E2F1 is Crucial for E2F-Dependent Apoptosis - Coursework Example

?E2F1 is crucial for E2F-dependent apoptosis Cell cycle regulates the cell proliferation and they must divide to enter the mitotic Before entering the mitotic phase, the cell must pass through a number of check points. Retinoblastoma protein (pRb) plays an important role in the epithelial cell differentiation and viability apart from this they also act as positive transcriptional co regulators. These retinoblastoma protein were phosphorylated and dephosphorylated in the mammalian cells and were found to play an important role as tumor suppressor gene. The tumor suppressor genes constitute a large group of genes that specify protein products mediating diverse cell-physiologic functions. These proteins have the ability to function at all parts of the cell. These genes also lead to the discovery of familial cancer syndromes. About 50 tumor suppressor genes were catalogued and pRb and p53 proteins were found to have preeminent importance in the human tumor pathogenesis. pRb is closely co ordinate with the cell cycle. pRb hyperphosphorylation occurs when the cells pass the M/G1 transition at the R point. pRb contains atleast 16 different sites for phosphorylation. Thus it proves that pRb is the molecular governor of the R point transition. After the findings that the DNA tumor-virus encoded oncoproteins can disrupt the regulation of cell growth, the importance of learning pRb became important. It was also found that 3 DNA tumor virus oncoproteins, though they are structurally unrelated, they targeted the pRb. pRb inactivation can be done in four pathways. Interaction with viral proteins, phosphorylation, gene mutation and caspase mediated deregulation. The binding of the onco proteins at the site of pRb also promote cellular proliferation. The cell cycle dependent transcription was associated with the co ordination of the pRb and the E2F as the central mechanism. The pRB which are also called as rocket proteins helps to silence the E2F regulated promoters. The complexes between the pRb and the E2F family members were found to be formed at various phases of the cell cycle. The pRb/E2F complex helps to regulate growth arrest and cell cycle reentry. This association is released by phosphorylation of the pRb by the cyclin dependent kinase. In the absence of pRb protein, the cell death was found to be higher. Hence the effect of pRb on the apoptosis was studied. The E2F protein and the pRb complex formation and dissociation was extensively studied. Apoptosis in the absence of the pRb was found to be dependent on the activity of E2F1. The cell cycle progression is dependent upon the release of the E2F mediated by the phosphorylation of pRb. The E2Fs contain a distinct domain at the C terminus which is more important for protein binding. The E2F1 mediated apoptosis is ascribed to two mechanisms both p53- dependent and p53- independent. When there occurs any DNA damage during the G0 and G1 phases, the p 53 tumor suppressor proteins induces apoptosis. E2F1 can proliferate and arrest the cell cycle at any stage. Thus it acts as both positive and negative regulator. The hypophosphorylation of pRb binds to the E2Fs and activates it through negative regulation. Thus E2Fs expression on regulated genes increases. Since E2F1 and E2F3 loss can induce apoptosis, this became the key factor in the study of human cancer. E2F transcription factor are associated with a number of promoters on the cell cycle at G1. These E2Fs are bound by many pocket proteins. These pocket proteins are the pRb proteins. When these pRbs undergo hyperphosphorylation, they lose their grip on E2Fs and they stimulate the transcription of the genes. The viral onco proteins target the pRb and they mimic pRb hyperhosphorylation and prevent pRb from binding to the E2Fs. E2Fs is a heterodimeric protein composed of E2F1, 2, 3, 4, 5 and 6 sub units. In this report, the ability of E2F3 to trigger apoptosis in vivo was analyzed and concluded that E2F3 deregulation will trigger apoptosis. It was also concluded that E2F1 and E2F3 association is required for apoptosis. Deregulated E2F3 activity induces apoptosis in vivo: E2F1 in the presence of E2F3 induces apoptosis. The ER- E2F3 under the tissue-specific promoter POMC and fusion protein are inactive without 4-hydroxy tamoxifen. In presence of OHT, activation of E2F3 increases the expression of EF genes and this proliferate the melanotrophs of the intermediate lobe of the pituitary gland. The increased expression was found not associated with the tumor formation. Hence deregulation ofE2F3 and its activity was studied. The