expression of anti-apoptotic genes.

Increased levels of hypoxia‐inducible factor‐1α are associated with Bcl‐xL expression, tumor apoptosis, and clinical outcome in chondrosarcoma

Hypoxia-inducible factor (HIF)-1α is a key nuclear transcription factor that regulates the cellular response to hypoxia, and is important for solid tumor growth and survival. However, the underlying role of HIF-1α in human chondrosarcoma has not been well characterized. This study aims to investigate the expression patterns of HIF-1α in chondrosarcoma, and its association with clinicopathologic features, Bcl-xL expression, apoptosis index (AI), and overall survival of patients with chondrosarcoma. Our results shown that the protein levels of HIF-1α were increased, and the mRNA and protein levels of Bcl-xL were also increased in SW1353 cells under hypoxic conditions. In eight patients with chondrosarcoma, increased expression of HIF-1α and Bcl-xL was detected in chondrosarcoma tissues compared with the paired adjacent normal tissues. Of 34 archival specimens of chondrosarcomas, 20 (58.8%) showed high HIF-1α protein expression as compared to benign cartilage tumors. Increased HIF-1α expression was correlated with a higher pathological grade and MSTS stage of chondrosarcoma. Moreover, HIF-1α expression was significantly associated with Bcl-xL expression and AI. More significantly, the survival rate of patients with HIF-1α high tumors was significantly lower than that of patients with HIF-1α low tumors. These findings suggest that increased HIF-1α levels mediated up-regulation of Bcl-xL play a prominent role in evasion of apoptosis and tumor progression, and can be predictive for the prognosis in human chondrosarcoma.

Inhibition of JAK2V617F Kinase Potently Induce Apoptosis In HEL Cells

AbstractAbstract 5062Myeloproliferative neoplasms (MPNs), formerly recognized as myeloproliferative disorders(MPDs), are a group of hematological conditions where a molecular defect in a multi-potent hematopoietic stem cell leads to increased production in one or more blood cell lineages. In early 2005, four different groups described an identical JAK2V617F mutation in exon 12 of JAK2 in a large number of patients with MPNs. Whereafter, it has been confirmed that the Gain-of-function mutations of JAK2V617F play crucial roles in the development of MPN. However, how this activated singaling pathway leads to overproduction of blood cells are not fully understood. Our experiment was designed and performed to address one aspect of this issue. Here we report that AG490, a JAK2 kinase inhibitor, is potently inducing apoptosis in HEL cells. HEL cell line, which harbor a homozygous JAK2V617F mutation confirmed by Allele-specific polymerase chain reactions (AS-PCR), restriction enzyme digestion and DNA sequencing, was selected as a cell model. HEL cells were treated with AG490 at different concentrations and for different time length. The Flow cytometry(FCM) with AnnexinV-FITC/PI double staining and genome DNA electrophoresis were performed to determine the apoptosis rate of the HEL cell, respectively; RT-PCR was performed to analyze the effect of AG490 on the expression of Bcl-x mRNA. The FITC-AnnexinV/PI double staining demonstrated that the apoptosis rate of HEL cell increased obviously in a time (F=267.226, P<0.0001)and dose(F=343.77, P<0.0001) dependent manner after treated by AG490. Similar results were observed by DNA ladder. The results of RT-PCR demonstrated that when treated by AG490, the mRNA of Bcl-xL gene was down-regulated obviously(Bcl-xL mRNA was the main species of Bcl-x found), and the down-regulation was strongly time(F=26.402, P<0.0001) and dose(F=39.247, P<0.0001) dependent. Thus, our results suggest that AG490 can induce apoptosis in HEL cells obviously, and JAK2V617F might inhibit cell apoptosis through upregulation of Bcl-xL. Disclosures:No relevant conflicts of interest to declare.

Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner

Resveratrol, a phytoalexin, reduced the viability of MH7A cells, a human rheumatoid arthritis synovial cell line. In the apoptosis assay, resveratrol increased TUNEL-positive cells and stimulated H2A.X phosphorylation. Resveratrol disrupted mitochondrial membrane potentials in MH7A cells and stimulated cytochrome c release from the mitochondria to the cytosol. Resveratrol activated caspase-3 and caspase-9 but not caspase-8 in MH7A cells. Resveratrol upregulated the expression of the NAD-dependent deacetylase sirtuin 1 mRNA and downregulated the expression of the Bcl-XL mRNA, and resveratrol-induced MH7A cell death, mitochondrial damage, and caspase-3/-9 activation were prevented by sirtinol, an inhibitor of sirtuin 1. The results of the present study show that resveratrol induces MH7A cell apoptosis by activating caspase-9 and the effector caspase-3 along mitochondrial disruption as a result of reduced Bcl-XL expression, allowing cytochrome c release from the mitochondria into the cytosol, in a sirtuin 1-dependent manner. This suggests that resveratrol could suppress hyperplasia of synovial cells, a critical factor of rheumatoid arthritis.

Glutaredoxin Regulates Apoptosis in CardiomyocytesviaNFκB Targets Bcl-2 and Bcl-xL: Implications for Cardiac Aging

Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by approximately 40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFkappaB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFkappaB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent ( approximately 50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFkappaB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFkappaB function.

Functional and biological analysis of Bcl-xL expression in human osteosarcoma

Bcl-xL, a member of Bcl-2 protein family functioned as dominant regulators of apoptotic cell death, has been reported to play important roles in malignant transformation and tumor development. In the present study, our aim was to explore the roles of Bcl-xL overexpression and determine its possibility as a therapeutic target in human osteosarcoma. Real-time quantitative RT-PCR and Western blot or immunohistochemistry assays were performed to detect the expression of Bcl-xL mRNA and protein in human osteosarcoma cell lines or tissue samples. The expression of other Bcl-2 family proteins (Bcl-2, Mcl-1, Bim and Bik) in osteosarcoma tissues was also detected by immunohistochemistry. The associations of Bcl-xL mRNA expression with clinicopathologic factors and prognosis of osteosarcoma patients were evaluated. RNA interference or gene overexpression technologies were employed to downregulate or upregulate endogenous Bcl-xL expression in osteosarcoma cells and the effects of Bcl-xL downregulation or upregulation on phenotypes and chemo- or radiosensitivity of human osteosarcoma cells were analyzed. Finally, the mechanism of synergistic effects of Bcl-xL downregulation and chemo- or radiotherapy was explored by detecting the activity of caspase-3. The expression levels of Bcl-xL mRNA and protein in high metastatic osteosarcoma cells showed higher than those in low metastatic osteosarcoma cells. Moreover, the levels of Bcl-xL mRNA expression were significantly higher in osteosarcoma tissues than those in chondroma or corresponding non-tumor tissues ( P < 0.01), and osteosarcoma tissues showed stronger immunostaining of Bcl-xL protein than non-tumor tissues. The stronger staining of Bcl-2 and Mcl-1 proteins was also observed, while the staining of pro-apoptotic proteins (Bim and Bik) was significantly weaker or not detected in osteosarcoma tissues. The higher levels of Bcl-xL mRNA expression were significantly correlated with advanced clinical stage ( P = 0.005) or hematogenous metastasis ( P = 0.001) of osteosarcoma patients. Osteosarcoma patients with higher Bcl-xL mRNA expression showed a poorer survival compared with those with lower expression ( P = 0.039). Bcl-xL downregulation or upregulation could significantly reduce or increase the proliferation capacity of osteosarcoma cells. Furthermore, Bcl-xL downregulation could significantly enhance in vitro chemo- or radiosensitivity of osteosarcoma cells, which might be associated with elevated activity of caspase-3. Taken together, overexpression of Bcl-xL may play important roles in osteosarcoma progression and this molecule will be a potential chemo- or radiotherapeutic molecular target for osteosarcoma therapy.

