Expression Of Cell Survival Genes Research Articles

Memantine treatment exerts an antidepressant-like effect by preventing hippocampal mitochondrial dysfunction and memory impairment via upregulation of CREB/BDNF signaling in the rat model of chronic unpredictable stress-induced depression

Mitochondrial and cognitive dysfunctions have long been associated with major depressive disorders (MDDs). Studies have shown that Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, possesses an antidepressant-like effect. Hence, the NMDA receptor can be a better therapeutic target for MDD. Therefore, the present study was designed to study the impact of Memantine on mitochondrial functional status and depression-like symptoms in the chronic unpredictable stress (CUS) model of depression. CUS for 28 days resulted in depression-like symptoms (as indicated by increased immobility time in the forced swim test) and a decline in the spatial learning and retention memory in the Morris water maze (MWM) test, which was prevented by Memantine (10 mg/kg/day) treatment. We observed elevated plasma corticosterone (CORT) levels, microdialysates glutamate concentration, and synaptosomal calcium (Ca2+) ion levels after 28 days of CUS. Memantine treatment prevented only increased plasma CORT and synaptosomal Ca2+ ion levels. Memantine treatment also restored CUS induced increase in oxidative stress parameters [increased neuronal nitric oxide synthase (nNOS) expression, nitric oxide (NO) levels, lipid peroxidation (LPO) and superoxide dismutase (SOD) activity], decrease in mitochondrial electron transport chain (ETC) enzymes activity and mitochondrial membrane potential (MMP). CUS also reduced the expression of cell survival genes, cyclic-AMP response element-binding protein (CREB), and brain-derived nerve growth factor (BDNF), which was reversed by treatment with Memantine. CUS, however, caused a non-significant decrease in the hippocampal adenosine triphosphate (ATP) levels and a non-significant increase in the expression of pro-apoptotic genes, Caspase 3, and the number of TUNEL positive cells, indicating that hippocampal mitochondrial dysfunction caused due to CUS was not severe enough to affect overall energy production, mitochondrial integrity, and cellular apoptosis status. Thus, Memantine treatment exerts an antidepressant-like effect by preventing CUS induced excitotoxicity, oxidative stress, and enhancing CUS induced decrease in mitochondrial functioning and expression of cell survival genes via upregulation of stress-responsive CREB/BDNF signaling.

Lycopene induces apoptosis by inhibiting nuclear translocation of β-catenin in gastric cancer cells.

Reactive oxygen species (ROS) promote the development and progression of cancer by their effects on several signaling pathways. Lycopene, a major carotenoid natural product, is known to display antioxidant activity and to induce apoptosis of cancer cells. The aim of the present study was to investigate the mechanism by which lycopene induces apoptosis of the human gastric cancer AGS cells. In the present study, we showed that lycopene reduces the viability of AGS cells by inducing DNA fragmentation and increasing the Bax/Bcl-2 ratio. To determine the mechanistic basis for these effects, studies were conducted to assess the effects of this carotenoid on activation and nuclear translocation of β-catenin, and the expression of β-catenin target genes in AGS cells. The results showed that lycopene reduces the levels of ROS. It also inhibits activation of β-catenin signaling by changing the Wnt/β-catenin multi-protein complex such as a reduction in phosphorylation of glycogen synthase kinase 3β [GSK3β] and an increase in adenomatous polyposis coli [APC] and β-transducin repeats-containing proteins [β-TrCP]). It suppresses nuclear translocation of β-catenin and the expression of the β-catenin target survival genes c-myc and cyclin D1. Lycopene induces apoptosis by reducing ROS levels and suppressing β-catenin-c-myc/cyclin D1 axis. Thus, lycopene induces apoptosis of gastric cancer cells by disrupting nuclear translocation of β-catenin and expression of key cell survival genes.

Von Hippel-Lindau Protein Maintains Metabolic Balance to Regulate the Survival of Naive B Lymphocytes.

