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Supplementary Data from BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds through the intrinsic mitochondrial apoptosis pathway. We identify the cascade of molecular events responsible for cell death induced by a prototypical proteasome inhibitor, MG132, starting with rapid accumulation of BH3-only proteins in the mitochondria, proceeding through mitochondrial membrane permeabilization and subsequent loss of ΔΨm, and leading to irreversible changes of mitochondrial ultrastructure, degradation of mitochondrial network, and detrimental impairment of crucial mitochondrial functions. Our results also establish a rationale for the broader use of proteasome inhibitors to kill apoptosis-resistant tumor cells that lack functional BAX/BAK proteins. (Mol Cancer Res 2009;7(8):1268–84)

A facile method to produce iron oxide nanoparticles (FeO NPs) has been introduced as a part of green approach. Here, we introduce a semi-green method to fabricate FeO NPs using Channa straitus (C. ...

In colorectal cancer, chemotherapy and/or radiotherapy can lead to the formation of resistant cells that become metastatic through Epithelial-Mesenchymal Transition (EMT). Invasive and metastatic characteristics of carcinoma cells in primary tumors are mediated by EMT. During EMT, the primary tumor cells lose cell-cell adhesion, have increased intercellular separation, and gain an elongated shape with pseudopodia. There is also dysregulation of Polycomb group proteins (such as BMI1, SUZ12, and EZH2), and changes in the expression of microRNA-200 (miR-200) family. In this study, we developed a chemoresistant colorectal cancer cell line (DLD-1-OxR) by exposing DLD-1 colorectal cancer cells to increasing concentrations of oxaliplatin (a chemotherapy drug used for colorectal cancer), and tested for EMT characteristics. We found that DLD-1-OxR exhibited EMT characteristics by morphologic, biochemical and molecular markers. SUZ12, a Polycomb repressive complex 2 subunit, was upregulated in DLD-1-OxR. The miRNA-200 family members that target SUZ12 were downregulated. Drug resistance is an impediment to chemotherapy and understanding the molecular mechanisms of chemoresistance can lead to its reversal and improvement of chemotherapy outcomes.

Simple methodology was developed to synthesize copper oxide nanoparticles (CuO NPs) using mucus of Channa striatus (C. striatus). The mucus of C. striatus is known for its biological properties due to the presence of numerous amino acids. This mucus was used as stabilizing agent for CuO NPs synthesis from copper acetate. The prepared CuO NPs were characterized by fourier transforms infrared spectrometer (FTIR), powder x-ray diffraction (XRD), x-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), energy dispersive x-ray analysis (EDX) and transmission electron microscope (TEM) coupled with selected area diffraction pattern (SAED). The FTIR study suggested the utilization of mucus in the synthesis of CuO NPs. The XRD data also confirmed formation of pure crystalline phase of CuO NPs. Fish mucus stabilized CuO NPs exhibited significant activity against HeLa cells. The results of cell death clearly indicated that the synthesized CuO nanoparticles could be served as a biomaterial for anticancer treatment.

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.

Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC–MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells.

Hydroxyapatite nanorods (HA) were fabricated using caffeine as a modifier at room temperature. Various concentrations of caffeine were utilized to synthesize the HA nanoparticles to analyze its effect on size, shape and morphology. The formation of phosphate ion was confirmed by Fourier Transform spectrophotometer (FTIR). Crystalline structure and phase of the nanoparticles was confirmed by x-ray diffraction study (XRD). The formation of HA nanorods with diversified morphology was confirmed by Transmission Electron Microscope (TEM) images. The results acquired in this study reveals that the HA nanoparticles synthesized using caffeine are uniform and evenly scattered nanorods. The average size of the nanorods obtained from the XRD study was in the range of 20–30 nm. The TEM and SAED investigation revealed the shape, morphology and arrangement of HA nanorods with higher crystallinity. This investigation claims that caffeine is a natural modifier to produce HA nanorods.

