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G Chinnadurai

Publications and source records attributed to G Chinnadurai.

At least 19 recordsLinked to original sources
PubMed2006

Changes in C-terminal binding protein 2 (CtBP2) corepressor complex induced by E1A and modulation of E1A transcriptional activity by CtBP2.

The N-terminal region of adenovirus E1A interacts with histone acetyl transferases (HATs) such as p300, P/CAF, and GCN5. The C-terminal region interacts with the transcriptional corepressors CtBP1 and CtBP2. The functional significance of co-recruitment of HATs and CtBPs by E1A is not well understood. In this study, we have shown that E1A enhanced acetylation of CtBP2 by recruitment of p300 to the CtBP2 complex. Additionally, E1A also displaced the histone methyltransferase G9a and the E-box repressor ZEB from the CtBP2 complex through the C-terminal CtBP-binding domain. A transcriptional activation function encoded by the E1A N-terminal region was efficiently inhibited by CtBP2 but not by a mutant with an N-terminal deletion or by a mutant deficient in interaction with E1A. Two isoforms of CtBP1 (CtBP1-L and CtBP1-S) poorly inhibited transcriptional activity of the E1A N-terminal region. Thus, the N-terminal domain of CtBP2 may contribute a unique transcriptional regulatory activity of CtBP2. Our results provide new insights by which CtBP might modulate the biochemical activities of E1A.

Acetylation↗
PubMed2006

Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells.

In contrast to the initial notion that the biological activity of p14(ARF) strictly depends on a functional mdm-2/p53 signaling axis, we recently demonstrated that p14(ARF) mediates apoptosis in a p53/Bax-independent manner. Here, we show that p14(ARF) induces breakdown of the mitochondrial membrane potential and cytochrome c release before triggering caspase-9- and caspase-3/7-like activities in p53/Bax-deficient DU145 prostate cancer cells expressing wild-type Bak. Re-expression of Bax in these cells failed to further enhance p14(ARF)-induced apoptosis, suggesting that p14(ARF)-induced apoptosis primarily depends on Bak but not Bax in these cells. To further define the role of Bak and Bax in p14(ARF)-induced mitochondrial apoptosis, we employed short interference RNA for the knockdown of bak in isogeneic, p53 wild-type HCT116 colon cancer cells either proficient or deficient for Bax. There, combined loss of Bax and Bak attenuated p14(ARF)-induced apoptosis whereas single loss of Bax or Bak was only marginally effective, as in the case of DU145. Notably, HCT116 cells deficient for Bax and Bak failed to release cytochrome c and showed attenuated activation of caspase-9 (LEHDase) and caspase-3/caspase-7 (DEVDase) upon p14(ARF) expression. These data indicate that p14(ARF) triggers apoptosis via a Bax/Bak-dependent pathway in p53-proficient HCT116, whereas Bax is dispensable in p53-deficient DU145 cells. Nevertheless, a substantial proportion of p14(ARF)-induced cell death proceeds in a Bax/Bak-independent manner. This is also the case for inhibition of clonogenic growth that occurs, at least in part, through an entirely Bax/Bak-independent mechanism.

Apoptosis↗
PubMed2006

Genetic identification of adenovirus type 5 genes that influence viral spread.

The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.

Adenovirus E1B Proteins↗
PubMed2005

Acetylation by p300 regulates nuclear localization and function of the transcriptional corepressor CtBP2.

CtBP family members, CtBP1 and CtBP2, are unique transcriptional regulators that adapt a metabolic enzyme fold, and their activities are regulated by NAD(H)-binding. CtBP1 is both cytoplasmic and nuclear, and its subcellular localization is regulated by sumoylation, phosphorylation, and binding to a PDZ protein. In contrast, we showed that CtBP2 is exclusively nuclear. CtBP1 and CtBP2 are highly similar, but differ at the N-terminal 20 amino acid region. Substitution of the N-terminal domain of CtBP1 with the corresponding CtBP2 domain confers a dominant nuclear localization pattern to CtBP1. The N-terminal domain of CtBP2 contains three Lys residues. Our results show that these Lys residues are acetylated by the nuclear acetylase p300. Although all three Lys residues of CtBP2 (Lys-6, Lys-8, and Lys-10) appear to be acetylated, acetylation of Lys-10 is critical for nuclear localization. CtBP2 with a single amino acid substitution at Lys-10 (K10R) is predominantly localized in the cytoplasm. The cytoplasmic localization of the K10R mutant is correlated with enhanced nuclear export that is inhibited by leptomycin B. Furthermore, lack of acetylation at Lys-10 renders CtBP2 to be more efficient in repression of the E-cadherin promoter. Our studies have revealed the important roles of acetylation in regulating subcellular localization and transcriptional activity of CtBP2.