Sensitization of SiHa cell to gemcitabine by CD40 activation and its overexpression in cervical carcinoma

CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types. Some studies show that CD40 expression is related to several carcinomas, where its function remains largely unknown. This study investigated the expression of CD40 on cervical carcinoma and evaluated the effect of agnostic anti-CD40 mAb (5C11) on tumor cell line (SiHa). CD40 expression on the primary cervical carcinoma samples was detected by immunohistochemistry. Results showed that CD40 is commonly expressed in human cervical carcinoma, which is higher than that of normal cervix, cervical precancerous tissue and chronic cervicitis. Treatment of the SiHa cell with the agonistic anti-CD40 monoclonal antibody or Gemcitabine alone did not inhibit the proliferation of the SiHa cell in vitro, but the activation of CD40 on SiHa could enhance its sensitivity to Gemcitabine. Furthermore, CD40 activation blocked SiHa in the S phase, stimulated proapoptotic Bax and inhibited antiapoptotic Bcl-XL mRNA synthesis in the SiHa cell. Apoptosis in SiHa was associated with an increasing ratio of Bax to Bcl-XL in mRNA levels. It is concluded that use of the anti-CD40 mAb 5C11 in combination with chemotherapy may have significant therapeutic potential.

Resistance to the development of stress-induced behavioral despair in the forced swim test associated with elevated hippocampal Bcl-xl expression

Stress may predispose individuals toward depression through down-regulation of neurogenesis and increase in apoptosis in the brain. However, many subjects show high resistance to stress in relation to psychopathology. In the present study, we assessed the possibility that individual-specific patterns of gene expression associated with cell survival and proliferation may be among the molecular factors underlying stress resilience. Brain-derived neurotrophic factor (BDNF), anti-apoptotic B cell lymphoma like X (Bcl-xl) and pro-apoptotic bcl2-associated X protein (Bax) expression were determined in the hippocampus and frontal cortex of rats naturally differed in despair-like behavior in the forced swim test. In the hippocampus, BDNF messenger RNA (mRNA) level was significantly down-regulated 2h after the forced swim test exposure, and at this time point, Bcl-xl mRNA and protein levels were significantly higher in stressed than in untested animals. The ratios of hippocampal Bcl-xl to Bax mRNA negatively correlated with the total time spent immobile in the test. When animals were divided in two groups according to immobility responses in two consecutive swim sessions and designated as stress resilient if their immobility time did not increase in the second session as it did in stress sensitive rats, it was found that resilient rats had significantly higher Bcl-xl/Bax ratios in the hippocampus than stress sensitive animals. The data suggest that naturally occurring variations in the Bcl-xl/Bax ratio in the hippocampus may contribute to individual differences in vulnerability to stress-induced depression-like behaviors.

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells

Angiotensin II (Ang II) is a key proapoptotic factor in fibrotic tissue diseases. However, the mechanism of Ang-II-induced cell death in endothelial cells has not been previously elucidated. Using the neutral comet assay and specific receptor antagonists and agonists, we found that Ang-II-mediated apoptosis in primary pulmonary endothelial cells required the AT2 receptor. Ang II caused cytochrome c release from the mitochondria concurrent with caspase-3 activation and DNA fragmentation, and apoptosis was suppressed by an inhibitor of Bax-protein channel formation, implicating mitochondrial-mediated apoptosis. There was no evidence that the extrinsic apoptotic pathway was involved, because caspase-9, but not caspase-8, was activated by Ang-II treatment. Apoptosis required phosphoprotein phosphatase activation, and inhibition of the SHP-2 phosphatase (encoded by Ptpn11) blocked cell death. Reduced levels of anti-apoptotic Bcl-2-family members can initiate intrinsic apoptosis, and we found that Ang-II treatment lowered cytosolic Bcl-x(L) protein levels. Because the protein nucleolin has been demonstrated to bind Bcl-x(L) mRNA and prevent its degradation, we investigated the role of nucleolin in Ang-II-induced loss of Bcl-x(L). RNA-immunoprecipitation experiments revealed that Ang II reduced the binding of nucleolin to Bcl-x(L) mRNA in an AU-rich region implicated in instability of Bcl-x(L) mRNA. Inhibition of SHP-2 prevented Ang-II-induced degradation of Bcl-x(L) mRNA. Taken together, our findings suggest that nucleolin is a primary target of Ang-II signaling, and that Ang-II-activated SHP-2 inhibits nucleolin binding to Bcl-x(L) mRNA, thus affecting the equilibrium between pro- and anti-apoptotic members of the Bcl-2 family.

