Role of the p53-homologue p73 in E2F1-induced apoptosis.

Most human cancers harbour aberrations of cell-cycle control, which result in deregulated activity of the E2F transcription factors with concomitant enhanced cell-cycle progression. Oncogenic signalling by E2F1 has recently been linked to stabilization and activation of the tumour suppressor p53 (refs 1,3,4). The p73 protein shares substantial sequence homology and functional similarity with p53 (refs 5-7 ). Hence, several previously considered p53-independent cellular activities may be attributable to p73. Here we provide evidence that E2F1 directly activates transcription of TP73, leading to activation of p53-responsive target genes and apoptosis. Disruption of p73 function by a tumour-derived p53 mutant reduced E2F1-mediated apoptosis. Thus, p73 activation by deregulated E2F1 activity might constitute a p53-independent, anti-tumorigenic safeguard mechanism.

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes.

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.

Immune responses to RHAMM in patients with acute myeloid leukemia after chemotherapy and allogeneic stem cell transplantation.

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients.

Evaluation of systemic targeting of RET oncogene-based MTC with tumor-selective peptide-tagged Ad vectors in clinical mouse models.

Significant advantage of targeted antitumoral treatment consists in the possibility to restrict maximum therapeutic efficacy to the malignant cell population by reducing toxicity in healthy tissues. Using different clinical models for aggressive medullary thyroid carcinoma (MTC), we have recently identified peptide ligands that bind highly selective to tumor cells. By linking the most convincing SRESPHP peptide to an adenoviral (Ad) vector expressing the MTC-related oncogene inhibitor RETΔTK, gene transfer was specifically directed to neoplastic tissue after systemic virus administration. We show that peptide-mediated delivery of RETΔTK significantly enhanced apoptosis, resulting in a strong inhibition of orthotopic and xenograft tumor growth. Conversely, tumors treated with controls expanded their initial size without notable cell death. According to the therapeutic effect, strong virus accumulation was found exclusively in thyroid carcinomas. Strikingly, application of native tropism depleted viral vector linked to tumor-selective peptide was accompanied by a substantial reduction of Ad binding to the liver. Of note, single systemic injection of a low dose (10e8 pfu/mouse) of MTC-specific Ad.RETΔTK induced regression of multiple tumors at different sites in all treated animals. In sum, our results open up the possibility for an efficient cancer cell-specific therapy of primary MTC, their migrating populations and potentially metastases.

Transcriptome analysis in mouse tumors induced by Ret-MEN2/FMTC mutations reveals subtype-specific role in survival and interference with immune surveillance.

Activating mutations in the Ret proto-oncogene are responsible for occurrence of multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (FMTC). A striking genotype-phenotype correlation between the mutated RET codon and clinical manifestation implies that tumorigenesis is conditioned by the type of mutation. We investigated gene expression profiles between and within distinct MEN2 subtypes through whole-genome microarray analysis in tumors induced by NIH-3T3 cells transformed with defined RET-MEN2A (C609Y, C634R), MEN2B, (A883F, M918T), and FMTC (Y791F) mutations. Expression profiling identified a statistically significant modification of 1494 genes, 628 down- and 866 upregulated in MEN2B compared with MEN2A/FMTC tumors. By contrast, no obvious alterations were observed among individual MEN2B and MEN2A type mutations, or between MEN2A and FMTC. Functional clustering of differential genes revealed RET-MEN2B specific upregulation of genes associated with novel growth and survival pathways. Intriguingly, RET-MEN2A/FMTC-specific tumors were characterized by a considerable number of genes involved in the host antitumor immune response via stimulation of natural killer/T-cell proliferation, migration, and cytotoxicity, which were completely absent in RET-MEN2B related cancers. QPCR on tumors versus cultured NIH-RET cell lines demonstrated that they are largely attributed to the host innate immune system, whereas expression of CX3CL1 involved in leukocyte recruitment is exclusively RET-MEN2A/FMTC tumor cell dependent. In correlation, massive inflammatory infiltrates were apparent only in tumors carrying MEN type 2A/FMTC mutations, suggesting that RET-MEN2B receptors specifically counteract immune infiltration by preventing chemokine expression, which may contribute to the different clinical outcome of both subtypes.

Evaluation of potential mechanisms underlying genotype-phenotype correlations in multiple endocrine neoplasia type 2.

