Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased proinflammatory cytokine response of human alveolar macrophages and more severe inflammatory lung disease in infected hamsters. This is the first evidence of a clinical relevance of differential expression of the small Ad E1B gene product. The results suggest that there is a low, critical threshold of E1B 19/20K expression that is needed for viral replication and infection transmission but that a higher level of E1B 19/20K expression is required for the usual repression and control of the Ad-triggered host innate immune response.

Experimental studies on treatment of pancreatic cancer with double-regulated duplicative adenovirus AdTPHre-hEndo carrying human endostatin gene.

BACKGROUND: Gene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. METHODS: A gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. RESULTS: Double-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. CONCLUSIONS: AdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.

Expression of anti-apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture.

In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a "high" glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in "high" glucose medium reached 690 mg/L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dual-regulated oncolytic adenovirus.

PURPOSE: A dual-regulated adenovirus variant CNHK500, in which human telomerase reverse transcriptase promoter drove the adenovirus 5 (Ad5) E1a gene and hypoxia-response promoter controlled the E1b gene, was engineered. This virus has broad anticancer spectrum and higher specificity compared with mono-regulated adenovirus CNHK300. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of human telomerase reverse transcriptase and hypoxia response promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subsequently, hepatocellular carcinoma xenografts were established to evaluate CNHK500 antitumor efficacy in vivo by different routes of virus administration and different dosages. RESULTS: Human telomerase reverse transcriptase and hypoxia response promoters were activated in a tumor-selective manner or under hypoxia treatment in a broad panel of cells. Selective adenoviral early gene expression, efficient viral replication, and oncolysis were observed in all tested cancer cells with more attenuated replication capacity in normal cells. Significant regression of hepatocellular carcinoma xenografts and prolonged survival were observed by either i.t. or i.v. administration. CONCLUSIONS: CNHK500 greatly reduced side effects in normal cells via dual control of adenoviral essential genes while still preserving potent antitumor efficacy on broad-spectrum cancer cells in vitro and in vivo. It can be used as a powerful therapeutic agent not only for liver cancers but also for other solid tumors.

Matching complementing functions of transformed cells with stable expression of selected viral genes for production of E1-deleted adenovirus vectors.

Production of E1-deleted adenovirus (rAd) vectors requires complementation by E1A and E1B functions provided by the production cell line. The two cell lines most commonly used for production of rAd vectors, 293 and Per.C6, were derived from human primary cells and contain contiguous E1A and E1B sequences from the Ad genome. As an alternative system, we tested complementation of rAd vectors using sequential transfection of individual E1A and E1B expression cassettes into A549 human lung tumor cells, which support highly efficient replication of wild type adenovirus. We found that E1A function could be complemented in A549 cells by the mutant E1Adl01/07, and that E1B function could be provided in such cells using only the 55K E1B gene. Production yields in the resulting producer cell line, designated SL0003, were similar to those obtained from 293 cells without generation of detectable recombinant replication competent adenovirus.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

[Analysis of transient G1/S arrest in E1A+E1B-19kDa transformed cells after ionizing radiation]. / Analiz mekhanizmov realizatsii kratkovremennogo bloka G1/S v transformantakh E1A+E1B-19kDa posle oblucheniia.

Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.

A novel targeting modality to enhance adenoviral replication by vitamin D(3) in androgen-independent human prostate cancer cells and tumors.

We report the development of a novel replication-competent adenoviral vector, Ad-hOC-E1, containing a single bidirectional human osteocalcin (hOC) promoter to drive both the early viral E1A and E1B gene. This vector selectively replicated in OC-expressing but not non-OC-expressing cells, with viral replication enhanced at least 10-fold on vitamin D(3) exposure. Both the artificial TATA-box and hOC promoter element in this bidirectional promoter construct were controlled by a common OC regulatory element which selectively activated OC expression in cells. The expression ofE1A and E1B gene by Ad-hOC-E1 can be markedly induced by vitamin D(3). Unlike Ad-sPSA-E1, an adenoviral vector with viral replication controlled by a strong super prostate-specific antigen (sPSA) promoter which only replicates in PSA-expressing cells with androgen receptor (AR), Ad-hOC-E1 retarded the growth of both androgen-dependent and androgen-independent prostate cancer cells irrespective of their basal level of AR and PSA expression. A single i.v. administration of 2 x 10(9) plaque-forming units of Ad-hOC-E1 inhibited the growth of previously established s.c. DU145 tumors (an AR- and PSA-negative cell line). Viral replication is highly enhanced by i.p. administration of vitamin D(3). Ultimately, enhancing Ad-hOC-E1 viral replication by vitamin D(3) may be used clinically to treat localized and osseous metastatic prostate cancer in men.