Acetylation↗
PubMed2005

Mitochondrial localization of p53 during adenovirus infection and regulation of its activity by E1B-19K.

Recent results have revealed that the p53 tumor suppressor protein possesses a direct transcription-independent apoptotic activity. During apoptosis induced by genotoxic stress, a small fraction of p53 is targeted to mitochondria where it initiates apoptosis by causing mitochondrial dysfunction. In adenovirus-infected cells, the expression of E1A protein enhances the accumulation of p53 during early phases of infection and during late times after infection, it is targeted for degradation by the combined action of E1B-55K and E4-orf6 proteins. The functional significance of E1A-mediated accumulation of p53 during early phases of viral replication is not known. Our studies with isogenic epithelial cell lines that differ only on the status of p53 indicate that Ad infection induces apoptosis by p53-dependent and -independent pathways and both pathways are suppressed by E1B-19K. We show that during early phase of Ad infection, a fraction of p53 is targeted to the mitochondria. In virus infected cells, a large fraction of the viral antiapoptosis protein E1B-19K is also localized in mitochondria during early and late phases of infection. Coimmunoprecipitation analysis has revealed that p53 and E1B-19K form a complex in mitochondria. The interaction of 19K involves two noncontiguous regions located around amino-acid residues 14-15 and 123-124. On p53, the mutations within the DNA-binding domain reduce interaction with E1B-19K. Our studies also suggest that 19K may additionally complex with the multidomain mitochondrial proapoptotic protein BAK, thereby reducing the level of p53 interaction with BAK. We suggest that p53-induced apoptosis may be important for efficient cell lysis and viral spread and that E1B-19K may neutralize the apoptotic activity of p53 at multiple levels.

Adenoviridae↗
PubMed2005

CtIP, a candidate tumor susceptibility gene is a team player with luminaries.

CtIP is a nuclear protein conserved among vertebrates that was discovered as a cofactor of the transcriptional corepressor CtBP. CtIP also interacts with the tumor suppressors such as BRCA1 and the pRb family members through binding sites that are frequently mutated in human cancers. CtIP is a target for BRCA1-dependent phosphorylation by the ATM kinase induced by DNA double strand breakage. CtIP plays a role in DNA-damage-induced cell cycle checkpoint control at the G2/M transition. Homozygous inactivation of the Ctip gene causes very early embryonic lethality during mouse development. The Ctip(-/-) embryo cells are arrested in G1 and do not enter S phase. Depletion of Ctip in established mouse embryo fibroblasts arrests cells in G1 and results in an accumulation of hypophosphorylated Rb and the Cdk inhibitor p21, suggesting that CtIP is also a critical regulator of G1/S transition of the cell cycle. The Ctip gene contains a mononucleotide (A9) repeat and one of the alleles is mutated at a high frequency in colon cancers with microsatellite instability. The Ctip(+/-) mice develop multiple types of tumors suggesting that haploid insufficiency of Ctip leads to tumorigenesis. Among the various tumor types observed in Ctip(+/-) heterozygous mice, large lymphomas are prevalent. Recent studies raise the possibility that Ctip may itself be a tumor susceptibility gene and suggest that it might be important for the activities of tumor suppressors BRCA1, pRb family proteins and Ikaros family members.

Alcohol Oxidoreductases↗
PubMed2004

Modulation of oncogenic transformation by the human adenovirus E1A C-terminal region.

The E1A oncogene of human adenoviruses cooperates with other viral and cellular oncogenes in oncogenic transformation of primary and established cells. The N-terminal half of E1A proteins that form specific protein complexes with pRb family and p300/CBP transcriptional regulators is essential for the transforming activities of E1A. Although the C-terminal half of E1A is dispensable for the transforming activities, it negatively modulates the oncogenic activities of the N-terminal region. Mutants of E1A lacking the C-terminal half or a short C-terminal region exhibit a hyper-transforming phenotype in cooperative transformation assays with the activated ras oncogene. The E1A C-terminal region implicated in the oncogenesis-restraining activity interacts with a 48-kDa cellular phosphoprotein, CtBP, that functions as a transcriptional corepressor. It appears that the C-terminal region of E1A may suppress E1A-mediated oncogenic transformation by a dual mechanism of relieving repression cellular genes by CtBP, and also by antagonizing the oncogenic activities of the N-terminal half of E1A.

Adenovirus E1A Proteins↗
PubMed2003

Pro-apoptotic activity of transiently expressed BCL-2 occurs independent of BAX and BAK.

