Oncolytic adenovirus-induced autophagy: tumor-suppressive effect and molecular basis.

Autophagy is a catabolic process that produces energy through lysosomal degradation of intracellular organelles. Autophagy functions as a cytoprotective factor under physiological conditions such as nutrient deprivation, hypoxia, and interruption of growth factors. On the other hand, infection with pathogenic viruses and bacteria also induces autophagy in infected cells. Oncolytic virotherapy with replication-competent viruses is thus a promising strategy to induce tumor-specific cell death. Oncolytic adenoviruses induce autophagy and subsequently contribute to cell death rather than cell survival in tumor cells. We previously developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301, which induces cell lysis in tumor cells with telomerase activities. OBP-301-mediated cytopathic activity is significantly associated with induction of autophagy biomarkers. In this review, we focus on the tumor-suppressive role and molecular basis of autophagic machinery induced by oncolytic adenoviruses. Addition of tumor-specific promoters and modification of the fiber knob of adenoviruses supports the oncolytic adenovirus-mediated autophagic cell death. Autophagy is cooperatively regulated by the E1-dependent activation pathway, E4-dependent inhibitory pathway, and microRNA-dependent fine-tuning. Thus, future exploration of the functional role and molecular mechanisms underlying oncolytic adenovirus-induced autophagy would provide novel insights and improve the therapeutic potential of oncolytic adenoviruses.

Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor.

One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.

A 49-kilodalton isoform of the adenovirus type 5 early region 1B 55-kilodalton protein is sufficient to support virus replication.

The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.

The adenovirus E1B 55-kilodalton and E4 open reading frame 6 proteins limit phosphorylation of eIF2alpha during the late phase of infection.

During a productive infection, species C adenovirus reprograms the host cell to promote viral translation at the expense of cellular translation. The E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins are important in this control of gene expression. As part of a ubiquitin-protein ligase, these viral proteins stimulate viral mRNA export, inhibit cellular mRNA export, promote viral gene expression, and direct the degradation of certain host proteins. We report here that the E1B-55K and E4orf6 proteins limited phosphorylation of eIF2alpha and the activation of the eIF2alpha kinase PKR. Phospho-eIF2alpha levels were observed to rise and fall at least twice during infection. The E1B-55K and E4orf6 proteins prevented a third increase at late times of infection. PKR appeared to phosphorylate eIF2alpha only in the absence of E1B-55K/E4orf6 function. PKR activation and eIF2alpha phosphorylation was unrelated to the cytoplasmic levels of the adenovirus inhibitor of PKR, VA-I RNA. Nonetheless, expression of a PKR inhibitor, the reovirus double-stranded RNA-binding protein sigma 3, prevented PKR activation and eIF2alpha phosphorylation. The sigma 3 protein largely corrected the defect in viral late protein synthesis associated with the E1B-55K and E4orf6 mutant viruses without affecting cytoplasmic levels of the late viral mRNA. The ubiquitin-protein ligase activity associated with the E1B-55K/E4orf6 complex was necessary to prevent activation of PKR and phosphorylation of eIF2alpha. These findings reveal a new contribution of the E1B-55K/E4orf6 complex to viral late protein synthesis and the existence of multiple layers of regulation imposed on eIF2alpha phosphorylation and PKR activation in adenovirus-infected cells.

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication.

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

RUNX1 permits E4orf6-directed nuclear localization of the adenovirus E1B-55K protein and associates with centers of viral DNA and RNA synthesis.

The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-DeltaN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1.

Lamin proteolysis facilitates nuclear events during apoptosis.