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.

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2.

The E1B-55K and E4orf6 oncoproteins of adenovirus type 5 are involved in the export of viral mRNAs. Previously, it was suggested that a complex composed of E1B-55K and E4orf6 serves as a nucleocytoplasmic transporter for viral mRNAs in which the E4orf6 protein directs both nuclear import and export. We now demonstrate that the E1B-55K protein itself shuttles efficiently in the absence of E4orf6. In addition, E1B-55K trafficking was independent of the defined shuttle proteins Mdm2 or p53, which interacts with E1B-55K. The identified N-terminal E1B-55K leucine-rich nuclear-export signal (NES) conferred rapid nuclear export even in a heterologous system in contrast to the postulated E4orf6NES. Interestingly, although shuttling was blocked by inhibitors of the CRM1 mediated export pathway, E1B-55K inhibited neither the activity nor the trafficking of the retroviral shuttle proteins HIV-1 Rev and HTLV-1 Rex. In contrast, Rev or Rex blocked the nuclear export of E1B-55K, most likely by competing for essential export factors. Our results provide new insights into the regulation of the adenovirus mRNA export system and the processes of adenovirus mediated transformation. Oncogene (2000) 19, 850 - 857.

Characterization of early region 1 and pIX of bovine adenovirus-3.

The mRNAs from early region 1 (E1) and pIX of bovine adenovirus type 3 (BAV-3) have been studied by Northern blot, S1 nuclease, and cDNA analysis and transcriptional maps for the regions were constructed. The transcriptional map for the E1 region of BAV-3 is different from those of mouse and human adenoviruses for which transcriptional maps for the regions have been constructed. The E1A region of BAV-3 is located between 0.8 and 10.5 map units and several different transcripts are produced from the region using alternative splice donor sites. The transcripts from the E1A region overlap with those of E1B and pIX. In BAV-3, the E1B region maps between 4.2 and 10.5 map units and encodes two major mRNA species. The mRNAs of E1B region differ from each other in that the smaller mRNA coding for the 157R protein has a large intron removed from a region corresponding to the coding region of E1B 420R protein. As in HAVs, the E1B 420R protein of BAV-3 could be translated only by internal initiation from the larger bicistronic mRNA as there are no transcripts produced exclusively for the production of 420R protein. The transcriptional unit of pIX is transcribed from an independent promoter and encodes a structural component of the adenovirus capsid. To identify and characterize the proteins produced from the region, antibodies were raised in rabbits that recognized specific proteins in Western blot and immunoprecipitation assays.

Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death.

Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, is an effective inhibitor of apoptotic cell death induced by many cytotoxic agents. Survival-promoting homologues of Bcl-2 include its close relative, Bcl-xL and the 19 kD protein encoded by the E1B gene of adenoviruses. Whether these proteins are functionally equivalent and whether they can antagonise all or only some pathways to apoptosis is unresolved. We have carried out a systematic comparison of Bcl-2, Bcl-xL and adenovirus E1B19kD activity, using several cell lines and a range of cytotoxic conditions. High levels of expression of each of these proteins inhibited apoptosis induced by growth factor deprivation or treatment with gamma-radiation, glucocorticoid and various cytotoxic drugs. In contrast, none of them could effectively counter apoptosis induced via the TNF receptor or Fas/APO-1 (CD95). Biochemical analysis revealed that all three proteins can associate with Bax and Bak, members of the Bcl-2 protein subfamily that can facilitate apoptosis. The results provide evidence that Bcl-2, Bcl-xL and adenovirus protein E1B19kD are indistinguishable in their ability to regulate the cell death effector machinery.