BCL-2 suppresses apoptosis induced by a wide variety of stimuli in multiple cell types. Most of the in vitro studies that have examined the activity of BCL-2 have employed stable cell lines that ectopically express BCL-2. We have reported that BCL-2 is expressed at high levels in the absence of the 5'- and 3'-UTRs of the Bcl-2 gene and transient high level of expression results in potent cell death (Uhlmann et al., [1998]: JBC 278:17926-17932). Expression of BCL-2 under the transcriptional control of the cognate 5'- and 3'-UTRs express lower levels of BCL-2 and does not cause cell death. Our present results suggest that in contrast to BCL-2, transient expression of BCL-xL does not induce cell death and coexpression of BCL-xL with the pro-apoptotic BCL-2 does not suppress cell death. The pro-apoptotic activity of BCL-2 appears to involve activation of the cytochrome c/caspase 9/caspase 3 pathway. Elevated levels of BCL-2 expression results in N-terminal cleavage of BCL-2 at a novel site different from a previously identified caspase cleavage site at Asp 34 by a non-caspase protease. Transient expression of a BCL-2 mutant lacking aa 51-85 within the loop region induces efficient cell death and N-terminal cleavage of BCL-2 while a different deletion mutant lacking aa 30-91 induces reduced levels of cell death in the absence of BCL-2 cleavage suggesting that N-terminal processing of BCL-2 may be an amplification event in BCL-2-mediated cell death. Overexpression of BCL-2 in a Bax-null human colon cancer cell line (HCT116Bax-/-) induces efficient cell death. The pro-apoptotic activity of BCL-2 is also observed in a Bax-null cells in which BAK expression is inhibited by stable RNAi expression. Our results suggest that BCL-2 contains an intrinsic pro-apoptotic activity and can induce apoptosis independent of BAX and BAK under specific conditions.

Apoptosis↗
PubMed2003

Association of class I histone deacetylases with transcriptional corepressor CtBP.

The C-terminal binding protein (CtBP) family proteins are transcriptional regulators that are conserved from worm to human. They function as corepressors of a wide array of DNA-binding transcriptional repressors. The mammalian CtBPs appear to mediate transcriptional repression in a histone deacetylase (HDAC)-dependent or -independent manner, depending on the context of the promoter. To identify the components of the CtBP corepressor complex, we isolated CtBP-containing protein complexes from the nuclear extracts prepared from HeLa cells infected with adenovirus vectors that expresses hCtBP1. Western blot analysis of these complexes suggests that hCtBP1 associates with class I HDACs, HDAC-1, HDAC-2 and HDAC-3. Some of these HDACs also interact with the Drosophila CtBP homolog, dCtBP. The CtBP protein complex exhibits significant HDAC activity in vitro suggesting that association of CtBP with HDACs may be functionally relevant.

Alcohol Oxidoreductases↗
PubMed2003

Nicotinamide adenine dinucleotide stimulates oligomerization, interaction with adenovirus E1A and an intrinsic dehydrogenase activity of CtBP.

The C-terminal region of adenovirus E1A interacts with the transcriptional corepressor, CtBP. The mechanism of transcriptional regulation by CtBP is not known. CtBP shares a significant homology with NAD(+)-dependent D2-hydroxy acid dehydrogenases. CtBP binds to NAD(+) and NADH. Both forms of the dinucleotide stimulate oligomerization of native CtBP and enhance complex formation with E1A. CtBP also has a slow dehydrogenase activity. Interaction of CtBP with E1A reduces the dehydrogenase activity. Our results raise the possibility that the oxidation/reduction reactions of CtBP may regulate transcription. Thus, CtBP is a unique transcriptional regulator with an enzymatic activity similar to metabolic dehydrogenases. The levels of intracellular nicotinamide adenine dinucleotide may modulate transcriptional activity of CtBP.

Adenovirus E1A Proteins↗
PubMed2003

CtBP family proteins: more than transcriptional corepressors.

CtBP family proteins predominantly function as transcriptional corepressors. Studies with mutant mouse suggest that the two mouse genes, Ctbp1 and Ctbp2, play unique and redundant gene regulatory roles during development.1 Ctbp1-deficient mice are viable, but are small and die early, while Ctbp2 deficiency leads to embryonic lethality. Ctbp2-null mutation causes defects in axial patterning, heart morphogenesis and neural development. The Ctbp2 mutant phenotype is more severe in the absence of Ctbp1. The studies with Ctbp2 mutant embryos suggest that CtBP can also activate transcription. A plant CtBP homolog, Angustifolia (AN), has recently been identified.2,3 AN controls polar elongation of leaf cells via the microtubule cytoskeleton. Microarray analysis suggests that AN also functions as a transcriptional repressor. Thus, the CtBP family proteins control cellular processes by serving as transcriptional activators and regulators of the cytoskeleton as well as transcriptional corepressors.