Expression of the adenovirus E1A oncogene stimulates both cell proliferation and p53-dependent apoptosis in rodent cells. p53 implements apoptosis in all or in part through transcriptional activation of bax, the product of which promotes cell death. The adenovirus E1B 19K product is homologous in sequence and in function to Bcl-2, both of which bind to and inhibit the activity of Bax and thereby suppress apoptosis. The E1B 19K protein also interacts with the nuclear lamins, but the role of this interaction in the regulation of apoptosis is not known. Lamins are, however, substrates for members of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases that are activated during apoptosis and function downstream of Bcl-2 in the cell death pathway. lamins are degraded during E1A-induced p53-dependent apoptosis. Lamin A and C are cleaved into 47- and 37-kD fragments, respectively, and the site of proteolysis is mapped to a conserved aspartic acid residue at position 230. The cleavage of lamins during apoptosis is consistent with the activation of an ICE-related cysteine protease down-stream of p53. No lamin protease activity was detected in cells expressing the E1B 19K protein, indicating that 19K functions upstream of protease activation in inhibiting apoptosis. Substitution of the aspartic acid at the cleavage site produced a mutant lamin protein that was resistant to proteolysis both in vitro and in vivo. Expression of uncleavable mutant lamin A or B attenuated apoptosis, delaying cell death and the associated DNA fragmentation by 12 h. Mutant lamin expressing cells failed to show the signs of chromatin condensation and nuclear shrinkage typical of cell death by apoptosis. Instead, the nuclear envelope collapsed and the nuclear lamina remained intact. However, the late stage of apoptosis was morphologically unaltered and formation of apoptotic bodies was evident. Thus, lamin breakdown by proteolytic degradation facilitates the nuclear events of apoptosis perhaps by facilitating nuclear breakdown.

Adenoviral E1B55K oncoprotein sequesters candidate leukemia suppressor sequence-specific single-stranded DNA-binding protein 2 into aggresomes.

Sequence-specific single-stranded DNA-binding protein 2 (SSBP2) is a candidate tumor suppressor for human acute myelogenous leukemia (AML). Inducible expression of SSBP2 causes growth arrest and partial differentiation in AML cells. Here, we report that the adenoviral oncoprotein E1B55K directly binds to endogenous SSBP2 protein and sequesters it into juxtanuclear bodies in adenovirally transformed human embryonic kidney (HEK) 293 cells. Similarly, transient expression of E1B55K in IMR90 fibroblasts and HeLa cells result in the formation of juxtanuclear bodies containing SSBP2. When nuclear export of E1B55K is prevented, SSBP2 remains associated with E1B55K in nuclear foci. A requirement for intact microtubules to retain the integrity of the juxtanuclear bodies suggests them to be E1B55K containing aggresomes. The adenoviral E1B55K protein has been shown to localize to the Mre11 complex and p53 to aggresome structures; together with the viral E4orf6 protein, E1B55K recruits a cellular E3 ubiquitin ligase that induces degradation of Mre11 and p53. However, our present studies reveal that E1B55K does not degrade SSBP2. These data demonstrate that E1B55K targets the candidate leukemia suppressor SSBP2 and suggest that subverting its function may contribute to cell transformation by viral oncoproteins.

The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication.

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.

Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic.

Adenoviruses encode proteins that block responses to interferons, intrinsic cellular apoptosis, killing by CD8(+) cytotoxic T lymphocytes and killing by the death ligands TNF, Fas ligand and TRAIL. The viral proteins are believed to prolong acute and persistent adenovirus infections. The proteins may prove useful in protecting adenovirus gene therapy vectors and transplanted cells from the immune system.

Control of programmed cell death by the baculovirus genes p35 and iap.

The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells.

The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB.

Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus.

Adenovirus continues to be an important model system for investigating basic aspects of cell biology. Interactions of several cellular proteins with E1A conserved regions (CR) 1 and 2, and inhibition of apoptosis by E1B proteins are required for oncogenic transformation. CR2 binds RB family members, de-repressing E2F transcription factors, thus activating genes required for cell cycling. E1B-19K is a BCL2 homolog that binds and inactivates proapoptotic BAK and BAX. E1B-55K binds p53, inhibiting its transcriptional activation function. In productively infected cells, E1B-55K and E4orf6 assemble a ubiquitin ligase with cellular proteins Elongins B and C, Cullin 5 and RBX1 that polyubiquitinates p53 and one or more subunits of the MRN complex involved in DNA double-strand break repair, directing them to proteosomal degradation. E1A CR3 activates viral transcription by interacting with the MED23 Mediator subunit, stimulating preinitiation complex assembly on early viral promoters and probably also the rate at which they initiate transcription. The viral E1B-55K/E4orf6 ubiquitin ligase is also required for efficient viral late protein synthesis in many cell types, but the mechanism is not understood. E1A CR1 binds several chromatin-modifying complexes, but how this contributes to stimulation of cellular DNA synthesis and transformation is not clear. E1A CR4 binds the CtBP corepressor, but the mechanism by which this modulates the frequency of transformation remains to be determined. Clearly, adenovirus has much left to teach us about fundamental cellular processes.