Adenovirus E1B 19K protein is required for efficient DNA replication in U937 cells.

The adenovirus E1B 19K gene plays an essential role in transformation of primary rodent cells in cooperation with E1A and in the inhibition of apoptosis during lytic infection. It has been shown that this E1B 19K protein is not necessary for viral DNA replication in human cell lines, such as HeLa and KB. We reported here that the E1B 19K mutant viruses were unable to replicate efficiently in a monocyte cell line, U937. Viral DNA synthesis and late gene expression were found to be defective in U937 cells infected with E1B 19K mutants compared with wild-type virus. Early viral RNA splicing patterns also differ between wild-type and dl337-infected cells. Furthermore, the defect in viral replication could be complemented by dl312 virus defective in E1A expression 4 days after infection with E1B mutants, suggesting persistence of the E1B mutant genome in the infected cells despite defective onset of the late phase of replication. These results imply that E1B 19K is required for efficient viral DNA replication in U937 cells. Inefficient DNA replication is also found in another monocyte cell line, THP-1.

Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity.

Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses.

Oncogenic potential of the adenovirus E4orf6 protein.

The group C adenovirus E4orf6 protein has previously been shown to bind to the p53 cellular tumor suppressor protein and block its ability to activate transcription. Here we show that the E4orf6 protein blocks the induction of p53-mediated apoptosis when AT6 cells, which harbor a temperature-sensitive p53, are shifted to the permissive temperature. The E4orf6 protein does not, however, prevent the induction of apoptosis in p53-deficient H1299 cells by treatment with tumor necrosis factor alpha and cycloheximide. The E4orf6 protein also cooperates with the adenovirus E1A protein to transform primary baby rat kidney cells, and it cooperates with the adenovirus E1A plus E1B 19-kDa and E1B 55-kDa proteins to increase the number of baby rat kidney cell transformants and enhance the rate at which they arise. The level of p53 is substantially reduced in transformed cells expressing the E4orf6 protein in comparison to adenovirus transformants lacking it. The E4orf6 gene also accelerates tumor formation when transformed baby rat kidney cells are injected subcutaneously into the nude mouse, and it converts human 293 cells from nontumorigenic to tumorigenic in nude mice. In addition to the well-studied E1A and E1B oncogenes, group C adenoviruses harbor a third oncogene, E4orf6, which functions in some respects similarly to the E1B oncogene.

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.

Parathyroid hormone-related protein gene expression in human squamous carcinoma cells is repressed by mutant isoforms of p53.

Parathyroid hormone-related protein (PTHrP) is a normal secretory product of a variety of squamous epithelia, including epidermal keratinocytes. Only a subset of squamous carcinomas, however, express the gene at levels sufficient to cause humoral hypercalcemia. In the present study, comparison of PTHrP expression levels with p53 functional status in a series of squamous carcinoma lines has revealed an association between expression of specific mutant isoforms of p53 and very low levels of PTHrP mRNA. Evaluation of p53 isoforms with mutations in codons 248 and 273 showed them to be capable of repressing PTHrP gene expression in a high-expressing, p53-negative squamous line by approximately 50%. Conversely, inactivation of an endogenous mutant p53 with E1B proteins resulted in an increase in PTHrP expression in a low-expressing cell line. Subsequent analysis of promoter-specific PTHrP transcripts in a p53-negative squamous line transfected with mutant p53 isoforms suggested that down-regulation occurred primarily at the two TATA-based promoters. Direct testing of a murine PTHrP reporter construct in transient transfection assays confirmed the capacity of the 248 and 273 mutants to repress this TATA-based promoter, although only about half as effectively as wild-type p53.

The high levels of p53 present in adenovirus early region 1-transformed human cells do not cause up-regulation of MDM2 expression.

The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.

The quaternary structure of the adenovirus 12 early region 1B 54K protein.

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.