SummaryB lymphocytes undergo metabolic reprogramming upon activation to meet the bioenergetic demands for proliferation and differentiation. Yet, little is known if and how the fate of naive B cells is metabolically regulated. Here, we specifically delete von Hippel-Lindau protein (VHL) in B cells using CD19-Cre and demonstrate that metabolic balance is essential for naive B cell survival. Loss of VHL disturbs glycolytic and oxidative metabolic balance and causes severe reduction in mature B cells. Mechanistically, the metabolic imbalance in VHL-deficient B cells, arising from over-stabilization of hypoxia-inducible factor-1α (HIF-1α), triggers reductive glutamine metabolism leading to increased Fas palmitoylation and caspase-8-mediated apoptosis. Blockade of reductive glutamine metabolic flux by lactate supplementation and ATP citrate lyase inhibition restores the metabolic balance and rectifies the impaired survival of VHL-deficient B cells. Hence, we unravel that the VHL/HIF-1α pathway is required to maintain the metabolic balance of naive B cells and ensure their survival.

Chronic Metabolic Derangement-Induced Cognitive Deficits and Neurotoxicity Are Associated with REST Inactivation.

Chronic metabolic alterations may represent a risk factor for the development of cognitive impairment, dementia, or neurodegenerative diseases. Hyperglycemia and obesity are known to imprint epigenetic markers that compromise the proper expression of cell survival genes. Here, we showed that chronic hyperglycemia (60days) induced by a single intraperitoneal injection of streptozotocin compromised cognition by reducing hippocampal ERK signaling and by inducing neurotoxicity in rats. The mechanisms appear to be linked to reduced active DNA demethylation and diminished expression of the neuroprotective transcription factor REST. The impact of the relationship between adiposity and DNA hypermethylation on REST expression was also demonstrated in peripheral blood mononuclear cells in obese children with reduced levels of blood ascorbate. The reversible nature of epigenetic modifications and the cognitive impairment reported in obese children, adolescents, and adults suggest that the correction of the anthropometry and the peripheral metabolic alterations would protect brain homeostasis and reduce the risk of developing neurodegenerative diseases.

Gelidiella acerosa inhibits lung cancer proliferation

BackgroundLung adenocarcinoma is the most common subtype of Non small cell lung cancer in which the PI3K/Akt cascade is frequently deregulated. The ubiquitous expression of the PI3K and the frequent inactivation of PTEN accounts for the prolonged survival, evasion of apoptosis and metastasis in cancer. This has led to the development of PI3K inhibitors in the treatment of cancer. Synthetic PI3K inhibitors undergoing clinical and preclinical studies are toxic in animals. Hence, there is a critical need to identify PI3K inhibitor(s) of natural origin.The current study aims to explore the efficacy of the red algae Gelidiella acerosaon inhibition of cell proliferation, migration and the expression of cell survival genes in lung adenocarcinoma cell line A549.MethodsThe phytoconstituents of Gelidiella acerosa were extracted sequentially with solvents of different polarity, screened qualitatively and quantitatively for secondary metabolites and characterized by GC-MS. The in-vitro studies were performed to check the efficacy of the extract on cell proliferation (MTT assay), cell invasion (scratch assay and colony formation assay), apoptosis (fluorescent, confocal microscopy and flow cytometry) and expression of apoptosis and cell survival proteins including PI3K, Akt and GSK3β and matrix metalloproteinase MMP2 and MMP9 by Western blot method. The antitumor activity of GAE was analyzed in a tumor model of Zebrafish.ResultsThe outcomes of the in vitro analysis showed an inhibition of cell proliferation, induction of apoptosis, inhibition of cell migration and colonization by the crude extract. The analysis of protein expression showed the activation of caspases 3 and Pro apoptotic protein Bax accompanied by decreased expression of Bcl-2 and Bcl-XL. On the other hand, subsequent activation of GSK3β and down regulation of PI3K, Akt were observed. The decreased expression of MMP2 correlated with the antimetastatic activity of the extract. The in vivo studies showed an inhibition of tumor growth by GAE in Zebrafish.ConclusionThe phytoconstituents of algal extract contributed to the anticancer properties as evidenced by in vitro and in vivo studies. These phytoconstituents can be considered as a natural source of PI3K/Akt inhibitor for treatment of cancers involving the PI3K cascade.