C-terminal binding protein (CtBP) family transcriptional corepressors include CtBP1 and CtBP2. While CtBP1 and CtBP2 share significant amino acid sequence homology, CtBP2 possesses a unique N-terminal domain that is modified by acetylation and contributes to exclusive nuclear localization. Although CtBP1 and CtBP2 are functionally redundant for certain activities during vertebrate development, they also perform unique functions. Previous studies have identified several CtBP1-interacting proteins that included other transcriptional corepressors, DNA-binding repressors and histone modifying enzymatic components such as the histone deacetylases and the histone demethylase LSD-1. Here, we carried out an unbiased proteomic analysis of CtBP2-associated proteins and discovered the association of several components of the CtBP1 proteome as well as novel interactions. The CtBP2 proteome contained components of the NuRD complex and the E2F family member E2F7. E2F7 interacted with the hydrophobic cleft region of CtBP1 and CtBP2 through a prototypical CtBP binding motif, PIDLS. E2F7 repressed E2F1 transcription, inhibited cell proliferation in a CtBP-dependent fashion. Our study identified CtBP as a corepressor of E2F7 and as a regulator of DNA damage response.

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells.

The balance of Double wheel self supporting robot is maintained by following the inverted pendulum concept. The PWM technique is adapted for controlling the DC motors which are connected to the robot wheels. In the existing robot models tilt sensor is used which makes them to move in a straight line only. Even with the connecting encoder tilt sensor does not reduced the error. The proposed method uses both Gyroscope and Accelerometer which are used to detect tilt angle & acceleration. As a result of this our robot will be able to move in curvilinear path. Any error from this sensor is overcome by coupling a kalman filter. By Using IOT and adopting port forwarding method multiple robots can be controlled. The application areas of this kind of robots are moving wheel chair, explosive detection and disposing and short range vehicles.

In this issue of Cell Cycle, Straza et al.1 report therapeutic targeting of the transcriptional compressor C-terminal binding protein (CtBP) with a small molecule to specifically induce apoptosis in transformed cells and in cancer cells. Although CtBP has been associated with tumor promotion as a negative regulator of several tumor suppressors,2 the present study is significant since it is the first report on exploiting CtBP as an anti-cancer target. The vertebrate CtBP family proteins consist of two major related proteins, CtBP1 and CtBP2 (collectively referred as CtBP) that function as transcriptional corepressors for a large number of repressors. Both proteins exhibit overall sequence and structural similarity to the D-isomer specific 2-hydroxy acid dehydrogenases (D2-HDH).3 The role of the dehydrogenase activity in CtBP-mediated transcriptional repression remains controversial. Although CtBP1 was shown to possess a slow dehydrogenase activity,4,5 the cognate “substrate” for the CtBP dehydrogenase activity remained obscure until an intermediate of the methionine salvage pathway, 2-keto-4-methylthio-2-oxo butyrate (MTOB) was identified as a good substrate for CtBP1 dehydrogenase.6 The analysis of the constituents of the CtBP1 protein complex has helped to establish a general mechanism of transcriptional repression by CtBP7 (Fig.1). A DNA-binding transcriptional repressor recruits CtBP to the target promoter through a PLDLS-like motif first identified in adenovirus E1A. Using the individual subunits in the CtBP dimer, CtBP simultaneously interacts with a transcriptional repressor and a chromatin-modifying protein complex that consists of enzymes such as histone deacetylases (HDAC) 1/2 and lysine specific demethylase-1 (LSD-1)4 that suppress gene transcription. By structural and biochemical studies, an NAD(H)-binding domain in CtBP has been found to be a redox sensor of the cellular NAD+/NADH ratio, altering CtBP conformation and/or dimerization to regulate CtBP interaction with repressors/chromatin modifying enzymes. Figure 1 (A) Transcriptional repression of tumor suppressors and apoptotic genes by the CtBP dimer under normal conditions. CtBP is recruited to the target promoter through interaction with the PLDLS-like motif in the repressor which is anchored to the target ... The present study by Straza et al. has revealed an important clue to the role of the dehydrogenase “substrate”-binding domain in CtBP function. In the CtBP structure, the substrate-binding domain and the PLDLS-binding cleft are physically very close to the dehydrogenase catalytic center.8,9 Although PLDLS-binding does not seem to compete with substrate binding in vitro,8 data by Straza et al. suggest that substrate loading can exert a significant impact on CtBP recruitment to target promoters. The CtBP substrate MTOB was shown to relieve CtBP2-mediated repression of a pro-apoptotic gene Bik at high concentrations, resulting in enhanced apoptosis of transformed cells and cancer cells in contrast to the relative resistance of normal cells to MTOB. This effect was correlated with reduced recruitment of CtBPs, especially CtBP2, to the Bik promoter. Although CtBP1/2 were previously reported to repress several pro-apoptotic genes including Noxa, Perp, Bax and Bik,10,11 Bik appears to be most critical for MTOB-induced apoptosis since shRNA-mediated depletion of Bik relieved most of MTOB-induced apoptosis in colorectal cancer cells. This result suggests that molecules that maximize the cell killing activity of Bik may constitute attractive anticancer agents. Although no detailed mechanism of MTOB-suppression of CtBP recruitment to target promoters has been provided by the authors, one possibility might be that MTOB loading to the CtBP substrate-binding domain causes conformational changes in the PLDLS-binding cleft, resulting in reduced CtBP interaction with promoter-bound transcriptional repressors (Fig.1). Should this be the case, then experiments with mutants in the substrate-binding dozmain of CtBP could be performed to show resistance of the mutants to inhibition by MTOB. It is of high significance that this study also showed that CtBP is over-expressed in many primary colon tumors and CtBP expression appears to be inversely correlated with expression of tumor suppressor ARF. More detailed expression profiling of CtBP, tumor suppressors, and apoptosis-regulatory genes including Bik in various tumor samples may help uncover new correlations between these genes and help better understand the mutual regulations and roles of these genes during tumorigenesis. The authors demonstrated the utility of MTOB as an anti-cancer agent in cell culture as well as in the mouse xenograft model. Since MTOB appears to inhibit the transcriptional activity of CtBP at relatively high concentrations, identification of new generation CtBP substrate molecules that function at relatively low concentrations might constitute more effective anti-cancer drugs. Should the biochemical mechanism of MTOB-mediated inhibition of CtBP be clarified further, there might be MTOB derivatives that could bind to the CtBP substrate-binding domain and inhibit CtBP function more specifically and more efficiently.