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.

p53 selective and nonselective replication of an E1B-deleted adenovirus in hepatocellular carcinoma.

An E1B gene-attenuated adenovirus (dl1520) has been proposed to have a selective cytolytic activity in cancer cells with a mutation or deletion in the p53 tumor suppressor gene (p53-null), a defect present in almost half of human hepatocellular carcinomas (HCCs). In this study, the in vitro and in vivo antitumor activity of dl1520 was investigated focusing on two human HCC cell lines, a p53-wild type (p53-wt) cell line and a p53-null cell line. dl1520 was tested for in vitro cytopathic effects and viral replication in the human HCC cell lines Hep3B (p53-null) and HepG2 (p53-wt). The in vivo antitumor effects of dl1520 were investigated in tumors grown s.c. in a severe combined immunodeficient mouse model. In addition, the combination of dl1520 infection with systemic chemotherapy was assessed in these tumor xenografts. At low multiplicities of infection, dl1520 had an apparent p53-dependent in vitro viral growth in HCC cell lines. At higher multiplicities of infection, dl1520 viral replication was independent of the p53 status of the target cells. In vivo, dl1520 significantly retarded the growth of the p53-null Hep3B xenografts, an effect augmented by the addition of cisplatin. However, complete tumor regressions were rare, and most tumors eventually grew progressively. dl1520 had no effect on the in vivo growth of the p53-wt HepG2 cells, with or without cisplatin treatment. The E1B-deleted adenoviral vector dl1520 has an apparent p53-dependent effect in HCC cell lines. However, this effect is lost at higher viral doses and only induces partial tumor regressions without tumor cures in a human HCC xenograft model.

Efficient replication of adenovirus despite the overexpression of active and nondegradable p53.

The adenoviral oncoproteins E1B-55 kDa and E4orf6 inactivate and destabilize the tumor suppressor protein p53, thereby contributing to malignant transformation. However, it is unclear whether the elimination of p53 also contributes to the efficiency of viral replication. Furthermore, it is controversial whether adenoviruses with a deletion in the E1B-55 kDa-coding region might selectively replicate in cells with a mutation or deletion of the p53 gene and, therefore, represent a tool in cancer therapy. To address the role of p53 in virus replication, amino acid substitutions were introduced into the NH(2)-terminal portion of p53, replacing residues 24-28 with the corresponding sequence of the human p53-homologue p73. This replacement leaves p53 transcriptionally active but renders the modified protein, termed p53mt24-28, completely resistant to inhibition and degradation by adenoviral oncoproteins. Surprisingly, even strong overexpression of p53 or p53mt24-28 allowed the virus to replicate as efficiently as in the absence of p53 proteins, both in tumor cells and in primary endothelial cells. Also, p53 or p53mt24-28 did not reduce the amount of virus released from infected cells. These observations were made in primary cells or in cell lines that were capable of expressing the p53-agonist p14ARF. Thus, active p53 does not inhibit the growth of adenovirus. Alternative strategies should be used to improve the utility of adenoviruses in cancer therapy.

The role of the E1B 55 kDa gene product in oncolytic adenoviral vectors expressing herpes simplex virus-tk: assessment of antitumor efficacy and toxicity.

In this study, we evaluated three herpes simplex virus-1 thymidine kinase (HSV-tk) carrying replication-competent adenoviral vectors with and without the Ad5 E1B 55 kDa gene to assess whether this gene product has an influence on their antitumor efficacy, replication kinetics, and potential hepatotoxicity. Furthermore, we assessed the efficacy of these vectors in combination with ganciclovir (GCV). When compared with wild-type adenovirus, the recombinant vectors, in particular the E1B 55 kDa-deleted vector Ad.TK(RC)(II), generated a more efficiently cytopathic effect in proliferating cells, independently of their p53 phenotype. In a s.c. A549 lung cancer xenograft model, the cytoreductive effect of Ad.TK(RC)(II) was enhanced when followed by GCV treatment. In contrast, the efficacy of both E1B 55 kDa-positive vectors could not be further improved by GCV. In an i.p. MDAH 2774 ovarian cancer xenograft tumor model, the survival of animals treated with a prototypical replication-deficient adenovirus expressing HSV-tk (Ad.TK) was improved compared to controls when followed by GCV. In contrast, the cytoreductive efficacy of the replication-competent vectors was diminished when combined with the virostatic GCV. However, the antitumor effect of all replication-competent vectors was superior to combination chemotherapy with paclitaxel and carboplatin. In both tumor models, the oncolytic effect of the E1B 55 kDa-positive vectors was greater than that of Ad.TK(RC)(II). In an attempt to assess the toxicity of these vectors in a nonpermissive host, the viruses were administered systemically to immunocompetent and immunodeficient mice. Greater hepatotoxicity was seen with i.v. administration of the replication-competent viruses than with Ad.TK and in immunocompetent hosts, suggesting involvement of the immune system in the induction of tissue damage. The E1B 55 kDa gene had no significant influence on the liver toxicity of the vectors in this system. At therapeutic doses, intratumoral or i.p. injection of all vectors was well tolerated. Importantly, these replication-competent HSV-tk-expressing vectors were highly susceptible to GCV, representing an effective fail-safe mechanism to abolish viral replication in a clinical setting. Controllable intratumoral viral replication holds promise as a new treatment modality for cancer.