IL-1β induces up-regulation of BIRC3, a gene involved in chemoresistance to doxorubicin in breast cancer cells

Epithelial to mesenchymal transition (EMT) of tumor cells facilitates their progress to metastasis. In the tumor microenvironment the inflammatory cytokine 1β (IL-1β) has been associated with tumor development and invasiveness. IL-1β-induced EMT triggers the expression of markers associated with malignancy. We have recently reported that an IL-1β-highly responsive clone (6D cells) from non-invasive MCF-7 breast cancer cells activates PI3K/Rac and IL-1RI/β-catenin pathways that up-regulate the transcription of genes involved in an EMT-like process. However, a correlation between the EMT program induced by a pro-inflammatory environment, and the acquisition of chemoresistance has not been yet determined in these cells. In this work, we report the expression of cell survival genes after IL-1β stimulation of 6D cells. The expression of CDKN1A, TP63, SFN and, particularly, BIRC3 was found to be up-regulated in a RNA-seq analysis and validated by qPCR. Cells stimulated with IL-1β when challenged with doxorubicin showed resistance to the drug, whereas silencing of BIRC3 decreased viability of the cells treated with the drug. Our present results show that IL-1β confers doxorubicin resistance to breast cancer cells, underlining the importance of an inflammatory environment in cancer malignancy.

MEK inhibition abrogates sunitinib resistance in a renal cell carcinoma patient-derived xenograft model

Background:Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model.Methods:RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA.Results:During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC.Conclusions:Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.

Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells

ABSTRACTPurpose: The development of a suitable storage method for retinal pigment epithelium (RPE) is necessary in the establishment of future RPE replacement therapy, and storage temperature has proven to be pivotal for cell survival. ARPE-19, a widely used model for RPE, has been shown to yield the greatest number of viable cells when stored at 16°C compared to other storage temperatures. In this study, we analyze the gene expression profile of cultured ARPE-19 cells after seven days of storage at different temperatures in an effort to predict the gene-level consequences of storage of RPE transplants.Materials and Methods: ARPE-19 cells were cultured until confluence and then stored in minimum essential medium at 4°C, 16°C, and 37°C for seven days. The total RNA was isolated and the gene expression profile was determined using DNA microarrays. The Results were validated using qPCR.Results: Principal component and hierarchical clustering analyses show that the gene expression profiles of cell cultures stored at different temperatures cluster into separate groups. Cultures stored at 4°C cluster closest to the control cultures that were not stored and display the least change in gene expression after storage (157 differentially expressed genes). Cultures stored at 16°C and 37°C display a much larger change in differential gene expression (1787 and 1357 differentially expressed genes, respectively). At 16°C, the expression of several genes with proposed tumor suppressor functions was markedly increased. Changes in regulation of several known signaling pathways and of oxidative stress markers were discovered at both 16°C and 37°C, and activation of the angiogenesis marker vascular endothelial growth factor (VEGF) was discovered at 37°C. There was no evidence of the activation of inflammatory processes in stored cell cultures.Conclusion: ARPE-19 cultures stored at 16°C show the greatest propensity to modulate their gene expression profile in a manner that supports cell survival during storage.

