mRNA Levels Of Bcl-xL Research Articles

EBV interferes with the sensitivity of Burkitt lymphoma Akata cells to etoposide

Burkitt lymphoma (BL) commonly exhibits Epstein-Barr virus (EBV) positivity associated with latent chronic infection. Models of acute EBV infection have been associated with cellular resistance to apoptosis. However, the effect of latent long-term EBV infection on apoptosis induced by drugs is not well defined. To determine this, we have studied the response of the Akata EBV+ cell line (type I latency) to etoposide, before and after downregulating EBV gene expression. We observed that downregulating EBV nuclear antigen-1 (EBNA-1) expression with siRNAs reverted cellular sensitivity to etoposide. In accordance with this finding, Akata EBV+ cells showed increased sensitivity to etoposide, when compared to the Akata EBV- cells. We also observed that Akata EBV+ cells presented increased apoptosis levels and decreased Bcl-xL mRNA and protein levels, when compared to the Akata EBV- cells. In addition, Akata EBV+ cells contained less endoplasmic reticulum (ER) than EBV- cells. Finally, downregulation of EBV with EBNA-1 siRNAs caused an increase in the expression of Bcl-xL indicating that EBV is responsible for the differences found between the Akata EBV+ and EBV- cell lines.

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.

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.

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.

Expression of Hedgehog signaling pathway genes in human oral squamous cell carcinoma and the effects of specific Hedgehog pathway inhibitor (Cyclopamine) on the proliferation and apoptosis of human oral epidermoid HSQ-89 cells

Objective To investigate the expression of Hedgehog signaling pathway genes in human oral squamous cell carcinoma (OSCC), and the effects of specific Hedgehog pathway inhibitor ( Cyclopamine) on the proliferation and apoptosis of human oral epidermoid HSQ-89 cells. Methods The expression of Hedgehog signaling pathway components ( Shh, Smo, Ptch, Gli-1 ) was detected in 80 cases of human OSCC by immunochemistry. CCK8 assay was used to investigate the inhibition of Cyclopamine on HSQ-89 cells proliferation in vitro. Flow eytometry (FCM) was used to examine the effects of Cyclopamine on apoptosis of HSQ-89 cells. Reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of bcl-2, bcl-xL, and Bid in HSQ-89 cells. Results The positive expression rate of Shh, Smo, Ptch and Gli-1 was 61.25%, 56. 25%, 47. 50%, 53.75% in 80 cases of human OSCC tissue slices, respectively. After the cells were treated with different concentrations of Cyclopamine (0, 5,10, 20, 40, 80 μmoL/L, respectively ), the growth inhibition rate was 0, 3.0%, 4. 5%, 28.2%,51.2%, 62. 7% respectively; the apoptosis rate 3.01% ,3. 36% ,5. 70% ,9.42% respectly. Cyclopamine significantly inhibited cell proliferation ( P < 0. 05 ). FCM revealed that the apoptosis rate of HSQ-89 cells was significantly higher after exposure to Cyclopamine than in control groups (P < 0. 05 ). The mRNA levels of bcl-2 were significantly decreased, but Cyclopamine had no significant effects on the mRNA level and protein secretion of bcl-xL, Bid. Conclusion The expression of main hedgehog signaling pathway components was detected in human OSCC. Cyclopamine specifically inhibited the proliferation and the mRNA expression of bcl-2 in HSQ-89 cells. Key words: Oral squamous cell carcinoma; Signal transduction; Apoptosis; Proliferation; Cyclonpamine

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.

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.

Serum-borne factors in cancer patients with advanced cachexia: influence on adipose cells

Background: The clinical syndrome cancer cachexia is recognized by a considerable weight loss being out of proportion to any reduction in energy intake. The underlying mechanisms are not completely known, but the marked weight loss is attributable to depletion of adipose tissue as well as skeletal muscle mass. Enhanced lipolysis in adipocytes, apoptosis of preadipocytes may be important for loss of adipose tissue. Results: Sera from cachectic cancer patients induced apoptosis in cultured human preadipocytes at a higher rate than sera from non-cachectic cancer patients (control group). There was a tendency towards increased mRNA levels of the pro-apoptotic Bcl-2 gene Bax after incubation of preadipocytes with cachectic sera. Moreover, the mRNA levels of anti-apoptotic Bcl-XL and pro-apoptotic Bcl-XS were increased and decreased, respectively, as compared to incubation with control sera. However, lipolysis was not enhanced in cultured human adipocytes after incubation with sera from cachectic cancer patients as compared to non-cachectic cancer patients. Methods: Serum samples from cachectic cancer patients (n=8) and non-cachectic cancer patients (n=6) were collected. Human SGBS (Simpson-Golabi-Behmel syndrome) preadipocytes and differentiated adipocytes were incubated in the presence of serum from cachectic and non-cachectic (control) cancer patients. Induction of apoptosis and necrosis was examined by cell staining with Hoechst 342 (HO342) and propidium iodide (PI), respectively. Expression of pro- and anti-apoptotic Bcl-2 genes was measured by quantitative RT-PCR. Lipolysis was monitored by measuring the release of radiolabeled fatty acids. Conclusion: Our in vitro data suggest that apoptosis of preadipocytes can be increased by serum-borne factors in cancer cachexia. Death or survival of preadipocytes may depend on the balance of pro- and anti-apoptotic mediators. Further studies of patients with cancer cachexia will be needed to reveal if the disease involves loss of adipose tissue due to apoptosis of preadipocytes. We could not show that serum-borne factors associated with cachexia have a major impact on lipolysis in cultured human adipocytes. Adipobiology 2009; 1: 57-66.