Alcohol Oxidoreductases↗
PubMed2003

A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

Active Transport, Cell Nucleus↗
PubMed2002

Critical requirement of BAX for manifestation of apoptosis induced by multiple stimuli in human epithelial cancer cells.

Studies with mouse embryo fibroblasts deficient for the BCL-2 family multidomain proapoptotic proteins BAX and BAK have revealed that both of these proteins are essential for apoptosis induced by multiple stimuli, suggesting that these proapoptotic proteins are functionally overlapping in these cells [M. C. Wei et al., Science (Wash. DC), 292: 727-730, 2001; W. X. Zong et al., Genes Dev., 15: 1481-1486, 2001]. We have determined the effect of several different apoptotic stimuli in a Bax-deficient human epithelial cancer cell line (HCT116BaxKO). We show that this cell line expresses functional BAK protein and is defective in manifestation of apoptosis induced by the BH3-only proteins BIK and BID as well as extrinsic stimuli that engage the death receptors, tumor necrosis factor receptor, tumor necrosis factor-related apoptosis-inducing ligand receptor, and Fas. In addition, this cell line is deficient for apoptosis induced by cytotoxic agents such as UV, staurosporine, and thapsigargin that induce either mitochondrial or endoplasmic reticulum stress. Our results suggest that BAX plays a critical role in the manifestation of apoptosis paradigms induced by multiple stimuli in human epithelial cancer cells. Our results also suggest that the integrity of BAX may have important consequences in the progression of epithelial tumors and in determining the outcome of chemotherapeutic regimens of such tumors.

Antibodies↗
PubMed2002

Sox9 transactivation and testicular expression of a novel human gene, KIAA0800.

The Sry and Sox9 sex-determination factors initiate and promote testis differentiation by gene transactivation through similar promoter elements. However, knowledge is limited concerning what genes are regulated by Sry/Sox9 in the testis. Identification and characterization of Sry/Sox9-regulated genes are critical for understanding sexual differentiation. We now demonstrate that a novel human gene, KIAA0800, is preferentially expressed in the testis and is transactivated by Sox9. The KIAA0800 promoter is repressed by an upstream element involving a polyT track and two Alu repeats. Two specific Sox9-bindings sites have been identified in the KIAA0800 promoter by using DNaseI footprinting assays and gel electrophoretic mobility shift assays. Sox9 transactivation of the KIAA0800 promoter appears to be exerted mainly through the relief of promoter repression. Genes homologous to the human KIAA0800 exist in organisms with differentiated sex tissues including mouse, Drosophila, and C. elegans, but not in unicellular organisms, including yeast and bacteria. Further, our recent sequence analysis shows that KIAA0800 protein is 97% identical between human and mouse. Thus, KIAA0800 is a novel Sox9-activated gene that is evolutionarily conserved and potentially involved in sexual differentiation.

Alu Elements↗
PubMed2002

CtBP, an unconventional transcriptional corepressor in development and oncogenesis.

CtBP family proteins are conserved among vertebrates and invertebrates and function as transcriptional corepressors. They repress transcription in a histone deacetylase-dependent or -independent manner. CtBPs play important roles during development and oncogenesis. In this review, their unusual properties, the mechanisms of transcriptional repression, regulation, and their biological functions are discussed.

Acetylation↗
PubMed2002

Requirement of BAX for efficient adenovirus-induced apoptosis.

Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax(+/-)) and HCT116BaxKO (Bax(-/-)) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax(+/-) cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax(-/-) cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax(+/-) cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.

Adenovirus E1B Proteins↗
PubMed2001

Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells.

The mechanism underlying apoptosis induced by proteasome inhibition in leukemic Jurkat and Namalwa cells was investigated in this study. The proteasome inhibitor lactacystin differentially regulated the protein levels of proapoptotic Bcl-2 family members and Bik was accumulated at the mitochondria. Bik overexpression sufficed to induce apoptosis in these cells. Detailed examination along the respiration chain showed that lactacystin compromised a step after complex III, and exogenous cytochrome c could overcome this compromise. Probably as a result, the succinate-stimulated generation of mitochondrial membrane potential was significantly diminished. Bcl-x(L) interacted with Bik in the cells, and Bcl-x(L) overexpression prevented cytochrome c leakage out of the mitochondria, corrected the mitochondrial membrane potential defect, and protected the cells from apoptosis. These results show that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members, Bik being one of them.

Acetylcysteine↗