Anti-Apoptosis and Cell Survival Gene Expression Profile in LAD2 Cells

Anti-Apoptosis and Cell Survival Gene Expression Profile in LAD2 Cells

Abstract P4-05-03: Cytoplasmic PELP1 promotes breast cancer initiation via NF-κB signaling

Abstract Progress in breast cancer prevention is limited by an inability to reliably predict which women will develop breast cancer and which high-risk women will respond to chemoprevention therapies. Thus, there is a critical need to determine the molecular mechanisms that promote breast cancer initiation in order to 1) identify biological markers of disease susceptibility and 2) to develop novel targeted chemoprevention agents. A promising target for preventing breast cancer initiation is proline, glutamic acid, and leucine rich protein 1 (PELP1). PELP1 was first identified as an estrogen receptor (ER) co-activator in ER+ breast cancer cell lines. Subsequent studies found that PELP1 functions in ER+ and ER- breast cancer cell lines and is overexpressed in 80% of invasive breast cancers. Interestingly, while PELP1 is primarily localized to the nucleus in the normal breast epithelial cells, in about 40% of invasive breast tumors a significant amount of PELP1 is localized in the cytoplasm. Interestingly, in a mammary gland specific transgenic mouse model, PELP1-cyto expression induced mammary gland hyperplasia. We therefore analyzed breast needle aspirates from asymptomatic high-risk women, and found cytoplasmic PELP1 in 4/11 (36%) samples. These findings suggest that altered localization of PELP1 may be an early event in breast cancer initiation. The objective of this research project is to identify the molecular mechanisms associated with PELP1-induced breast cancer initiation. We performed global gene expression analyses of our in vitro human mammary epithelial cell (HMEC) models to identify potential downstream pathways regulated by PELP1-cyto. Interestingly, genes regulated by PELP1-cyto differ greatly from those regulated by nuclear PELP1. Our results showed that PELP1-cyto induces expression of cell survival genes, and inflammatory cytokines and chemokines, and activates the NF-κB signaling pathway. Thus, we hypothesize that cytoplasmic PELP1 induces activation of inflammatory cytokines and chemokines via NF-κB, which promotes tumor initiation and a pro-tumorigenic microenvironment. In support of our hypothesis we have found that PELP1-cyto expression promotes phosphorylation of the non-canonical IKK, TBK1, and the NF-κB subunit p65. Additionally, we found that conditioned medium from PELP1-cyto cells, induced expression of IL-1β, TNFα, and IL-8 in THP-1 cells that had been differentiated into macrophages, suggesting that PELP1-cyto HMECs release cytokines that promote the activation of macrophages. The goal of future studies is to validate these findings in vitro and in vivo and identify the cytokines and/or chemokines induced by PELP1-cyto that promote breast cancer initiation and activation of tumor associated macrophages. Citation Format: Julie H Ostrander, Brian J Girard, Todd Knutson, Bethanie Kuker, Victoria Seewaldt. Cytoplasmic PELP1 promotes breast cancer initiation via NF-κB signaling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-03.

Abstract LB-65: Protein kinase Cε, which is linked to development of squamous cell carcinomas, potentiates ultraviolet radiation-induced expression of novel transcription factors GATA and PAX, which regulates expression of cell survival genes