Distinct dominant activating mutations in the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2 (MEN 2). Concise examination of the mutated codons led to the detection of a striking genotype-phenotype correlation between the mutated codon and the MEN 2 phenotype in terms of onset and aggressiveness of the disease, suggesting that manifestation and clinical progression is conditioned by the type of mutation. To gain insight into the molecular basis for this genotype-phenotype correlation, we analysed the impact of common and rare mutations identified in MEN 2A (C609Y, C634R), MEN 2B (A883F, M918T) and familial medullary thyroid carcinoma (Y791F) patients on several aspects of cell transformation, including proliferation, apoptosis, anchorage-independent growth and signaling. We found that tumor cells arising from distinct extracellular or intracellular MEN 2 mutations clearly differ in their proliferation properties owing to the activation of different molecular pathways, but importantly, also in resistance to apoptosis. Whereas MEN 2A mutants resulted in accelerated cell proliferation, MEN 2B-RET mutants significantly enhanced suppression of apoptosis, which may account, at least partially, for some of the clinical differences in MEN 2 patients.

Selection of novel mediators of E2F1-induced apoptosis through retroviral expression of an antisense cDNA library.

The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (approximately 30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis.

A novel mitochondrial protein DIP mediates E2F1-induced apoptosis independently of p53.

The transcription factor E2F1 does not only induce cell proliferation but also shows the strongest proapoptotic effect of all E2F family members as part of an antitumor safeguard mechanism. We have recently identified KIAA0767 as a novel p53-independent target of E2F1. Here, we investigated the biological function of interaction. Overexpression studies of KIAA0767, termed D(eath)-I(nducing)-P(rotein), revealed its strong proapoptotic effect. DIP greatly reduced cell viability in several in vitro systems accompanied by typical apoptotic features such as caspase-3 activation and cleavage of poly(ADP-ribose)-polymerase. Endogenous DIP levels increased following E2F1 activation. Yet, inhibition of endogenous DIP function by small interfering RNA rescued p53-negative cells from E2F1-induced apoptosis, indicating that DIP is an essential mediator of the p53-independent E2F1 death pathway. Localization studies showed that DIP localizes to the mitochondria, where endogenous DIP is upregulated following E2F1 induction. These results provide new insights to the incompletely understood regulatory mechanisms of E2F1-induced apoptosis.

Large nontransplanted hepatocellular carcinoma in woodchucks: treatment with adenovirus-mediated delivery of interleukin 12/B7.1 genes.

BACKGROUND: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. METHODS: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x 10(9) plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. RESULTS: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P =.006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4(+) and CD8(+) T cells and interferon gamma (IFN gamma). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4(+) and CD8(+) T cells and IFN gamma) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (> or = 95%) 7 weeks after treatment. CONCLUSION: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans.

Therapeutic efficacy of E2F1 in pancreatic cancer correlates with TP73 induction.

Pancreatic cancer is particularly resistant to apoptosis by antineoplastic agents, which is partly attributable to the lack of functional p53. Here we show that E2F1 in combination with the most clinically efficient drug, gemcitabine, resulted in a strong induction of apoptosis independent of functional p53, whereas the effect of either therapy alone varied between different cell lines. Intratumoral injection of a helper-dependent adenovirus vector expressing E2F1 plus drug treatment resulted in a significant reduction of tumor volume. The therapeutic effect is directly correlated with the induction of the p53 homologue p73, suggesting that the recently discovered E2F1/p73 pathway plays a critical role in cancer therapy.

E1A overcomes the apoptosis block in BCR-ABL+ leukemia cells and renders cells susceptible to induction of apoptosis by chemotherapeutic agents.

A crucial function of the BCR-ABL chimeric gene in chronic myeloid leukemia is the prolongation of cell survival by inhibition of apoptosis. BCR-ABL expression confers cross-resistance to multiple genotoxic anticancer drugs by inhibition of the apoptotic response to DNA damage in association with cell cycle arrest at the G2-M restriction point. Previous reports indicated that BCR-ABL exerts its antiapoptotic effect against various apoptotic stimuli upstream to the cleavage and activity of caspase-3. Here we show that the adenovirus E1A protein induces substantial apoptosis in BCR-ABL expressing K562 and LAMA-84 leukemia cells. This apoptotic activity of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose) polymerase and can be significantly blocked by z-VAD-fmk Z-Val-Ala-Asp(OCH3)-CH2F and the caspase-3-specific inhibitor Z-DEVD-FMK Z-Asp(OCH3)-Glu-Val-Asp(OCH3)-CH2F. Moreover, E1A renders K562 cells, which are particularly resistant to cell death irrespective of the inducing agent, susceptible to induction of apoptosis by the chemotherapeutic agents etoposide and daunorubicin. Counteracting the DNA damage-induced inactivation of cdc2 kinase, E1A reverses the drug-induced G2-M arrest These results indicate that solitary delivery of E1A significantly antagonizes BCR-ABL-induced antiapoptotic functions and circumvents the inherent resistance to DNA damage-induced apoptosis, supporting the use of E1A in combination with chemotherapeutic agents as a promising therapeutic strategy for successful treatment of Philadelphia chromosome-positive leukemia in vivo.

