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Plated 1-Oleoyl lysophosphatidic acid at equal density. Cells were counted and re-plated on the indicated days. Values are mean ?SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student's t-test comparing eGFP to EWS/ WT1 TS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.Bandopadhayay et al. BMC Cancer 2013, 13:585 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11011031 http://www.biomedcentral.com/1471-2407/13/Page 5 ofHaving established reliable, inducible EWS/WT1 expression, we examined the effect of EWS/WT1 expression on proliferation of MEFs transformed either with SV40 large T antigen or E1A/RAS. Rate of cell proliferation was measured by cell counts over a 14-day time course and fold-change determined (Figure 1C). Expression of either EWS/WT1 isoform increased proliferation rates compared to eGFP controls in multiple, independent pools of SV40 large T antigen transformed MEFs (p value <0.05) but not E1A/RAS transformed MEFs. The magnitude of the increase was modest because these cells are already transformed, but robustly repeatable. In all cell lines tested and in all independent experiments, EWS/WT1 functioned to promote proliferation in SV40 large T antigen transformed but not E1A/RAS transformed MEFs. These data indicate that both isoforms of EWS/WT1 increase cell proliferation specifically in cells transformed with SV40 large T antigen. This suggests EWS/WT1 may co-operate with the loss of specific tumor suppressor pathways.EWS/WT1 oncogenic functions are evident in cells lacking one or both alleles of pThe increased cell proliferation observed in MEFs transformed by SV40 expressing EWS/WT1 led us to hypothesize that EWS/WT1 co-operates with loss of p53 to promote proliferation. To investigate this, we infected freshly isolated MEFs from E14.5 embryos derived from crosses of p53+/- mice, with lentivirus encoding either EWS/WT1 isoform or eGFP. For these, and subsequent experiments the doxycycline repressible lentiviral expression system was used, due to increased infection efficiency compared to the 4-OHT-inducible system. Cells were continuously cultured, counted and replated every third day over a three-week time course (Figure 1D). In wild-type MEFs, neither EWS/WT1 isoform was sufficient to permit unrestricted division or the development of foci (Figure 1D upper panel), and wild type cells were unable to be maintained in culture beyond three weeks. In contrast, in p53+/- MEFs both EWS/WT1 + KTS and EWS/WT1 TS induced the development of foci and significantly increased the rate of proliferation compared to eGFP controls (Figure 1D middle panel). eGFP expressing p53+/- cells eventually stopped proliferating while those expressing EWS/WT1 did not. In p53-/- cells, whilst foci formed independently of infection with EWS/WT1, they were more frequent and the proliferation rate was increased in cells expressing either EWS/WT1 isoform (Figure 1D lower panel). p53+/- cells expressing EWS/WT1-KTS or EWS/WT1 + KTS could be maintained in culture indefinitely while those expressing eGFP could not. These data confirm the ability of EWS/WT1 to induce foci formation and increase proliferation rates of MEFs is revealed by the loss of at least one copy of p53.We next determined whether EWS/WT1 could promote colony formation after serum deprivation. Equal numbers of wild-type, p53+/- and p53-/- MEFs expressing EWS/ WT1 + KTS, EWS/WT1-KTS or eGFP were cultured.