Head and neck squamous cell carcinomas (HNSCC) are one of the leading causes of cancer deaths world wide. Up-regulation of the epidermal growth factor receptor (EGFR) and BCL-2 family anti-apoptosis proteins in these cancers is linked to aggressive tumor growth, metastasis and chemoresistance. Infection of two HNSCC cell lines, SCC25 and CAL27 by an Ad5 mutant (lp11w) defective in coding for the viral anti-apoptosis protein, E1B-19K efficiently induced apoptotic cell death. In cells infected with lp11w there was a dramatic down-regulation of EGFR by apoptosis-dependent and -independent mechanisms. The levels of the anti-apoptotic proteins BCL-2, BCL-xL and MCL- 1 were also down-regulated in lp11w-infected cells compared to uninfected or Ad5 wt infected cells. Infection with lp11w also enhanced sensitivity of the HNSCC cells to the chemotherapeutic drug cisplatin. Our results suggest that adenoviral vectors defective in E1B-19K would be valuable for efficient down-regulation of cell survival proteins and EGFR in epithelial cancers and could be exploited as oncolytic agents to treat HNSCCs.

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds through the intrinsic mitochondrial apoptosis pathway. We identify the cascade of molecular events responsible for cell death induced by a prototypical proteasome inhibitor, MG132, starting with rapid accumulation of BH3-only proteins in the mitochondria, proceeding through mitochondrial membrane permeabilization and subsequent loss of ΔΨm, and leading to irreversible changes of mitochondrial ultrastructure, degradation of mitochondrial network, and detrimental impairment of crucial mitochondrial functions. Our results also establish a rationale for the broader use of proteasome inhibitors to kill apoptosis-resistant tumor cells that lack functional BAX/BAK proteins. (Mol Cancer Res 2009;7(8):1268–84)

CtBP1 and CtBP2 are closely related and evolutionarily conserved transcriptional corepressors. There is strong evidence linking CtBPs to tumorigenesis and tumor progression. CtBPs promote epithelial-mesenchymal transition and function as apoptosis antagonists. Also, CtBPs mediate repression of several tumor suppressor genes. Certain tumor suppressors also target CtBPs to restrain their tumor-promoting activity. Down-regulation of CtBPs mediated by some tumor suppressors results in p53-independent apoptosis and reduced tumor cell migration and invasion. The role of CtBPs in modulating the activities of different tumor suppressors is reviewed here. The results discussed here suggest that CtBPs may constitute a novel p53-independent anticancer target. [Cancer Res 2009;69(3):731–4]