experiment was done using two groups. One group of transgenic mice and another group of wild-type mice. They were given tamoxifen for 3 days. It was observed from active 3 caspase staining and TdT-mediated dUTP nick end labeling (TUNEL) assay that E2F3 induces apoptosis. E2F3 can induce p53 Independent apoptosis: An assay was performed to assess the effect of E2F3 induction on p53- independent apoptosis. The TUNEL assay and caspase 3 staining showed that the loss of p53 did not have any effect on E2F3 to induce apoptosis. These results coincided with the previous findings. Deregulated E2F3 activity was induced independently in p53. E2F3 induces E2F1-dependent apoptosis in MEFs: In the E2F family members E2F1 is an E2F responsive gene and is highly characterized. It was found that the activation of E2F3 can help to increase the E2F1 levels to induce apoptosis. This was concluded by after the test for expression of ER-E2F3 in mouse embryo fibroblasts. Both the wild type and the E2F -/- MEFs were infected and the viability of the cells was found by the common technique tryphan blue assay. Both the cells types were infected in this process. At normal stage, if the E2F3 were activated then the wild type cells attained apoptosis but in this experiment the E2F3 activation resulted in the decrease of the death rate thought the MEFs lacked the E2F1 protein. The E2F3 activation was caused by E2F3-induced senescence, which resulted in the decrease of the proliferation rate. In both the cell lines, Cyclin E1 and PCNA (proliferating cell nuclear antigen) were induced at the same level as a result of which the functionality of E2F3 was not changed. The expression level of the caspase 3 was analyzed in both wild type and E2F1-/- cells as an independent marker. When E2F3 was activated it lead to the increase in activated caspase 3 in the wild type and not in the E2F1-/- MEFs. As expected this increase in the expression of E2F3 resulted in the up regulation of hE2F1 expression gene in the wild type MEFs. Thus it was confirmed that activation of E23F3 resulted in the E2F1 dependent apoptosis through direct transactivation. The Wild type and E2F1-/- MEFs were then infected with the retrovirus expressing ER-E2F1 and ER-E2F3 or both. It was found that E2F1 protein production increased the apoptosis in the wild type and E2F1-/- MEFs. Thus it agrees with the previous reports that apoptosis was triggered more by E2F1 than E2F3. The importance of E2F1 and E23F3 in apoptosis was analyzed together and separately. It was observed that E2F3 induction did not affect the apoptotic level of E2F1. The relationship between E2F3 and E2F1 is that accumulation of E2F1 by E2F3 results in apoptosis. To confirm the relationship reciprocally, the floxed E2F3 alleles were used and the cell cycle was proceeded. It was noted that E2F3 absence did not affect apoptosis and its level. The over expression and under expression of E2F3 did not alter the apoptotic level. Whereas, if E2F1 is over expressed, apoptotic level was also found to be higher. E2F3 induces E2F1- dependendt apoptosis in vivo: A transgenic mice having the ER E2F3 in an E2F1-/- back ground was analysed. It was found that the expression level of ER-E2F3 in the E2F1-/- back ground failed to induce apoptosis whereas the wild type mice had the expression level using the caspase 3 assay and the TUNEL assay. The E2F3 activation and its expression were not affected by the lack of the E3F1 gene. The 5-bromodeoxyuridine incorporation and PCNA staining showed the E2F3 activation level at the unscheduled proliferation in both the wild type and the E2F1-/-. Thus it was confirmed in vivo that the E2F3 activation triggers the E2F1 dependent apoptosis. E2F3 –induced proliferation is enhanced in E2F1-/- mice: Mice’s were given tamoxifen and the pituitary homeostasis was performed for 6 months and the results were monitored. It was found that the E2F3 at the pituitary gland has lead to hyperplasia. There was no tumor caused for any of the mice during the experimental period. Thus it was concluded that even after the deregulation, there were no chance for the tumor formation among the mice. Conclusion: It was concluded that E2F3 can induce apoptosis similar to the E2F1 both in vivo and in MEFs. It was also found that E2F3 expression in the cell leads to the activation of the E2F1 that leads to apoptosis. The increased activity of the E2F1 leads to apoptosis ARF-p53 depende3nt and p53 independent pathway. They also concluded that E2Fs can induce all apoptosis that depends on E2F1. Thus E2F1 activity triggers apoptosis rather than the total E2F activity. Read More