bfl-1, a bcl-2 homologue, suppresses p53-induced apoptosis and exhibits potent cooperative transforming activity.

The bcl-2 family of genes code for proteins that contain anti-apoptotic or pro-apoptotic activity. The human bfl-1 gene contains an open reading frame for a 175-amino acid Bcl-2 family protein. Among the various Bcl-2 family members, the Bfl-1 protein shares the highest homology with the mouse A1 protein. These two proteins share three conserved domains, Bcl homology (BH)1, BH2, and BH3, with other Bcl-2 family proteins. Unlike other Bcl-2 family members, Bfl-1 contains a GIn-rich NH2-terminal region and lacks an NH (19K homology) domain 1. We demonstrate that the Bfl-1 protein suppresses apoptosis induced by the p53 tumor suppressor protein in a manner similar to other Bcl-2 family members such as Bcl-2, Bcl-xL and EBV-BHRF1. In addition, the bfl-I gene cooperates efficiently with the Ela oncogene in transformation of primary rodent epithelial cells. Our results suggest that the human bfl-1 gene may play an important role in carcinogenesis.

Sp100A is a tumor suppressor that activates p53-dependent transcription and counteracts E1A/E1B-55K-mediated transformation.

Human adenoviruses (HAdV) are used as a model system to investigate tumorigenic processes in mammalian cells where the viral oncoproteins E1A and E1B-55K are absolutely required for oncogenic transformation, because they simultaneously accelerate cell cycle progression and inhibit tumor suppressor proteins such as p53, although the underlying mechanism is still not understood in detail. In our present study, we provide evidence that E1B-55K binding to the PML-NB component Sp100A apparently has an essential role in regulating adenovirus-mediated transformation processes. Specifically, when this E1B-55K/Sp100A complex recruits p53, Sp100A-induced activation of p53 transcriptional activity is effectively abolished. Hence, Sp100A exhibits tumor-suppressive activity, not only by stabilizing p53 transactivation but also by depressing E1A/E1B-55K-mediated transformation. E1B-55K counteracts this suppressive activity, inducing Sp100A SUMOylation and sequestering the modified cellular factor into the insoluble matrix of the nucleus or into cytoplasmic inclusions. These observations provide novel insights into how E1B-55K modulates cellular determinants to maintain growth-promoting activity during oncogenic processes and lytic infection.

Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells.

The adenovirus early region 1B (Ad E1B) genes have no transforming capability of their own but markedly increase the transformation frequency of Ad E1A following co-transfection into mammalian cells. The larger E1B proteins of both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptionally activate other genes. We have previously demonstrated that synthetic peptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro. In the work presented here we have examined the effects of complex dissociation on Ad E1-transformed human cells. It has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-transformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocated to the nucleus. In Ad12 E1-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized, 'flecked' distribution to one in which nuclear staining was homogeneous and diffuse. Peptides added to either Ad5 E1 or Ad12 E1 transformed cells resulted in the release of transcriptionally active p53. Interestingly, the level of p53 then fell presumably as a result of proteasomal action - this was probably a reflection of the short half-life of 'free' (i.e. dissociated) p53 compared to that of the bound protein. Free p53 did not cause apoptosis in target cells probably due to the presence of the smaller (19K) E1B proteins. However, addition of peptide leads to a significant reduction in cell growth rate. We have further demonstrated that a significant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger EIB protein during transformation is considered in view of these data.