Abstract Protein kinase C epsilon (PKCε) is among the six PKC isoforms (α, δ, ε, η, μ, ξ) expressed in both mouse and human skin. We have reported that epidermal PKCε levels dictate the susceptibility of PKCε transgenic mice (TG) to the development of squamous cell carcinomas (SCC) elicited by repeated exposures to ultraviolet radiation (UVR). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. To find clues about the mechanism by which PKCε may impart susceptibility to UVR-induced development of SCC, we compared the effects of UVR treatment of TG mice with their wildtype (WT) littermates on global transcription factors (TFs) activation profile. In this experiment, mice were exposed to UVR (2kj/m2) four times (Monday, Wednesday, Friday and Monday). At 1hr and 3hrs post last UVR exposure, these mice were sacrificed. Nuclear lysates were prepared from the scraped epidermis of these mice. To screen for unknown TFs, we employed the Panomics Combo Protein array, which contains consensus complimentary sequences of 345 TFs binding sites. Array results from samples of UVR exposed mice were directly compared to the unexposed mice. TFs that had increased over 2-fold in the UVR exposure experiments were scored as positive for activation. We observed (>2-fold) activation of 70 TFs as early as 1hr post UVR exposure. Among all seventy TFs, highly activated TFs were GATA-1, GATA-2, PAX-4, PAX-6, PAX-8, Thy-1BP, HFH-2, HFH-3, HIF-1, CEA, HOXD8/9/10, AhR/Amt, and MUSF. We performed the validation of various activated TFs and among them we found that GATA and PAX families of transcription factors were found activated. UVR-induced expression level of GATA-1, GATA-2, PAX-4 was enhanced in PKCε over-expressing transgenic mice as compared to wild-type littermates. We employed the Genego program and found that VEGFR-2, CyclinD1, Bcl-2, c-Myc, BMP-4, WT1 and survivin are the downstream target genes of GATA1 and GATA-2. All of these genes are known to play role in the cell survival pathway. We have previously shown that PKCε overexpressing transgenic mice also exhibit constitutive activation of Stat3. Taken together, we conclude that PKCε-mediated activation of Stat-3, GATA-1, GATA-2 and PAX-4 may contribute to the susceptibility of PKCε transgenic mice to UVR-induced development of SCC. (Support: NIH Grant CA 35368) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-65. doi:1538-7445.AM2012-LB-65

Abstract 4204: Chronic ultraviolet radiation exposure of mouse skin activates GATA and PAX families of transcription factors, which regulate expression of cell survival genes

Abstract Chronic exposure to ultraviolet radiation (UVR) is the most common etiologic factor linked to the development of squamous cell carcinoma (SCC), a non-melanoma form of skin cancer that can metastasize. UVR-induced development of SCC has been shown to be associated with activation of transcription factors Stat3, AP-1 and NF-κB. Here, we report the activation of several novel transcription factors (TFs) following exposure of mouse skin to UVR. In this experiment, FVB/N mice were exposed to UVR (2kj/m2) four times (Monday, Wednesday, Friday and Monday). At 1hr and 3hrs post last UVR exposure, these mice were sacrificed. Nuclear lysates were prepared from the scraped epidermis of these mice. To screen for unknown TFs, we employed the Panomics Combo Protein/DNA array, which contains consensus complimentary sequences of 345 TFs binding sites. Array data was analyzed according to the vendor's suggestions. TFs spots that show more than two fold induction were scored as positive for activation. We observed activation of 70 TFs as early as 1hr post UVR exposure. Twenty four TFs (GATA-1, GATA-2, Thy-1BP, AhR/Amt, Sp-1, HFH-2, HFH-3, HIF-1, CEF-2, TEF-1, PU-1, c-Rel, E47, Anti-oxidant-RE, HOXD8/9/10, PAX-4, LH-2/lim-1, NF-4FA, NCAM BP, WTI, E12, EBP40/45, CEA, MUSF) were activated compared to unexposed mice. Forty six out of seventy were new TFs, which were absent in unexposed mice. These were MHC W box, MT Box, Freac-2, CTCF, MyoD, KBF-A, PPAR, HOXD9/10, Myb, RSRFC4, PYR, WTI, MZF, SAA, COUP-TF, CCAC, EGR, E2F-1, NF-E1/YY-1, NF-1, NF-1/L, PAX-1, PAX-2, PAX-8, PAX-6, NF-ATp, CEBP, AAF, HMG, ADD-1, L-IIIBP, GATA-6, Ets, AP-3, KKLF, HNF-1A, Lyf, EGR-1, XBP-1, HFH-1, Surf-1, YB-1, Fast-1, MSP-1, PPUR, T3R. Among all seventy TFs, highly activated TFs were GATA-1, GATA-2, PAX-4, PAX-6, PAX-8, Thy-1BP, HFH-2, HFH-3, HIF-1, CEA, HOD8/9/10, AhR/Amt, and MUSF. Literature search revealed that there is no report about the role of these TFs in UVR-induced skin carcinogenesis. We employed the Genego program to determine the downstream target genes for all these TFs and found that VEGFR-2, CyclinD1, Bcl-2, Bcl-xl, p53, c-Myc, BMP-4, WT1, survivin are the downstream target genes of GATA1, GATA-2, PAX-4 and PAX-8. All these genes have been shown for their role in anti-apoptosis, cell survival and metastasis of various types of cancers including skin. Importantly, we also observed activation of PAX-8 in UVR exposed mice, which inhibits the transcriptional activation of the tumor suppressor p53 gene. Evidence indicates that GATA-1 controls the expressions of miRNA-144 and miRNA-451. Other study has been shown that inhibition of miRNA-144 causes a decrease in proliferation of Hela cells. We conclude that GATA and PAX family of TFs may have an important role in UVR-induced skin carcinogenesis via modulation of various downstream target genes and microRNAs. (Support: RO1CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4204. doi:10.1158/1538-7445.AM2011-4204

