Identification of E2F-3B, an alternative form of E2F-3 lacking a conserved N-terminal region

We have identified a novel form of the full-length E2F-3 protein that we term E2F-3B. In contrast to full-length E2F-3, which is expressed only at the G1/S boundary, E2F-3B is detected throughout the cell cycle with peak levels in GO where it is associated with Rb. Transfection and in vitro translation experiments demonstrate that a protein identical to E2F-3B in size and iso-electric point is produced from the E2F-3 mRNA via the use of an alternative translational start site. This alternative initiation codon was mapped by mutagenesis to codon 102, an ACG codon. Mutation of the ACG codon at position 102 abolished E2F-3B expression, whereas the conversion of ACG 102 to a consensus ATG led to the expression of a protein indistinguishable from E2F-3B. Given these results, E2F-3B is missing 101 N-terminal amino acids relative to full-length E2F-3. This region includes a moderately conserved sequence of unknown function that is present only in the growth-promoting E2F family members, including E2F-1, 2 and full-length E2F-3. These observations make E2F-3B the first example of an E2F gene giving rise to two different protein species and also suggest that E2F-3 and E2F-3B may have opposing roles in cell cycle control.     

E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

Background  

PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells.     

TAL1/SCL induces leukemia by inhibiting the transcriptional activity of E47/HEB

Activation of the basic-helix-loop-helix (bHLH) gene TAL1 (or SCL) is a frequent gain-of-function mutation in T cell acute lymphoblastic leukemia (T-ALL). To provide genetic evidence that tal1/scl induces leukemia by interfering with E47 and HEB, we expressed tal1/scl in an E2A or HEB heterozygous background. These mice exhibit disease acceleration and perturbed thymocyte development due to repression of E47/HEB target genes. In tal1/scl thymocytes, we find the corepressor mSin3A bound to the CD4 enhancer, whereas an E47/HEB/p300 complex is detected in wild-type thymocytes. Furthermore, tal1/scl tumors are sensitive to pharmacologic inhibition of HDAC and undergo apoptosis. These data demonstrate that tal1/scl induces leukemia by repressing E47/HEB and suggest that HDAC inhibitors may prove efficacious in T-ALL patients who express TAL1/SCL.     

HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity

HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we show that HMGA2 interacts with pRB and induces E2F1 activity in mouse pituitary adenomas by displacing HDAC1 from the pRB/E2F1 complex-a process that results in E2F1 acetylation. We found that loss of E2F1 function (obtained by mating HMGA2 and E2F1(-/-) mice) suppressed pituitary tumorigenesis in HMGA2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas.     

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas. Oncogene (2000).     

E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.     

转录因子E2F-1的研究进展

目的:总结国内外关于E2F-1在细胞增殖、凋亡及其在肿瘤中的作用的研究进展。方法:应用Medline及CNKI期刊全文数据库系统,以"E2F-1、细胞增殖、凋亡及肿瘤为关键词,检索1999-01-2009-12的相关文献,共检索英文文献1 453篇、中文文献29篇。纳入标准:1)E2F-1的来源和分子结构特征;2)E2F-1与细胞周期的关系;3)E2F-1和细胞增殖的关系;4)E2F-1和凋亡的关系;5)E2F-1和肿瘤的关系。根据纳入标准,附合分析条件的文献有52篇,最后纳入分析33篇。结果:E2F-1是细胞周期调节的关键因子,具有调节细胞增殖和凋亡的双重作用,在肿瘤中表现出抑癌基因和癌基因的功能。结论:E2F-1可能是抗肿瘤研究的靶点。