Abstract 4979: The role of Tel in repression of BCL-XL transcription in tumor cells

Abstract Study of Bcl-xl and hepatocyte growth factor (HGF) receptor c-Met demonstrates that ETS transcription factors influence the transcriptional control of Bcl-xl in multiple tumors. The phosphorylation of Tel, a transcriptional repressor in the ETS family, by activated c-Met is required to increase Bcl-xl transcription in addition to activation of ETS2, a transcriptional activator. Our data indicate that stimulation by HGF causes the activation of ERK and leads to the nuclear export of Tel. In order to determine whether ERK is responsible for the phosphorylation of Tel after HGF stimulation, physical interaction between Tel and ERK was detected using I.P. (Tel)-western blotting (ERK). To determine if blocking MAP kinase could prevent Tel's phosphorylation after HGF exposure, we again used I.P. Western to compare the phosphorylation of Tel under specific ERK kinase inhibitors, after HGF exposure, to the phosphorylation of Tel in the absence of inhibitors, after HGF exposure. A His-Tel 213mutant (phosphorylation sites mutants) was created. The Tel 213 mutant did not undergo phosphorylation induced by c-Met activation, as demonstrated by I.P. Western blot. Nuclear localization of mutated His-Tel was detected under HGF/EGF exposure via fluorescent microscopy using FITC labeled anti-his antibody, indicating that phosphorylation of Tel is also required for nuclear export. A luciferase reporter assay shows that the transient expression of Tel 213 mutant in cancer cell lines results in further repression of Bcl-xl promoter activity. Indeed, Tel 213's repressive function is independent of c-Met stimulation, as determined by comparing with wild type Tel cDNA transfected cells. Consequently, Tel 213 transfection results in much stronger reduction in Bcl-xl mRNA and protein expression levels in the presence of HGF, as compared to transfection with wild-type Tel cDNAs in the presence of HGF. Furthermore, a CHIP assay demonstrates that the activation of c-Met reduced the binding of wild-type Tel to the Bcl-xl promoter, while the binding of Tel 213 to bcl-xl promoter was not affected. Finally, transfection of Tel 213 leads to increased cell proliferation inhibition and apoptosis. Taken together, these findings are supportive of a role for Tel, through a c-Met aixs, as an inhibitor of Bcl-xl transcription. 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 4979.

Abstract 5023: Down-regulating interferon regulatory factor-1 by siRNA promotes endurance to fulvestrant in sensitive breast cancer cells partially via Ras/Raf/MEK/ERK pathway

Abstract AIM: Interferon Regulatory Factor 1 (IRF1) is a tumor suppressor gene that has an antitumor role in some cancers. In this study, we showed that reducing IRF1 expression by siRNA promotes resistance to Fulvestrant in MCF7/LCC1 cells at least partly by acting though the Ras/Raf/MEK/ERK pathway. METHODS: After transfection with IRF1 siRNA by Amaxa Nuclofection, the MCF7/LCC1 cells were treated with 100 nM Fulvestrant for 2 days for monitoring gene expression, and for 3 and 6 days for apoptosis and cell proliferation assays, respectively. Five μM U0126 or 10 μM PD98059, the inhibitors of MEK, were used when necessary. Transactivation of IRF1, Bcl-2, Cyclin D1, and ERE were detected by luciferase assays after transfection with the appropriate reporter plasmid. Caspase 7, 8 and 9 activities were detected by cleavage of their respective substrates. RESULTS: After transfected with IRF1 siRNA, the mRNA and protein levels of IRF1 expression were greatly reduced in MCF7/LCC1 cells. Knocking-down IRF1 expression resulted in increased proliferation, increased S-phase ratio, and decreased apoptosis of MCF7/LCC1 cells compared to mock or control siRNA groups. mRNA and protein levels of Bcl-2, Bcl-w, Bcl-xL, Survivin and CyclinD1 were increased by IRF1 siRNA transfection. Luciferase assays showed that the Bcl-2 P2 (but not P1) promoter is inhibited by IRF1 siRNA transfection. At same time, the activities of Caspase 7 and 8 cannot be increased by Fulvestrant treatment after IRF1 siRNA transfection. Knocking down IRF1 expression partially restores Fulvestrant's inhibition of ERE-drive transcription. Total and phosphor-ER α expression were reduced by IRF1 siRNA transfection. Further, we found that the phosphor-ERK1/2 expression levels were increased by IRF1 siRNA transfection. Inhibition MEK with inhibitors could partially reverse the effect of IRF1 siRNA transfection on proliferation via inhibiting the effect of cell cycle other than apoptosis. Using the inhibitors has no effect on the expression of ER α and the transcriptional activity of ERE. CONCLUSIONS: Knocking down IRF1 expression promotes resistance to Fulvestrant in MCF/LCC1 breast cancer cells partially by increasing the expression of phosphor-ERK1/2. The reduced of ERα expression and increased transcriptional activity of ERE has also involved in the mechanism. 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 5023.

The let-7 family of microRNAs inhibits Bcl-xL expression and potentiates sorafenib-induced apoptosis in human hepatocellular carcinoma

Bcl-xL, an anti-apoptotic member of the Bcl-2 family, is over-expressed in human hepatocellular carcinoma, conferring a survival advantage to tumour cells. The mechanisms underlying its dysregulation have not been clarified. In the present study, we explored the involvement of microRNAs that act as endogenous sequence-specific suppressors of gene expression. The expression profiles of microRNAs in Huh7 hepatoma cells and primary human hepatocytes were compared by microarray analysis. The effect of let-7 on Bcl-xL expression was examined by Western blot and a reporter assay. The involvement of let-7 microRNAs in human tissues was analysed by western blot and reverse transcription-PCR. Microarray analysis, followed by in silico target prediction, identified let-7 microRNAs as being downregulated in Huh7 hepatoma cells in comparison with primary human hepatocytes, as well as possessing a putative target site in the bcl-xl mRNA. Over-expression of let-7c or let-7g led to a clear decrease of Bcl-xL expression in Huh7 and HepG2 cell lines. Reporter assays revealed direct post-transcriptional regulation involving let-7c or let-7g and the 3'-untranslated region of bcl-xl mRNA. Human hepatocellular carcinoma tissues with low expression of let-7c displayed higher expression of Bcl-xL protein than those with high expression of let-7c, suggesting that low let-7 microRNA expression contributes to Bcl-xL over-expression. Finally, expression of let-7c enhanced apoptosis of hepatoma cells upon exposure to sorafenib, which downregulates expression of another anti-apoptotic Bcl-2 protein, Mcl-1. let-7 microRNAs negatively regulate Bcl-xL expression in human hepatocellular carcinomas and induce apoptosis in cooperation with an anti-cancer drug targeting Mcl-1.

