E2F and p53 Induce Apoptosis Independently during Drosophila Development but Intersect in the Context of DNA Damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1

E7 proteins from oncogenic human papillomavirus types transactivate p73: role in cervical intraepithelial neoplasia

In common with other E2F1 responsive genes such as p14ARF and B-myb, the promoter of p73 is shown to be positively regulated in cell lines and primary human keratinocytes by E7 proteins from oncogenic human papillomavirus (HPV) types 16, 18, 31 and 33, but not HPV 6. Mutational analysis revealed that transactivation of the p73 promoter by HPV 16E7 requires association with pRb. Expression of p73 in normal cervical epithelium is confined to the basal and supra-basal layers. In contrast, expression in neoplastic lesions is detected throughout the epithelium and increases with grade of neoplasia, being maximal in squamous cell cancers (SCC). Deregulation of expression of the N-terminal splice variant p73Δ2 was observed in a significant proportion of cancers, but not in normal epithelium. The frequent over-expression of p73Δ2, which has recognized transdominant properties, in malignant and pre-malignant lesions suggests a role in the oncogenic process in cervical epithelium

Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro

E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs) reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s) other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation

Mutation analysis of the p73 gene in nonastrocytic brain tumours

Loss of heterozygosity (LOH) involving the distal chromosome 1p36region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73is a novel gene that has high sequence homology and similar gene structure to thep53 gene; it has been mapped to 1p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1p36–p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development. © 2001 Cancer Research Campaign http://www.bjcancer.co

Targeting histone deacetyalses in the treatment of B- and T-cell malignancies

HDAC inhibitors (HDACI) are now emerging as one of the most promising new classes of drugs for the treatment of select forms of non-Hodgkin’s lymphoma (NHL). They are particularly active in T-cell lymphomas, possibly hodgkin’s lymphoma and indolent B cell lymphomas. Presently, two of these agents, vorinostat and romidepsin, have been approved in the US for the treatment of relapsed and refractory cutaneous T cell lymphomas (CTCL). Initially, these agents were developed with the idea that they affected transcriptional activation and thus gene expression, by modulating chromatin condensation and decondensation. It is now clear that their effects go beyond chromatin and by affecting the acetylation status of histones and other intra-cellular proteins, they modify gene expression and cellular function via multiple pathways. Gene expression profiles and functional genetic analysis has led to further understanding of the various molecular pathways that are affected by these agents including cell cycle regulation, pathways of cellular proliferation, apoptosis and angiogenesis all important in lymphomagenesis. There is also increasing data to support the effects of these agents on T cell receptor and immune function which may explain the high level of activity of these agents in T cell lymphomas and hodgkin’s lymphoma. There is ample evidence of epigenetic dysregulation in lymphomas which may underlie the mechanisms of action of these agents but how these agents work is still not clear. Current HDAC inhibitors can be divided into at least four classes based on their chemical structure. At present several of these HDAC inhibitors are in clinical trials both as single agents and in combination with chemotherapy or other biological agents. They are easy to administer and are generally well tolerated with minimal side effects. Different dosing levels and schedules and the use of isospecific HDAC inhibitors are some of the strategies that are being employed to increase the therapeutic effect of these agents in the treatment of lymphomas. There may also be class differences that translate into specific activity against different lymphoma. HDAC inhibitors will likely be incorporated into combinations of targeted therapies both in the upfront and relapsed setting for lymphomas

p53 and p73: seeing double?

Whereas p73 is closely related to the tumour-suppressor protein p53, its contribution to tumour suppression and the spatial and temporal regulation of its isoforms is unclear. It has now been established that p73 is a transcriptional target of E2F1. Its ability to induce apoptosis in TP53(-/-) cells indicates a tumour-control mechanism that runs parallel to but independent of that mediated by p53. The new results illustrate a complex cross-talk between p53, E2F1 and p73

ASPP1 and ASPP2 are new transcriptional targets of E2F

The E2F family of transcription factors regulates the expression of a number of genes whose products are involved in cell cycle control, DNA replication and apoptosis. We show here that E2F-1 binds in vivo the promoters of ASPP1 and ASPP2 genes, two activators of p53-mediated apoptosis, E2F-1, E2F-2 and E2F-3 all activate the isolated ASPP1 and ASPP2 promoters. Overexpression or deregulation of E2F-1 increased the expression levels of ASPP1 and ASPP2 mRNA and proteins. The identification of ASPP1 and ASPP2 genes as transcriptional targets of E2F provides another mechanism by which E2F cooperates with p53 to induce apoptosis

Differential regulation of E2F1 apoptotic target genes in response to DNA damage

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation