Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.

p73-dependent apoptosis through death receptor: impairment by human cytomegalovirus infection.

The discovery of p73, a p53-related protein with various isotypes resulting from different promoter usage or splicing events, provided new insights into regulation of neurogenesis and tumorigenesis. Among p73 isoforms described thus far, TA-truncated molecules (DeltaN) appeared as key proteins according to their antagonistic activity against transcription factor activity of p53 family members. We previously showed that infection by human cytomegalovirus (HCMV) induced drug resistance and altered p53- and p73-dependent apoptosis of infected cells through accumulation of DeltaN-p73alpha. In accordance with the ability of p53 to induce apoptosis through death receptors, we asked whether p73 activation could compensate for p53 deficiency. We showed that p73 transcriptional activity sensitized cells to apoptosis through death receptors in a caspase-dependent pathway. Expression of the death-inducing signaling complex (DISC) proteins was unchanged, whereas p73 activation through either cisplatin treatment or ectopic overexpression induced up-regulation of Fas transcription and expression at cell surface. According to its ability to flood cells with DeltaN-p73alpha, HCMV inhibited p73-dependent Fas-mediated apoptosis, gaining an additional trick to favor its survival in the host cell. Owing to the involvement of p53- and p73-dependent death receptor signaling in development of the central nervous system, immune surveillance of neural cells, and sensitivity of tumors to drugs, our previous and present data prompt us to consider stabilization of DeltaN-p73alpha by HCMV as a possible mechanism in impairment of embryogenesis and in tumorigenesis.

P73 functionally replaces p53 in Adriamycin-treated, p53-deficient breast cancer cells.

p53-Related genes, p73 and p63, encode 2 classes of proteins, TA-p73/p63 and DeltaN-p73/p63. TA-p73/p63 demonstrate p53-like properties including gene transactivation and cell death promotion, whereas DeltaN-p73/p63 lack these p53-like functions. Although p53-deficient cancer cells are often less responsive to chemotherapy, they are not completely drug resistant, suggesting that other apoptotic pathways are at work. Here, we compared for the first time to our knowledge p73 and p63 activation in various breast cancer (BC) cell lines after Adriamycin (ADR) treatment, an agent considered as mandatory in breast cancer chemotherapy. Our study was carried out using 1 p53-proficient BC cell line (MCF7 cells) and 3 BC cell lines deficient in p53 response (MCF7/ADR(IGR), MDA-MB157 and T47D) after ADR-induced genotoxic stress. We report that in cells with no p53 response after ADR treatment, TAp73, but not TAp63 or DeltaN-p73/p63, may replace p53 in triggering not only apoptosis but also cell cycle arrest or DNA repair effectors such as p21, GADD45, 14-3-3sigma and p53R2. We also demonstrate that TAp73 siRNA inhibits the accumulation of TAp73 in response to ADR treatment in MDA-MB157 cells and confers protection against ADR. ADR-induced downregulation of the DeltaNp73 isoform in the T47D cell line with nonfunctional mutant p53 further supports anti-apoptotic function of the isoform antagonistic to both p53 and TA-p73/p63. Exogenous TAp73 and DeltaNp73 overexpression in p53-response-deficient cell lines further confirms these results. cDNA microarray techniques demonstrated that the cellular response induced by p73 during ADR treatment could involve specific genes.

The neurogene BTG2TIS21/PC3 is transactivated by DeltaNp73alpha via p53 specifically in neuroblastoma cells.

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of DeltaNp73alpha, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, DeltaNp73alpha inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the DeltaNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by DeltaNp73alpha depends on both p53 status (as it is not observed in a p53-/- neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that DeltaNp73alpha may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete DeltaNp73alpha C-terminus sequence as it is not observed with DeltaNp73beta, which lacks most of the C-terminal domain. We have previously shown that DeltaNp73alpha is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that DeltaNp73alpha not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line.

p73, the first p53 gene homologue, encodes an array of p73 proteins including p73 alpha full-length (TAp73 alpha) and amino-truncated isoforms (Delta Np73 alpha), two proteins with opposite biological functions. TAp73 alpha can induce tumor suppressive properties, while Delta Np73 alpha antagonizes p53 as well as TAp73 in a dominant-negative manner. In human malignant neuroblasts, p53 protein is wild-type but known to be excluded from the nucleus, therefore disabling its function as a tumor suppressor. The present study investigates whether there is a functional link between p73 isoforms and p53 in neuroblastoma. Experiments were performed on two neuroblastoma cell lines differing in their p53 status, e.g. wild-type p53 SH-5Y5Y cells and mutated p53 IGR-N-91 cells. Data indicate that (i) both TA- and Delta N-p73 alpha enhance p53 protein level in SH-SY5Y cells, whereas level remains unchanged in IGR-N-91 cells; (ii) only in SH-SY5Y cells does forced TAp73 alpha overexpression markedly induce nuclear accumulation of p53 protein; (iii) p21 protein expression is increased in both cell lines infected with TAp73, suggesting that, in IGR-N-91 cells, p21 is induced by p73 through a p53-independent pathway; (iv) in the SHSY5Y cell line, Btg2 expression is strongly enhanced in cells overexpressing TA, and to a lesser extent in cells overexpressing Delta N. Taken together our results suggest that TAp73 may restore p53 function in NB with wild-type nonfunctional p53, but not in NB with mutated p53.