B23 acts as a nucleolar stress sensor and promotes cell survival through its dynamic interaction with hnRNPU and hnRNPA1

The nucleolus is one of the functional nuclear compartments in which the ribosome biogenesis takes place. Proteomic analysis revealed a large number of nucleolar proteins that are involved in diverse cellular processes, including biogenesis of ribonucleoprotein particles, stress responses, cell proliferation and cell cycle progression. Here, we report that in response to transcription repression-induced nucleolar segregation, B23, a nucleolar protein required for rRNA processing, is localized to the cytoplasm and forms a complex with the mRNA-binding proteins hnRNPU and hnRNPA1 in a sequential manner. In addition, RNA Polymerase I, but not RNA Polymerase II inhibition, was found to account for the translocation-dependent assembly of B23/hnRNPU/hnRNPA1. We also showed that interactions among these proteins are regulated by hnRNPU-bound mRNAs such as the 3'-untranslated region (UTR) of Bcl-xL mRNA. Ectopically expressed 3'-UTR of Bcl-xL mRNA, which disrupted the interactions among B23, hnRNPU and hnRNPA1, led to enhanced cell apoptosis induced by either actinomycin D treatment or mitotic arrest. Overall, these data highlight a novel function of B23 as a stress sensor in the assembly of B23/hnRNPU/hnRNPA1 complex to promote cell survival.

Upregulation of the pro-apoptotic genes BID and FAS in septic shock patients.

IntroductionLymphocyte apoptosis has been suggested to play a central role in sepsis pathophysiology, and studies in animal models demonstrated that blocking this pathway improves outcome. However, no routine biomarkers of apoptosis are so far available in patients. Thus, the aim of our study was to assess the different biomarkers of apoptosis putatively usable on a routine basis in septic shock.MethodsThirteen septic shock patients (sampled twice between days 1 to 2 and days 3 to 5 after diagnosis of shock) and 15 sex-matched and age-matched healthy controls were prospectively enrolled. Apoptosis was measured in lymphocyte subpopulations using flow cytometry (Annexin-V binding, activated caspase-3 and Bcl-2 expressions). Representative pro-apoptotic and anti-apoptotic gene expressions were assessed by quantitative reverse-transcription PCR. Monocyte HLA-DR expression and lymphocyte subpopulation cell counts were measured as markers of sepsis-induced immune dysfunctions. To test for statistical significance, the Mann-Whitney U test was used with correction by the number of tests performed.ResultsFlow cytometric measurements of apoptosis in septic shock patients showed an increased Annexin-V binding on CD4+ T cells and an increased active caspase-3 expression on B cells only at days 3 to 5 (sixfold change and twofold change, respectively). Gene expression analysis showed an increased BCL-XL mRNA and an upregulation of the pro-apoptotic genes BID and FAS in septic shock patients (10-fold change and fivefold change, respectively) compared with healthy controls.ConclusionsThe present study highlights the difficulties encountered in monitoring apoptosis on a routine basis in septic patients, whereas in the same sampling conditions and on the same patients, HLA-DR expression and lymphocyte subpopulation cell counts showed characteristics described in the literature. However, pro-apoptotic genes BID and FAS appear to constitute promising apoptosis markers in our hands.

Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma

The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P = .04) and a shorter disease-free survival (P = .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre-miR-135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a-mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients.

Does elevated intraocular pressure reduce retinal TRKB-mediated survival signaling in experimental glaucoma?