STAT3 Silencing as a Novel Therapeutic Strategy for Activated B Cell-Type Diffuse Large B-Cell Lymphoma.

Abstract 926Among the non-Hodgkin's lymphomas, the diffuse large B cell lymphoma (DLBCL) represents the most frequent (30%) of the aggressive lymphomas. Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. IL-6 and IL-10 are major activators of STAT3 signaling and are important in the pathophysiology of DLBCL. STAT3 has been found to be persistently active in activated B cells (ABC), which are non-germinal center-derived DLBCL cells. We studied the consequences of STAT3 inhibition on multiple biological functions in two representative human cell lines of this group, Ly3 and Ly10 cells.For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells, control lentivirus Ly3 cells, STAT3 shRNA-expressing lentivirus Ly10 cells and control lentivirus Ly10 cells. The stable expression of STAT3 shRNA results in 40-50% reduction of total STAT3 protein levels in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus cells. STAT3 down-regulation induced inhibition of cell proliferation (approximately 40%). Ly3 cells respond to IL-10 more than to IL-6 in terms of proliferation; both cytokines induced less proliferation in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus Ly3 cells. Similar results were obtained in Ly10 cells, which respond more to IL-6 than to IL-10 in terms of proliferation.We analyzed by quantitative real-time PCR the mRNA levels of different STAT3 target genes and observed significant reduction in mRNA levels of Mcl-1, Bcl-xL and Survivin in STAT3 shRNA lentivirus Ly3 cells, as well as significant reduction of Cyclin D2 and up-regulation of STAT1 in shRNA lentivirus Ly10 cells. Comparison of these gene expression profiles with data obtained from other B-cell lymphoma cell lines revealed that silencing of STAT3 resulted in down-regulation of different STAT3 target genes in a cell-dependent manner. We also observed that both STAT3 and control lentivirus Ly3 cells have the same protein levels of c-Myc; nevertheless STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc.We next investigated the effect of STAT3 inhibition on adhesion to bone marrow stroma and chemotaxis. STAT3 shRNA lentivirus Ly3 cells adhered less to the stroma layer than control cells, and the longer they were cocultured with the stroma cells in the presence of serum-free media the more they lost the ability to adhere. Moreover, STAT3 shRNA lentivirus Ly3 cells had decreased capacity to migrate toward SDF-1 alpha, an important factor that mediates proliferation, survival, chemotaxis, migration and adhesion into bone marrow stroma.Radiation, in combination with chemotherapy, is one of the therapies used for DLBCL patients. We therefore investigated whether STAT3 down-regulation sensitized Ly3 cells to radiation. Radiation induced a higher accumulation of phospho-H2A.X (first sentinel event following DNA damage such as DSBs) and apoptosis in STAT3 shRNA lentivirus cells compared to control cells. Moreover, IL-6 and IL-10 protected the STAT3 shRNA lentivirus Ly3 cells less than the control cells from the induction of phospho-H2A.X following radiation.We further investigated the effect of STAT3 silencing in animal models of Ly3 lymphoma (Nude or NOD-SCID mice). Tumors in control lentivirus Ly3-bearing mice grew robustly, whereas tumors in STAT3 shRNA lentivirus Ly3-bearing mice regressed 5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis, significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin (approximately 40% to 60% inhibition), and reduction of cytokine production such as IL-10, IL-15, Leptin and Thrombopoietin. Taken together, these results suggest that inhibition of STAT3 is a potential promising approach in the therapy of ABC-type DLBCL. Disclosures:No relevant conflicts of interest to declare.

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.

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.

Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels

The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 mumol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G(0)/G(1) phase and decrease in S phase. After treatment with 0, 20, 60, 100 mumol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00+/-1.25)% to (58.84+/-0.32)% in G(0)/G(1) phase, whereas it was decreased from (61.45+/-1.04)% to (35.82+/-1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G(0)/G(1) phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.

Effects of clonidine and yohimbine on the levels of bax, Bcl-XL, and caspase-3 mRNAs in the brain of neonatal rats

Administration of clonidine, an agonist of alpha2-adrenoceptors at a dose of 0.4 mg/kg to a 3-day-old rat pups decreased the amount of proapoptotic protein Bax mRNA in the hippocampus and increased the content of antiapoptotic protein Bcl-XL mRNA in the brainstem 120 h after treatment. Administration of yohimbine, an antagonist of alpha2-adrenoceptors, at a dose of 2 mg/kg increased the level of Bcl-XL mRNA in the hippocampus at the same time point. These effects were specific for each drug. However, both these substances decreased the amount of mRNAs of Bax and the key executive apoptotic protease, caspase-3, in the cerebral cortex 24 and 120 h after treatment, respectively. Similar effects of clonidine and yohimbine suggest that the effect of the antagonist of alpha2-adrenoceptors was possibly mediated by endogenous norepinephrine released in response to the antagonist. These results may explain the neuroprotective features of agonists and antagonists of alpha2-adrenoceptors, which have been previously reported.