BIK is the founding member of the BH3-only family pro-apoptotic proteins. BIK is predominantly localized in the ER and induces apoptosis through the mitochondrial pathway by mobilizing calcium from the ER to the mitochondria and remodeling the mitochondrial cristae. BIK-mediated apoptosis is mediated by selective activation of BAX. BIK also induces non-apoptotic cell death in certain cell types by unknown mechanisms. BIK is non-essential for animal development, but appears to be functionally redundant for certain developmental functions with BIM. BIK is implicated in the selection of mature B cells in humans. BIK is a pro-apoptotic tumor suppressor in several human tissues and its expression in cancers is prevented by chromosomal deletions encompassing the Bik locus or by epigenetic silencing. BIK appears to be a critical effector in apoptosis induced by toxins, cytokines and virus infection. Several anti-cancer drugs transcriptionally activate Bik gene expression through transcriptional pathways dependent on factors such as E2F and p53 or by removal of epigenetic marks on the chromatin. BIK appears to be a prominent target for anti-cancer drugs that inhibit proteasomal functions. BIK has also been used as a therapeutic molecule in gene therapy-based approaches to treat difficult cancers.

BH3-only BCL-2 family proteins are effectors of canonical mitochondrial apoptosis. They discharge their pro-apoptotic functions through BH1-3 pro-apoptotic proteins such as BAX and BAK, while their activity is suppressed by BH1-4 anti-apoptotic BCL-2 family members. The precise mechanism by which BH3-only proteins mediate apoptosis remains unresolved. The existing data are consistent with three mutually non-exclusive models (1) displacement of BH1-3 proteins from complexes with BH1-4 proteins; (2) direct interaction with and conformational activation of BH1-3 proteins; and (3) membrane insertion and membrane remodeling. The BH3-only proteins appear to play critical roles in restraining cancer and inflammatory diseases such as rheumatoid arthritis. Molecules that mimic the effect of BH3-only proteins are being used in treatments against these diseases. The cell death activity of a subclass of BH3-only members (BNIP3 and BNIP3L) is linked to cardiomyocyte loss during heart failure. In addition to their established role in apoptosis, several BH3-only members also regulate diverse cellular functions in cell-cycle regulation, DNA repair and metabolism. Several members are implicated in the induction of autophagy and autophagic cell death, possibly through unleashing of the BH3-only autophagic effector Beclin 1 from complexes with BCL-2/BCL-xL. The Chapters included in the current Oncogene Review issues provide in-depth discussions on various aspects of major BH3-only proteins.

C-terminal binding protein (CtBP) family proteins CtBP1 and CtBP2 are highly homologous transcriptional corepressors and are recruited by a large number of transcription factors to mediate sequence-specific transcriptional repression. In addition to DNA-binding repressors, the nuclear protein complex of CtBP1 consists of enzymatic constituents such as histone deacetylases (HDAC1/2), histone methyl transferases (HMTases; G9a and GLP), and the lysine-specific demethylase (LSD1). Additionally, CtBPs also recruit the components of the sumoylation machinery. The CtBPs contain two different unique structural elements, a hydrophobic cleft, with which factors that contain motifs related to the E1A PLDLS motif bind, and a surface groove that binds with factors containing motifs related to the sequence RRTGXPPXL (RRT motif). By structure-based functional dissection of CtBP1, we show that the PLDLS-binding cleft region functions as the primary recruitment center for DNA-binding factors and for the core and auxiliary enzymatic constituents of the CtBP1 corepressor complex. We identify HDAC1/2, CoREST/LSD1, and Ubc9 (E2) as the core constituents of the CtBP1 complex, and these components interact with the PLDLS cleft region through non-PLDLS interactions. Among the CtBP core constituents, HDACs contribute predominantly to the repression activity of CtBP1. The auxiliary components include an HMTase complex (G9a/Wiz/CDYL) and two SUMO E3 ligases, HPC2 and PIAS1. The interaction of auxiliary components with CtBP1 is excluded by PLDLS (E1A)-mediated interactions. Although monomeric CtBP1 is proficient in the recruiting of both core and auxiliary components, NAD(H)-dependent dimerization is required for transcriptional repression. We also provide evidence that CtBP1 functions as a platform for sumoylation of cofactors.

Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major E1A proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.

The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. IMPORTANCE The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

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.

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 ( dl 327; 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.

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.

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.

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.

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.

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 ( dl 250) 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 dl 250 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.

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.

BIK is a pro-apoptotic BCL-2 family member and is the founding member of a subfamily of pro-apoptotic proteins known as "BH3-alone" proteins. Ectopic expression of BIK induces apoptosis in variety of mammalian cells. BIK complexes with various anti-apoptotic BCL-2 family proteins such as adenovirus E1B-19K and BCL-2 via the BH3 domain. However, the heterodimerization activity of BIK alone is insufficient for its apoptotic activity. Previous studies have shown that phosphorylation regulates the functional activity of both anti-apoptotic and pro-apoptotic members of the BCL-2 family. Here, we have examined phosphorylation of BIK and its effect on the apoptotic activity of BIK. We show that BIK exists as a phosphoprotein and is phosphorylated at residues 33 (threonine) and 35 (serine). Mutation of the phosphorylation sites, in which the Thr and Ser residues were changed to alanine residues, reduced the apoptotic activity of BIK without significantly affecting its ability to heterodimerize with BCL-2. Our results suggest that phosphorylation of BIK is required for eliciting efficient apoptotic activity. Partial purification of the protein kinase from HeLa cell cytoplasmic extracts suggest that BIK may be phosphorylated by a casein kinase II-related enzyme.

The human Bik gene codes for a strong pro-apoptotic protein BIK. We have used fluorescent in-situ hybridization to establish the chromosomal localization of the Bik gene to 22q13.3. Genomic clones of the Bik gene were identified from a cosmid library of chromosome 22. Detailed analysis of the Bik gene revealed that it spans a region of about 19 kb and comprises of five exons. Sequence analysis indicated that the 5′ flanking region of Bik lacks canonical TATA and CAAT boxes but directs transcriptional initiation from a single site. A 1.9 kb region containing the promoter elements of the Bik gene was identified and was found to direct expression of the reporter cat gene in transient transfection studies. By mutational analysis, the minimal Bik promoter was localized to a region between −211 to +153. Northern blot analysis showed a ubiquitous expression profile of the Bik mRNA with elevated levels of expression in heart and skeletal muscle.

CtIP (CtBP-interacting protein) is emerging as a key new player in the maintenance of genome stability. CtIP was discovered as a cofactor of the ubiquitous transcriptional corepressor CtBP.1 by virtue of its association with CtBP, it is presumed to function in transcriptional regulation. CtIP also interacts with the pRb family proteins and BRCA1 and has been linked to the transcriptional repression activities mediated by these tumor suppressors. While interaction of CtIP with pRb appears to be important for G1/S regulation,2 CtIP activity is also required for G2/M DNA damage checkpoint via transient association with BRCA1 and Chk1 activation during treatment with ionizing radiation.3 Recently, orthologs of CtIP that participate in DNA double strand repair have been identified in yeast, fungi, worm and plants.4–7 Specifically, CtIP and its orthologs have been shown to be critical for dsDNA break repair by homologous recombination. These orthologs share a distinct region of homology with the C-terminal region of CtIP. CtIP and its orthologs have been shown to associate with the Mre11/Rad50/NSB1 (MRN) complex and mediate DNA end resection to generate ssDNA intermediates for recombination. While CtIP orthologs appear to function primarily in DNA repair, the vertebrate CtIP appear to be a multifunctional modular adapter protein recruiting multiple protein complexes to mediate concerted cell cycle regulation and DNA damage checkpoint control and DNA repair. In the current issue of Cell Cycle, Gu and Chen8 report that CtIP is targeted to DNA replication foci by direct interaction with PCNA. The interaction between PCNA and CtIP is mediated by a 42-amino acid region of CtIP known as Replication Foci Targeting Sequence (RFTS) that contains a consensus PCNA-interacting motif.9 Endogenous CtIP and that expressed by transient transfection colocalized with PCNA in replication foci and mutations in the RFTS abolished such colocalization. Interfering with the interaction between CtIP and PCNA via overexpression of an RFTS-GFP chimeric construct (dominant negative) induced cell cycle arrest at S/G2 transition and cessation of cell proliferation. Cells expressing the chimeric RFTS construct induced DNA damage and activation of DNA damage checkpoint while a mutant RFTS construct defective in interaction with PCNA did not. These results suggest that the CtIP-PCNA complex might play a role in stabilization of the stalled replication forks. Since CtIP plays a role in DNA repair, it is possible that the normal function of the PCNA-CtIP complex might be involved in surveillance of the replication forks for potential stalling. Considering the established role of CtIP in DNA repair and the present results that PCNA directs CtIP to the replication foci, new investigations on the role CtIP in repair of DNA damage at the replication fork should be forthcoming.