Reduced retrograde transport of neurotrophins (NT) and their receptors has been hypothesized to contribute directly to retinal ganglion cell (RGC) loss in glaucoma. However, strategies of supplementing NT and NT receptors have failed to avert ultimate RGC death in experimental glaucoma. This study examines the response of major components of the NT system and their interacting proteins in a rat glaucoma model. Unilateral chronic intraocular pressure (IOP) elevation was produced by episcleral vein injection of hypertonic saline ( N = 99). Retinas were collected and grouped by extent of optic nerve injury. Quantitative reverse transcription PCR, western blot analysis and immunohistochemistry were used to determine mRNA and protein levels and protein localization. Out of three RGC-specific Brn3 proteins (Brn3a, b, and c), only Brn3a was significantly downregulated at the message level to 35 ± 4% of fellow values with the severest nerve injury. With IOP elevation, no significant alterations were found in retinal mRNA levels for BDNF, NGF, NT-4/5 or NT-3. The abundance of mature retinal BDNF protein was not significantly affected by elevated IOP, while proBDNF protein decreased linearly with increasing injury grade ( r 2 = 0.50). In retinas with the severest nerve injury, TrkB and TrkC receptor mRNA levels significantly declined to 67 ± 9% and 44 ± 5% of fellow values, respectively. However, the levels of TRKB protein and its phosphorylated form were unchanged. Message level for p75 NTR was linearly upregulated up to 219 ± 26% with increasing injury ( r 2 = 0.46), but no alteration was detected at protein level. The mRNA expression of p75 NTR apoptosis adaptor proteins NADE, NRIF, and Lingo1 were significantly downregulated in retinas with the greatest nerve injury. A positive correlation was found between injury extent and message levels for Jun ( r 2 = 0.23) as well as Junb ( r 2 = 0.27), and RGC labeling of activated JUN protein increased. Atf3 mRNA levels demonstrated a positive linear correlation to the extent of injury ( r 2 = 0.53), resulting in a nearly five-fold increase (482 ± 76%) in eyes with the greatest nerve damage. Among downstream pro-survival signaling components, Erk5 mRNA expression was linearly upregulated ( r 2 = 0.32) up to 157 ± 15% of fellow values in retinas with the severest nerve injury ( p < 0.01). A slight positive correlation was found between NF-κB message levels and injury extent ( r 2 = 0.12). Bcl-xl mRNA levels in the most severely injured retinas were significantly reduced to 83 ± 7% by elevated IOP exposure. Message levels for Erk1/2, Akt1-3 or Bcl2 appeared unaffected. Elevated IOP did not alter mRNA levels of pro-apoptotic Bim, Bax, or p53. This study demonstrates that elevated IOP exposure does not result in a dramatic decrease in retinal levels of either BDNF or its receptor, TrkB. It shows that the responses of NT pathways to elevated IOP are complex, particularly with regard to the role of p75 NTR and Atf3. A better understanding of the roles of these proteins in IOP-induced injury is likely to suggest informed strategies for neuroprotection in glaucoma.

Treatment of K562 cells with 1,25-dihydroxyvitamin D3 induces distinct alterations in the expression of apoptosis-related genes BCL2, BAX, BCLXL, and p21

Apoptosis, or programmed cell death, is a very important phenomenon in cytotoxicity induced by anticancer treatment. 1α,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), the active metabolite of vitamin D, inhibits the growth of multiple types of cancer cells including breast, colon, and prostate cancer cell lines. We studied alterations in the mRNA expression levels of BCL2, BAX, CYC, BCL-XL, and VDR genes in the K562 chronic myeloid leukemia cell line in response to treatment with 1,25-(OH)(2)D(3). Morphological observation of K562 cells was evaluated by the staining with Wright's solution. Cell percentage at different phases of the cell cycle was measured, and apoptosis was measured by flow cytometry. The expression levels of the apoptosis-related genes were analyzed by real-time reverse transcription polymerase chain reaction. We found that treatment with 1,25-(OH)(2)D(3) down-regulates BCL2 and BCL-XL mRNA expressions, as well as up-regulates expressions of BAX and p21 mRNA. The expression pattern of CYC and VDR genes were not influenced. However, K562 cells treated with 1,25-(OH)(2)D(3) caused an arrest of cell cycle progression in G1 phase resulting in a decreased number of cells in the S phase, complemented by an accumulation of cells in the G0-G1 phases. Our data show the modulatory effects of 1,25-(OH)(2)D(3) treatment in apoptosis-related genes in K562 cells.