Abstract 3550: Role of TNFα in the cytotoxicity of docetaxel and in docetaxel resistance in MCF-7 cells

Abstract Taxanes such as paclitaxel and docetaxel are widely used in cancer chemotherapy regimens due to their ability to stabilize microtubules and to arrest cell division in mitosis (Chazard, 1994). While the events leading to mitotic arrest are well understood, the mechanism by which the drug induces cytotoxicity is unclear. Paclitaxel has previously been shown to promote secretion of TNFα in macrophages; however this has not been observed in other cell lines (Bogdan, 1992). TNFα induces cell death in many cell lines through interaction with its receptor TNFR1 (Hsu, 1996). TNFα also binds to a second receptor, TNFR2, which does not possess a death domain. TNFR2 does however retain the ability to induce expression of NFκB-dependent survival genes. Our laboratory group recently selected MCF-7 cells for survival in increasing concentrations (doses) of paclitaxel or docetaxel (MCF-7TAX and MCF-7TXT, respectively). Resistance was only achieved at or above selection dose 9 (3.33nM docetaxel) (Hembruff et al., 2008). MCF-7 cells were also “selected” in the absence of these drugs to control for changes simply associated with extended cell propagation. The ABC transporter inhibitor cyclosporin A either had no effect on docetaxel sensitivity at selection dose 9, or partially restored sensitivity at selection dose 12 (Hembruff, 2008). These observations suggest there are likely additional mechanisms of docetaxel resistance at selection doses 9 through 12. Interestingly, we observed that docetaxel concentrations between 1.11nM and 3.33nM induced a >100-fold increase in TNFα secretion from MCF-7 cells, which likely contributes to cytotoxicity. Consistent with this view, a neutralizing TNFR1 antibody decreased cytotoxicity of docetaxel in MCF-7 cells. We also discovered that MCF-7TXT and MCF-7TAX cells at or above selection dose 9 were resistant to TNFα-induced cell death. Moreover, MCF-7TXT cells displayed downregulation of TNFR1 upon the acquisition of resistance to taxanes. Increased levels of TNFα were observed within the media of MCF-7TXT cells selected to dose levels 9 and 10, but not dose levels 11 or 12. We further provide evidence that the increased levels of TNFα in the medium of MCF-7TXT cells contributes to increased cell survivability through a TNFR2-dependent activation of NFκB and its resulting increase in the expression of cell survival genes. 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 3550.

IGF-1 Dependent Changes in Histone H3 Modifications Are Associated with Akt Signaling and the Expression of Cell Survival Genes.

IGF-1 Dependent Changes in Histone H3 Modifications Are Associated with Akt Signaling and the Expression of Cell Survival Genes.