Role of p73 in malignancy: tumor suppressor or oncogene?

The recently identified p53 family member, p73, shows substantial structural and functional homology with p53. However, despite the established role of p53 as a proto-type tumor suppressor, a similar function of p73 in malignancy is questionable. Overexpression of p73 can activate typical p53-responsive genes, and activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human tumors, led to the hypothesis that p73 has tumor suppressor activity just like p53. However, unlike p53-/- mice, p73 knockout mice do not develop tumors. Extensive studies on primary tumor tissues have revealed overexpression of wild-type p73 in the absence of p73 mutations instead, suggesting that p73 may augment, rather than inhibit tumor development. In contrast to p53, differential splicing of the TP73 gene locus gives rise to a complex pattern of interacting p73 isoforms with antagonistic functions. In fact, induction of apoptosis by increased levels of p73 can be blocked by both p53 mutants and the N-terminally truncated p73 isoforms, which were recently shown to possess oncogenic potential. In the light of these new findings the contradictory role of p73 in malignancy will be discussed.

E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products.

Induction of apoptosis seems to be a key function in maintaining normal cell growth by exerting negative controls on cell proliferation and suppressing tumorigenesis. The adenovirus E1A oncogene shows both cell cycle progression and apoptotic functions. To understand the mechanism of E1A-induced apoptosis, the apoptotic function of E1A 13S was investigated in p53-null cells. We show here that E1A is sufficient by itself to induce substantial apoptosis independent of p53 and other adenoviral genes. The apoptotic function of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose)-polymerase. Cell death is significantly blocked by the caspase inhibitor zVAD-fmk and when coexpressed with E1B19K, Bcl-2 or the retinoblastoma protein (RB). Analyses of E1A mutants indicated that the apoptotic activity of E1A correlates closely with the ability to bind the key regulators of E2F1-induced apoptosis, p300 and RB. Finally, in vivo relevance of down-modulation of p53-independent apoptosis for efficient transformation is demonstrated.

Analysis of adenovirus gene transfer into adult neural stem cells.

Adult neural stem cells (aNSCs) represent an attractive source for the production of specific types of neurons in degenerative CNS diseases and for the development of new regenerative gene therapies. However, the use of adult NSCs for transplantation and gene replacement strategies requires efficient gene expression in the cells. Due to the low pathogenicity of adenovirus (Ad) for humans, its large delivery capacity, and long-term transgene expression, Ad vectors are widely used. Here, we tested the potential of the Ad vector system to transduce adult NSCs. Analysis of Ad receptor expression in primary aNSCs revealed a complete lack of the coxsackie-adenovirus receptor and no or low expression of alphanu- and beta5-integrins, respectively, on mRNA and protein level. Consistently, transduction at different multiplicities of infection using an Ad vector expressing the enhanced green fluorescent protein (GFP) showed that adult NSCs are particularly resistant to Ad infection even at highest MOI (1000) in contrast to differentiated types of neural cells.

Adenoviral p53 gene transfer inhibits human Tenon's capsule fibroblast proliferation.