Optimisation and molecular signalling of apoptosis in sequential cryotherapy and chemotherapy combination in human A549 lung cancer xenografts in SCID mice

We define the optimal parameters for combination of cryotherapy (nitrous oxide) with chemotherapy (vinorelbine ditartrate, VNB) treatment and characterise some of the signals involved for apoptosis activation. No advantage appeared when cryotherapy and VNB were combined simultaneously compared to cryosurgery alone. In contrast, tumour volumes were reduced after a sequential treatment schedule, where each individual treatment was separated by 48 h. No significant benefit appeared when the sequential treatment was separated by 24 h, although some individual mice showed a good response. The sequence of treatment had no impact on the observed tumour growth inhibition in mice. The number of apoptotic cells was significantly augmented in the sequential treatment schedule where VNB was administered 48 h before cryotherapy. In this sequential treatment, the number of apoptotic cells correlated with heightened expression of the BH3-only Puma, Noxa and Bim-EL, at both the mRNA and protein levels. No significant change in Bax, Bcl-xL and Bcl-2 mRNA expression was apparent, whereas Mcl-1 expression increased only slightly to a much lower level than BH3-only mRNAs. Our data indicate that 48 h sequential rather than simultaneous cryotherapy with VNB in future cancer cryochemotherapy schedules will enhance the tumour response, and argue that VNB administration, 48 h before cryotherapy, will provoke apoptosis more efficiently.

C-Myb is a novel regulator of Bcl-xL in the CD4+ and CD8+ double positive stage of T cell development (85.2)

Abstract The lifespan of quiescent CD4+ and CD8+ double positive (DP) thymocytes regulates the composition of the peripheral T cell repertoire. However, the molecular mechanisms controlling DP thymocyte survival remain poorly understood. The c-Myb transcription factor is highly expressed in quiescent pre-selection DP thymocytes. Using a conditional deletion mouse model, we demonstrate that pre-selection DP thymocytes lacking c-Myb undergo premature apoptosis due to decreased expression of the key anti-apoptotic factor Bcl-xL. Moreover, c-Myb deficient DP thymocytes display a skewed TCRα repertoire, with a bias towards 3' Jα?usage, as a consequence of premature apoptosis occurring shortly after the initiation of TCRα rearrangements in vivo. Finally, re-introduction of c-Myb to c-Myb deficient DP thymocytes restored control levels of Bcl-xL mRNA and protein. Taken together, our study identifies c-Myb as a novel regulator of Bcl-xL expression in the DP stage of T cell development. We demonstrate that c-Myb acts through a novel pathway, independently of RORγt and TCF-1, two transcription factors previously shown to promote Bcl-xL expression in DP thymocytes. This study sheds new light on the regulation of the survival of pre-selection DP thymocytes and provides a novel mechanism by which c-Myb guards the balance between life, death and differentiation during hematopoiesis.

Downregulation of Notch signaling by γ-secretase inhibition can abrogate chemotherapy-induced apoptosis in T-ALL cell lines

Activation of Notch1 signaling plays an important role in the pathogenesis of precursor T-cell lymphoblastic leukemia (T-ALL). The Notch1 receptor is cleaved and activated via the gamma-secretase complex. Downregulation of Notch1 signaling by gamma-secretase inhibitors (GSIs) thus represents a potential novel therapeutic approach. In this study, we analyzed the response of four T-ALL cell lines to compound E, a potent gamma-secretase inhibitor, and to the combination of compound E with vincristine, daunorubicin, L-asparaginase (L-ASP), and dexamethasone (DEX). We identified two distinct types of responses: In type 1 cell lines, represented by TALL1 and HSB2, GSI-induced apoptosis followed cell cycle arrest and enhanced the induction of apoptosis caused by DEX and L-ASP. In type 2 cell lines, represented by CEM and Jurkat J6, GSI caused neither cell cycle block nor cell death. Notably, the combination of GSI with chemotherapy-induced resistance by decreasing apoptosis. In type 2 cells, GSI induced the upregulation of Bcl-xl mRNA and protein, which was thus identified as a candidate mechanism for the inhibition of apoptosis. In conclusion, the data presented here caution against clinical use of a combination treatment of GSI and chemotherapy in T-ALL.