Reactive electrophile species

The interest in reactive electrophile species (RES) stems largely from the fact that they can have powerful biological activities. RES stimulate the expression of cell survival genes as well many other genes commonly upregulated in environmental stress and pathogenesis. RES levels must be carefully controlled in healthy cells but their formation and destruction during stress is of great interest. Unlike many 'classical' signals and hormones, RES can potentially affect gene expression at all levels by chemically reacting with nucleic acids, proteins and small molecules as well as by indirectly lowering pools of cellular reductants. Recent works involving genetic approaches have begun to provide compelling evidence that, although excess RES production can lead to cell damage, lower levels of RES may modulate the expression of cell survival genes and may actually contribute to survival during severe stress.

Overexpression of MEKK3 Confers Resistance to Apoptosis through Activation of NFκB

Many cancers have constitutively activated NFkappaB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFkappaB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFkappaB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFkappaB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFkappaB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2-4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFkappaB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFkappaB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis.

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Bryostatin and CD40-ligand enhance apoptosis resistance and induce expression of cell survival genes in B-cell chronic lymphocytic leukaemia.

Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B-cell chronic lymphocytic leukaemia (B-CLL) cells were treated in vitro with CD40-ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin-1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti-Fas antibody; (b) expression of apoptosis-regulatory proteins (Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAG-1, Flip, XIAP); (c) expression of cell surface co-stimulatory antigens (CD80 [B7.1]; CD54 [ICAM-1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L- and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-XL in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L- and Bryostatin-treated B-CLL cells. This Fas-resistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation.

Little is known about the regulation of apoptosis in the adipocyte. Using the 3T3-L1 cell model of adipogenesis, we investigated whether induction of apoptosis by serum-starvation is differentiation-specific. Apoptosis was assessed by phase-contrast microscopy, Hoechst staining and DNA fragmentation. Protein expression levels were evaluated by immunoblot analysis. Following serum deprivation, Hoechst staining revealed chromatin condensation and formation of apoptotic bodies in 3T3-L1 preadipocytes, but not in differentiated adipocytes. Similarly, although cell cultures of serum-starved 3T3-L1 preadipocytes displayed extensive apoptotic DNA fragmentation, this was barely detectable for cell cultures subjected to differentiation medium, in which 70-90% of the cells had assumed the mature phenotype. Consistent with the ability of the adipocytes to resist apoptosis, immunoblot analysis revealed that differentiated cell cultures expressed Bcl-2 and the endonuclease DNase I at higher and lower levels, respectively. Expression of cell survival genes is modulated during 3T3-L1 adipocyte differentiation, potentially contributing to the state of resistance to apoptosis observed for mature 3T3-L1 adipocytes upon growth factor deprivation.

Cloning, characterization and expression of two Xenopus bcl-2-like cell-survival genes

We describe two cloned cDNAs, termed xR1 and xR11, isolated from a Xenopus laevis stage 28–30 embryonic head cDNA library. Comparison of amino acid (aa) sequences derived from nucleotide (nt) sequences of xR1 and xR11 cDNAs revealed substantial homology with bcl-2-related genes, especially with bcl-x spl . In particular, there was a marked conservation of the BH1 and BH2 domains considered to be important for the anti-cell death and heterodimerisation properties of bcl-2. Constitutive expression of xR11 in cultured rat fibroblast (Rat-1) cells conferred a strong protection against cell death induced by the cytotoxic agents staurosporine and cycloheximide, by serum deprivation and specific deregulation of c-myc. Measurement of xR1 and xR11 mRNAs by RNase protection assay revealed similar widespread expression in Xenopus embryos and tadpoles. Except for an abrupt increase in the accumulation of xR1 and xR11 mRNAs in brains of mid-metamorphic and post-metamorphic tadpoles and adults, there was insignificant modulation of their expression in tissues undergoing total regression (tail) or morphogenesis (limb) during natural or thyroid hormone-induced metamorphosis. These findings raise the possibility of continuing expression of cell survival genes in tissues undergoing total regression during post-embryonic development.