BACKGROUND/AIM: Although antiproliferative drugs have been used successfully to prevent scarring after filtration surgery in patients with glaucoma, complications associated with their use (such as hypotony or endophthalmitis) energise the search for an alternative treatment. Single application of beta radiation leads to long term growth arrest and expression of p53 in human Tenon's capsule fibroblasts (hTf). The authors assume that the activation of p53 is one of the cellular triggers. Their aim was to analyse the effect of p53 overexpression on hTf and to determine which pathways are involved. METHODS: A recombinant adenoviral vector (rAd.p53) containing transgenes encoding for human p53 and green fluorescent protein (GFP) was used to induce overexpression of p53 in hTF and a control vector (rAd.GFP). Transgene expression was detected by western blot (p53 and p21WAF-1/Cip1). Cell proliferation and viability were investigated using cell counts, 5'-bromodeoxyuridine incorporation (BrdU assay) and tetrazolium reduction (MTT assay). RESULTS: Infection of hTf with rAd.p53 resulted in significant inhibition of cell proliferation, DNA synthesis, and metabolic activity in vitro. Western blot showed increased levels of p53 and p21WAF-1/Cip1 in rAd.p53 infected cells, but not in rAd.GFP and uninfected cells. Apoptosis was excluded with flow cytometry. CONCLUSIONS: Adenoviral p53 gene transfer leads to significant growth inhibition in hTf. P53 induces p21(WAF-1/Cip1) expression and does not cause apoptosis in hTf in vitro. p53 as an antiproliferative drug has the potential to replace mitomycin C and 5-fluorouracil in glaucoma surgery.

A balancing act: orchestrating amino-truncated and full-length p73 variants as decisive factors in cancer progression.

p73 is the older sibling of p53 and mimics most of its tumor-suppressor functions. Through alternative promoter usage and splicing, the TP73 gene generates more than two dozen isoforms of which N-terminal truncated DNp73 variants have a decisive role in cancer pathogenesis as they outweigh the positive effects of full-length TAp73 and p53 in acting as a barrier to tumor development. Beyond the prevailing view that DNp73 predominantly counteract cell cycle arrest and apoptosis, latest progress indicates that these isoforms acquire novel functions in epithelial-to-mesenchymal transition, metastasis and therapy resistance. New insight into the mechanisms underlying this behavior reinforced the expectation that DNp73 variants contribute to aggressive cellular traits through both loss of wild-type tumor-suppressor activity and gain-of-function, suggesting an equally important role in cancer progression as mutant p53. In this review, we describe the novel properties of DNp73 in the invasion metastasis cascade and outline the comprehensive p73 regulatome with an emphasis on molecular processes putting TAp73 out of action in advanced tumors. These intriguing insights provoke a new understanding of the acquisition of aggressive traits by cancer cells and may help to set novel therapies for a broad range of metastatic tumors.

Antitumor capacity of a dominant-negative RET proto-oncogene mutant in a medullary thyroid carcinoma model.

Gain-of-function mutations in the RET proto-oncogene resulting in a constitutively active receptor tyrosine kinase have been identified as responsible for three subtypes of multiple endocrine neoplasia type 2 (MEN-2) and the development of sporadic medullary and papillary thyroid carcinoma. An important strategy in cancer gene therapy is the inhibition of oncogenic signal transduction by interfering with the molecular mechanisms of activation. In the present study, we tested the therapeutic capacity of an adenovirus expressing a dominant-negative (dn) RET mutant, RET(51).flag, under the control of a synthetic C cell-selective calcitonin promoter (TSE2.CP1) against human medullary thyroid cancer (MTC). Infection of human MTC-derived TT cells with Ad-TSE2.CP1-dn-RET(51).flag resulted in the accumulation of immature RET protein in the endoplasmic reticulum and a strong reduction of oncogenic RET receptor on the cell surface, indicating that RET(51).flag exhibits a dominant-negative effect over endogenous oncogenic protein. Analysis of potential downstream mechanisms associated with the inhibition of oncogenic RET signaling by overexpression of mutant RET(51).flag revealed a significant loss of cell viability in TT cells due to the induction of apoptosis. Finally, we examined the antitumor activity of the dominant-negative RET approach in vivo. Inoculation of Ad-TSE2.CP1- dn-RET(51).flag-expressing MTC cells into nude mice led to complete suppression of tumor growth. Moreover, a single intratumoral injection of Ad-TSE2.CP1-dn-RET(51).flag into established thyroid tumors resulted in prolonged survival of treated mice compared with the controls. Our data suggest that adenoviral delivery of dn-RET(51).flag may be a reliable strategy of effective molecular intervention for RET oncogene-related MTC.

Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer.

