The PI proposes to analyze the roles of SV40 T antigen in transformation. The Tevethia laboratory has previously generated an extensive series of deleted and mutated forms of large T antigen that have been used by their group and in collaborative studies with others to further understand how T antigen alters the growth state of cells. They have learned that T possesses two separable regions that can cooperate with ras to transform cells. Recently they also made the unexpected observation that the C-terminal region of T antigen (250-708) is responsible for the interaction of T with p300/p400 in cells. The first 4 aims proposed remain largely the same, with some minor alterations in response to the previous critique. although a new aim (#5) is added:
Aim 1 plans to further refine the regions and activities of T antigen that are involved in cooperation with ras. Their experiment suggest that a region between 121 and 128 confers upon the N-terminal portion of T the ability to cooperate with ras. It is planned to pinpoint even more precisely what the limits of the ras-cooperation domain is. They will examine whether the conserved hexapeptide region in the N-terminus or the RB binding region is involved in cooperation. If they can narrow down the ras-cooperation region to a few residues it is also planned to affix this region to a segment of the extreme C-terminus to see if this can also cooperate with ras in that context. They have evidence that a similar approach works for RB and that the RB-binding (""""""""pocketbinding"""""""") domain of T when fused to the C-terminus (601-708) actually confers upon this inert fragment the ability to bind RB and alter growth properties. They will now examine further the effects on RB in cells eg. E2F activation, etc. The PI also has evidence that the region between 251 and 272 are required for ras cooperation with the C-terminal T antigen fragment. They will determine the role of p53 binding by combining this region with various mutants generated in Dr. Simmons' lab that alter the interaction of T with p53. To continue with the more recent observation that T antigen's more C-terminal portion interacts with p300 the PI will test the mutants for their effects on interaction with p300. The goal here is to determine whether ras cooperation co-segregates with p300 binding.
Aim 2 follows up on a report that a ts mutant T antigen (438) induces apoptosis at the non-permissive temperature, Dr Tevethia hypothesizes that in contrast to what was previously concluded from studies with conditional mutant T antigens, namely that T is needed to initiate but not maintain the immortalized state, these new results mandate a new look at the question of T in initiation vs maintenance. To test this it is planned to establish cells containing an excisable T antigen i.e. one which is flanked by the FLP recombinase excision sequences, to introduce such T antigens into cells and after either early or late passage transfect a construct encoding the FLP recombinase into cells and then measure whether removal of T (by recombination out of the expression construct at various stages) affects the ultimate number of immortal cells (scored as G418-resistant colonies). If there is a significant reduction in those transfectants which received an excisable T antigen (to be subsequently confirmed by PCR analysis of T sequences in clones) this will mean that T antigen is needed for maintenance of the immortal phenotype.
Aim 3 uses both the T antigen mutant constructs generated previously as well as additional mutants obtained from others such as Dr Dan Simmons it is planned to conduct a thorough investigation of the role of T antigen in apoptosis. One set of experiments will involve transfecting various T antigen constructs along with wild-type p53 into HeLa cells and then monitoring the cells for apoptotic signs by TUNEL assay, duel chromogenic staining of nuclei, and a DMNA fragment assay. Since it is likely that the region of T antigen necessary for binding to p53 will abrogate any apoptotic effects seen with other regions, experiments will be conducted with these other fragments in the presence or absence of the p53-binding region. Based on the observations of S. Lowe and others working with adenovirus, in which it was shown that E1a sensitizes primary cells to DNA damage-induced p53-mediated apoptosis, a number of B6MEF cell lines expressing T antigen fragments plus or minus E1a have been generated and will be treated with DNA damaging agents and apoptosis measured. It is also planned to examine whether, like E1a, there is an activity of T antigen that can also sensitize cells to TNFa mediated apoptosis (an apoptotic pathway that is p53 independent, based on Lowe's findings). It is possible that TNFa-mediated apoptosis involves p300/400 binding and therefore it may be that this form of apoptosis, if it is controlled at all by T antigen, will require the same regions of T antigen that are distinct from the N-terminus.
In aim 4, the ability of T antigen to induce tumors in nude mice and in transgenic models will be examined using the fragments generated by Dr Tevethia and such experiments are expected to complement results from aims 1 and 3. The transgenic model to be explored involves expression of T antigen in pancreatic cells driven by the elastase promoter or in a broader spectrum of cells, using the CMV promoter. Preliminary results were presented showing that T1-128 transgenic mice produce malignant acinar cell tumors in the pancreas that have the same characteristics as mice expressing full length T antigen. One goal is to produce mice bearing p53-binding fragments and mice bearing Rb-binding fragments and then cross them, and characterize the tumors produced (or not) in the three conditions. It was observed that p300 is underphosphorylated in cell expressing full length but not C-terminal portions of T antigen. This suggest that the N-terminus may be required for this effect on p300 and, with the different N-terminal constructs that have been and will be generated, it is hoped that further insight (aim 5) will be obtained into what functions/ interactions associated with the N-terminus contribute to p300 hypophosphorylation.
| Tatum, Angela M; Mylin, Lawrence M; Bender, Susan J et al. (2008) CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors. J Immunol 181:4406-17 |