We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival.

The human Bfl-1 gene codes for a 175-amino acid BCL-2 family protein that has an anti-apoptosis activity and is overexpressed in certain human epithelial and hematopoietic malignancies. Bfl-1 efficiently suppresses apoptosis induced by the p53 tumor suppressor protein and cooperates with a dominant nuclear oncogene, E1A, in transformation of primary epithelial cells in vitro. Unlike other BCL-2 family proteins, expression of BFL-1 permits limited cell proliferation over an extended period of time when cells are induced to undergo apoptosis. We have carried out mutational analysis to dissect the various activities encoded by Bfl-1 and to determine the sequence requirements for these activities. BFL-1 shares four conserved domains, BH1, BH2, BH3 and BH4 with other BCL-2 family proteins. Mutations within BH1, BH2 and BH4 domains abolish or greatly attenuate the anti-apoptotic, oncogene cooperation and proliferation facilitating activities of BFL-1. In contrast, a mutation within the BH3 domain (which is essential for the activity of pro-apoptotic members of the BCL-2 family) does not significantly affect the BFL-1 functions. Although BFL-1 does not contain a well-defined C-terminal transmembrane domain, deletion of the C-terminal 24 amino acid region (corresponding to the transmembrane domain of other BCL-2 family proteins) partially reduces the various activities of BFL-1. All the mutants defective in the anti-apoptosis activity are also defective in the oncogene cooperation activity suggesting that these two activities may be linked. A unique feature of BFL-1 is the presence of a Gln-rich N-terminal region that overlaps with the BH4 domain. The Gln residues appear to be essential for the proliferation permitting activity of BFL-1. Since mutations of the Gln residues located within the BH4 domain appear to confer an extended cell survival activity in the absence of cell proliferation, our results suggest that BFL-1 communicates with both cell proliferation and apoptosis machineries and suggest a link between these two activities.

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.

Adenovirus infection induces apoptosis by multiple paradigms that involve viral proteins coded by three different early gene regions, E1A, E3, and E4. These cell death programs are antagonized by a different set of viral proteins coded by early gene blocks E1B, E3, and E4. The E1A proteins activate the cellular transcription factor E2F and also increase accumulation of the p53 tumor suppressor protein, both proteins with known apoptotic activity. The E1B-19K protein, a distantly related member of the BCL-2 family of antiapoptosis proteins, efficiently suppresses both p53-dependent and -independent apoptosis induced during adenovirus infection. Two other proteins, E1B-55K and E4-36K, which inactivate p53 by physical protein complex formation suppress p53-dependent apoptosis. The mechanisms by which the E1B-19K protein acts are not fully known, but at least one appears to involve antagonizing the activity of various cellular pro-apoptotic proteins such as BAX, BAK, and BIK. One of the two major E1A proteins, 289R, also appears to induce cell death via transactivation of the cellular transcription factor NF-κB as well as two early gene blocks, E3 and E4. NF-κB is believed to induce expression of TNF and various inflammatory cytokines in virus-infected cells. The E3 and E4 gene blocks each code for a cell death protein, E3-11.6K (ADP) and E4-orf4. The E1A proteins also sensitize infected cells for TNF-induced apoptosis and this activity is linked to the cell cycle regulatory activities of E1A. In addition to the E1B-19K protein, three E3 proteins, 14.7K, 10.4K, and 14.5K, specifically protect cells against TNF-induced cell death. One of the mechanisms by which the E3-14.7K and E3-10.4K/14.5K complex suppress TNF-induced toxicity appears to be mediated by the release of arachidonic acid by the cytosolic phospholipase A2. The E3 proteins also inhibit Fas-agonist-induced apoptosis by endosome-mediated internalization and degradation of Fas from the cell surface. Suppression of E1A-induced apoptosis in nonpermissive cells by E1B-19K, E1B-55K, and E4-36K proteins leads to oncogenic transformation.

Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9, 10-dimethyl-1, 2-benzanthracene (DMBA). These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55K (which are defective in the expression of either E1B-19K alone or both E1B-19K and E1B-55K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intra-tumoral administration of HAdV5 E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers.

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.

The adenovirus large E1A (L-E1A) protein is a prototypical transcriptional activator, and it functions through the action of a conserved transcriptional activation domain, CR3. CR3 interacts with a mediator subunit, MED23, that has been linked to the transcriptional activity of CR3. Our unbiased proteomic analysis revealed that human adenovirus 5 (HAdv5) L-E1A was associated with many mediator subunits. In MED23-depleted cells and in Med23 knockout (KO) cells, L-E1A was deficient in association with other mediator subunits, suggesting that MED23 links CR3 with the mediator complex. Short interfering RNA (siRNA)-mediated depletion of several mediator subunits suggested differential effects of various subunits on transcriptional activation of HAdv5 early genes. In addition to MED23, mediator subunits such as MED14 and MED26 were also essential for the transcription of HAdv5 early genes. The L-E1A proteome contained MED26-associated super elongation complex. The catalytic component of the elongation complex, CDK9, was important for the transcriptional activity of L-E1A and HAdv5 replication. Our results suggest that L-E1A-mediated transcriptional activation involves a transcriptional elongation step, like HIV Tat, and constitutes a therapeutic target for inhibition of HAdv replication.

The early transcribed adenovirus proteins E1A and E1B display a variety of functions in the transformation of primary rodent cells and the regulation of apoptosis and transcription. We have recently shown recently that the E1B 19 kDa protein from Adenovirus 5 (Ad 5) can functionally antagonize the stimulatory effect of E1A 13S on the human transcription factor NF-kappaB. Here we show that expression of E1B 19 kDa negatively interfered with the activation of NF-kappaB by different stimuli, such as the E1A 13S protein, and treatment with phorbol ester and tumor necrosis factor alpha. This suggests that E1B 19 kDa acts on a common upstream signaling event. Band shift experiments showed that expression of E1B 19 kDa impaired the generation of the nuclear, DNA-binding form of NF-kappaB. Domain mapping experiments employing various E1B 19 kDa mutants revealed the necessity of a hydrophobic Bcl-2 homology region between amino acids 90 and 96 for NF-kappaB inhibition. Co-transfection experiments showed that the inhibitory effect of E1B 19 kDa on E1A 13S-activated NF-kappaB transcription was gradually lost in the course of time. Thus the continuous stimulatory action of E1A 13S can finally override the antagonistic effects of E1B 19 kDa on NF-kappaB activity. In contrast to E1B 19 kDa, expression of the E1B 55 kDa protein did not result in a de novo activation of NF-kappaB, but co-stimulated the transcriptional potential of activated NF-kappaB.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.

The BHRF1 open reading frame of Epstein-Barr virus (EBV) codes for a 191-amino-acid protein that is expressed at high levels in productively infected cells and in certain latently infected cells. The BHRF1-coding sequences are conserved among all EBV isolates examined. BHRF1 shares a distant colinear amino acid sequence homology with the protein coded by the Bcl-2 protooncogene which suppresses apoptotic cell death induced by multiple stimuli. We have established Chinese hamster ovary cell lines which constitutively express the BHRF1 protein and show that this viral protein can protect against apoptosis induced by the DNA-damaging agents cisplatin, etoposide, and mitomycin C. The BHRF1 protein also strongly suppresses DNA fragmentation induced by an adenovirus E1B 19K deletion mutant. These results suggest that the BHRF1 protein is functionally similar to Bcl-2 and adenovirus E1B 19-kDa proteins. Since BHRF1 efficiently suppresses apoptosis induced by anti-cancer agents, its expression may have important implications for the chemotherapy of EBV-induced malignancies.

Review on RBBP8 (retinoblastoma binding protein 8), with data on DNA, on the protein encoded, and where the gene is implicated.