p73alpha expression induces both accumulation and activation of wt-p53 independent of the p73alpha transcriptional activity.

The p53 tumor suppressor gene belongs to a multigene family that includes two paralogues, p63 and p73. p73alpha has common activities with p53, such as DNA binding and transactivation, and can thus activate the transcription of p53-responsive genes. Using the adenoviral system, we report that an overexpression of either wt-p73alpha or one of the two transcriptional inactive mutants, deltaNp73alpha or p73alphaR292H, induces an accumulation of the endogenous wt-p53 expressed in the three transformed cell lines, SK-N-SH, MCF-7 and U-2OS, without stimulating the p53 gene transcription. p73-mediated accumulation of p53 protein coincides with an increase of p53-target gene expression in cells expressing either wt-p73alpha or the transcriptional inactive mutant p73alphaR292H, but not deltaNp73alpha that encodes a dominant-negative mutant of both p73 and p53. The fact that an ectopic expression of p73alphaR292H leads to both accumulation of p53 and stimulation of p53 target gene expression strongly suggests that p73alpha is able to induce activation of p53. This was confirmed by showing that p73alphaR292H no longer stimulated Waf1/p21 expression in MCF7/R-A1 cells that expressed a transcriptional inactive mutant of p53. We thus conclude that p73alpha protein was able to both stabilize and activate wt-p53 protein, independent of the p73alpha transcriptional activity.

When p53 needs p73 to be functional - forced p73 expression induces nuclear accumulation of endogenous p53 protein.

In human neuroblastoma (NB), wild type p53 protein does not elicit its archetypal human tumor suppressive activity so far described. To elucidate this alteration, substantial investigations using NB cell lines have underscored p53 protein nuclear localization defect and/or inappropriate conformation, but no definitive evidence has been provided so far. p73, the first homologue of the p53 gene, locates at the 1p36.3 locus, which is known to be deleted in various human tumors including NB. Unlike p53 mRNA, which specifies a single protein, p73alpha mRNAs encode two types of isoform (TAp73alpha and DeltaNp73alpha) resulting from the use of two different promoters, and eliciting or lacking NH(2)-terminal transactivation domain, respectively. DeltaNp73alpha inhibits p53 pro-apoptotic function in murine developing neurons and is abundantly expressed in human undifferentiated NB tumors. However, critical issues have been raised regarding p73alpha isoform roles, and their possible link to p53 are yet to be clarified in human NB using adenoviral infection approach.

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function.

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.

Regression of AK7 malignant mesothelioma established in immunocompetent mice following intratumoral gene transfer of interferon gamma.

Malignant mesothelioma (MM) is a lethal tumor linked with a prior exposure to asbestos in which limited progress has been made so far using conventional therapies. MM is an example of a "nonimmunogenic" tumor characterized by a fibrous stroma and an absence of infiltrating T lymphocytes. High levels of transforming growth factor-beta (TGF-beta) produced by mesothelioma cells have been related to the immune tolerance towards the tumor. In order to evaluate the effect of local delivery of cytokines such as interferon gamma (IFN-gamma) by gene transfer, we characterized and used a murine model, AK7, which appeared very similar to human mesothelioma. AK7 cells expressed low levels of major histocompatibility class I and class II antigens and secreted high levels of latent TGF-beta. The TGF-beta pathway in AK7 cells is operative but inefficient because endogenous TGF-beta is predominantly inactive. Treatment of pre-established AK7 tumors by direct intratumoral injection of an adenovirus vector expressing murine IFN-gamma, Ad.mIFN-gamma, led to significant tumor regression. Peripheral tumor infiltration by CD4+ and CD8+ T lymphocytes in the treated tumors appeared to be because of the induction of an immune response. Tumor relapse was observed, which could be due to local TGF-beta secretion by remaining tumor cells.

Wild-type p53 induced sensitization of mutant p53 TNF-resistant cells: role of caspase-8 and mitochondria.

In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome c to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the cross-talk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.