A murine model for human immune thrombocytopenic purpura and comparative analysis of multiple gene expression in bone marrow and spleen

Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other immunological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura (ITP) has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the minimum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h (6 days). Final homeostasis reached at approximately 408 h (17 days), following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes between bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum (144 h). In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.

C-myc Antisense Oligonucleotides Sensitize Human Colorectal Cancer Cells to Chemotherapeutic Drugs

Background/Aims: Overexpression of the c-myc oncogene frequently occurs in both colon tumors and colon carcinoma cell lines. We examined the sensitization of human colorectal cancer cells to chemotherapeutic drugs using c-myc antisense (AS) phosphorothioate oligonucleotides ([S]ODNs). Methods: Cancer cells were treated with c-myc [S]ODNs, taxol, 5-fluorouracil (5-FU), doxorubicin and vinblastine individually and in combination. The antiproliferative effects, type of interaction between c-myc [S]ODNs and cytotoxic drugs, cell cycle, apoptosis and expression of cell-cycle- and apoptosis-regulatory genes were evaluated. Results: After treatment withc-myc AS[S]ODNs, the growth of cancer cells was markedly inhibited in a dose- and time-dependent manner and the levels of c-myc mRNA and protein were greatly decreased (p < 0.0001). The combinations of c-myc AS[S]ODNs and cytotoxic drugs produced greater growth inhibition of human colorectal cancer cells compared to single treatment with either c-myc AS[S]ODNs (p < 0.006) or cytotoxic drugs (p < 0.0001). These combinations exhibited time- and dose-dependent additive and/or synergistic antiproliferative effects. Cancer cells treated with cytotoxic drugs were growth arrested in the S phase. In contrast, cells treated with either c-myc AS[S]ODNs or by the combination of c-myc AS[S]ODNs and cytotoxic drugs were growth arrested in the G₂/M and S phases. The combination treatments also exhibited a marked apoptotic effect compared to single treatments. c-myc AS[S]ODN treatment reduced the mRNA levels of Bcl2, BclxL, cdk2, cyclin E1, cdk1 and cyclin B1, while increasing the mRNA levels of p21, p27, bax and caspase-3. Conclusion: This two-hit approach may be important in the quest to overcome drug resistance in cancer patients whose tumors carry an overexpressed c-myc gene.

Effects of ligands of α2-adrenoceptors on mRNA level of apoptotic proteins in developing rat brain

The effects of antagonist of alpha2-adrenoceptors yohimbine and their agonist clonidine on Bax and Bcl-XL mRNA levels in neonatal rat brain were studied. Yohimbine decreased Bax mRNA level in the cerebellum, increased the ratio between Bcl-XL and Bax mRNA levels in the cerebellum, cortex, and hippocampus, and increased Bcl-XL mRNA level in the cortex and hippocampus of 6-day-old-rat pups 24 h after injection. Administration of clonidine 20 min after yohimbine administration abolished its effect on Bcl-XL mRNA level in the hippocampus. The data obtained indicate that the blockade of alpha2-adrenoceptors induces antiapoptotic changes in the developing rat brain, some of which can be abolished after coadministration with the agonist of these receptors.

Low-level laser therapy decreases levels of lung neutrophils anti-apoptotic factors by a NF-κB dependent mechanism

Background and objectiveLow-level laser therapy (LLLT) is a known modulator of inflammatory process. Herein we studied the effect of 660 nm diode laser on mRNA levels of neutrophils anti-apoptotic factors in lipopolysaccharide (LPS)-induced lung inflammation. Study design/methodologyMice were divided into 8 groups (n=7 for each group) and irradiated with energy dosage of 7.5 J/cm2. The Bcl-xL and A1 mRNA levels in neutrophils were evaluated by Real Time-PCR (RT-PCR). The animals were irradiated after exposure time of LPS. ResultsLLLT and an inhibitor of NF-κB nuclear translocation (BMS 205820) attenuated the mRNA levels of Bcl-xL and A1 mRNA in lung neutrophils obtained from mice subjected to LPS-induced inflammation. ConclusionLLLT reduced the levels of anti-apoptotic factors in LPS inflamed mice lung neutrophils by an action mechanism in which the NF-κB seems to be involved.

Evaluation of Bcl-2 family gene expression and Caspase-3 activity in hippocampus STZ-induced diabetic rats.

We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2, Bcl-xL, and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA and protein levels of Bcl-2 and Bcl-xL were lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA and protein levels (P < .01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P < .001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-xL, and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.

Emetine Regulates the Alternative Splicing of Bcl-x through a Protein Phosphatase 1-Dependent Mechanism

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.