Although cytokine gene transfer for cancer treatment can stimulate immune recognition and tumor regression in animal models, there is still a need for improvements to these strategies. In this study, we examined the efficacy of a combination gene therapy using adenovirus (Ad) 5 vectors expressing human interleukin-2 and the wild-type (wt) human p53 gene under control of the human cytomegalovirus immediate early promoter (AdIL-2 and Adp53wt, respectively). Infected murine cell lines and primary mouse tumor cells secreted high levels of IL-2 and over expressed the p53 protein for at least 9 days. After infection of cells with Adp53wt, DNA synthesis was significantly inhibited and apoptosis was induced within 3-5 days. Both vectors were tested in a transgenic mouse mammary adenocarcinoma model for antitumor response. Following a single intratumoral injection of mice bearing PyMT induced tumors, the combination of Adp53wt (1 x 10(9) pfu) plus a relatively low dose of AdIL-2 (1.5 x 10(8) pfu) caused regressions in 65% of the treated tumors without toxicity. Fifty percent of the treated mice remained tumor free and were immune to rechallenge with fresh tumor cells. In contrast, injection of either vector alone at this does resulted in only a delay in tumor growth. Only mice co-injected with Adp53wt and AdIL-2 showed specific antitumor cytolytic T lymphocyte (CTL) activity, indicating that the immune response involved in tumor regression was promoted by the combination therapy. These results suggest that cancer treatment strategies involving combined delivery of immunomodulatory and antiproliferative genes may be highly effective.

A multiprotein complex consisting of the cellular coactivator p300, AP-1/ATF, as well as NF-kappaB is responsible for the activation of the mouse major histocompatibility class I (H-2K(b)) enhancer A.

Major histocompatibility complex (MHC) class I genes encode highly polymorphic antigens that play an essential role in a number of immunological processes. Their expression is activated in response to a variety of signals and is mediated through several promoter elements among which the enhancer A is one of the key control regions. It contains binding sites for several transcription factors, for example: (i) a well-characterized binding site for rel/NF-kappaB transcription factors in its 3'-end (the H2TF1 or kappaB1 element), (ii) a second kappaB site (the kappaB2 element), which is located immediately adjacent 5' to the H2TF1 element and which is recognized by p65/relA in the human HLA system, and (iii) an AP-1/ATF recognition sequence in the 5' end (EnA-TRE). Here we demonstrate that latter element is bound by at least two distinct heterodimers of the AP-1/ATF transcription factor family, namely c-Jun/ATF-2 and c-Jun/Fra2. Moreover, our data reveal that the enhancer A is simultaneously bound by AP-1/ATF and rel/NF-kappaB transcription factors and that the cellular coactivator p300, which enhances enhancer A-driven reporter gene expression if cotransfected, is recruited to the enhancer A through this multiprotein complex. In contrast to the complete enhancer A, neither the EnA-TRE nor the H2TF1 element on their own are able to confer activation on a heterologous promoter in response to the phorbol ester tumor promoter TPA or the cytokine TNFalpha. Moreover, deletion of any one of the enhancer A control elements results in a dramatic loss of its inducibility by TNFalpha, and point mutations in either the EnA-TRE or the H2TF1 element lead to the loss of AP-1/ATF or NF-kappaB binding, respectively, and to the loss of enhancer A inducibility. Therefore, we conclude that the enhancer A is synergistically activated through a multiprotein complex containing AP-1/ATF, NF-kappaB transcription factors as well as the cellular coactivator p300.

GRAMD4 mimics p53 and mediates the apoptotic function of p73 at mitochondria.

p73, a member of the p53 family, shares high sequence homology with p53 and shows many p53-like properties: it binds to p53-DNA target sites, transactivates p53-responsive genes and induces cell cycle arrest and apoptosis. Apart from this transcription-dependent effect, less is known about the downstream mechanism(s) by which p73 controls cell fate at the mitochondria. We have previously identified GRAMD4 (alias KIAA0767 or Death-Inducing-Protein) as a novel p53-independent pro-apoptotic target of E2F1, which localizes to mitochondria. In this study, we found that p73-induced apoptosis is mediated by GRAMD4 expression and translocation to the mitochondria. We showed that this protein physically interacts with Bcl-2, promotes Bax mitochondrial relocalization and oligomerization, and is highly efficient in inducing mitochondrial membrane permeabilization with release of cytochrome c and Smac. Overexpression of p73α and p73ß isoforms, but not p53, leads to direct GRAMD4 promoter transactivation. In addition, GRAMD4 induces changes in Bcl-2 and Bax protein levels. GRAMD4 transcription is activated in response to cisplatin (cDDP) in a manner dependent on endogenous p73. Using solid tumor xenografts, ectopic expression of GRAMD4 together with cDDP resulted in enhanced cancer killing. Our findings demonstrate that p73 is able to trigger apoptosis via the mitochondrial pathway by a new mechanism using pro-apoptotic GRAMD4 as mediator, and strongly support its p53-like function.