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    Mechanisms of Responses of Cancer Cells to Mutant-Template Telomerase RNA.

    Scientific Abstract:
    PI: Blackburn, E. Tracking ID: PDF0403278 Mechanisms of responses of cancer cells to mutant-template telomerase RNA. Telomeres are specialised functional complexes at the ends of chromosomes, consisting of the terminal stretch of chromosomal DNA and associated proteins. Synthesis of telomeric DNA is accomplished by the ribonucleoprotein telomerase, the core components of which are the reverse transcriptase protein subunit TERT and telomerase RNA, WT-hTER. We aim to investigate the molecular mechanisms of effects, in cancer cells, of mutant template RNA (MT-hTer) constructs which were created by mutating the template sequence in TER. Expression of seven different MT-hTers have been shown to decrease cell viability and increase apoptosis (within 3 days). This "toxic telomerase" effect could be due to repeats from the mutant template being copied onto the telomere tip, disturbing the association of telomere terminal binding proteins, such as Pot1, hence disrupting the telomere "cap" and signalling to the cell to undergo cell cycle arrest or apoptosis. Another explanation is that mutant telomerase may cause telomere uncapping in MT-hTer expressing cells by remaining aberrantly associated with a telomere.This research proposal is specifically directed towards probing the mechanisms of mutant template RNA (MT-hTer) effects in human cancer cells. We aim to examine by high resolution deconvolution light microscopy the levels and localisations of the telomere-binding proteins TRF1, TRF2, hRAP1, hPot1 and hTERT in response to the telomere-uncapping caused by mutant template expression. We will determine critical points in the cell cycle when mutant templates exert their effects by following the subnuclear localisation of DNA repair and checkpoint proteins in real time throughout the cell cycle in response to telomere uncapping. Additionally we will study the requirement of p53 and/or ATM in cellular effects of MT-hTers and investigate the type of cell death pathway elicited by MT-hTer expression. In this study we will make GFP-fusion proteins of the above telomere-associated proteins, and use lentiviral vectors to transiently or stably express them in both primary and cancer cell lines after they have been infected with mutant templates. We will examine their precise localisations in fixed cells by deconvolution microscopy and live cell microscopy. Live cell microscopy will also be used to follow the recruitment of DNA damage proteins to the telomere to address the question of when in the cell cycle damaged telomeres signal to the cell. An additional biochemical approach will be to assess the temporal pattern of these proteins, binding to telomeres by chromatin immunoprecipitation (ChIP) using cells at different cell cycle stages. We will determine signalling requirements for ATM and p53 in mutant template expressing cells using dominant negative constructs of p53 and ATM, and siRNAs directed against their mRNAs, in cell lines deficient in p53 and/or ATM. Cell proliferation and viability assays will be performed, and these results will be compared with wild type cell lines. Caspase-specific inhibitors will be used to investigate the type of cell death pathway and apoptosis assays will be performed. We will also test whether overexpression of anti-apoptotic proteins Bcl-2 or Bcl-XL protects against apoptosis. Many normal human somatic cells have diminished telomerase and the progressive shortening of their telomeres has been proposed to be a major mechanism determining cellular senescence and conferring a finite replication potential to normal cultured human cells. However, telomerase is highly active in 85% of human cancers, conferring immortality on these cells by stabilising telomere length. A greater understanding of telomeres and telomerase should guide the rational development of new therapeutics for cancer and other disorders. Given that a low threshold of MT-hTer expression has a potent killing effect on cancer cells, its exploitation has potential as an anti-neoplastic strategy.

    Lay Abstract:
    PI: Blackburn, E. Tracking ID: PDF0403278 Mechanisms of responses of cancer cells to mutant-template telomerase RNA. Telomeres exist at both ends of every chromosome, which contain the DNA comprising the genetic information of the cell. Telomeres both protect the chromosomes from damage, and prevent the chromosomal DNA from shortening too much each time the cell divides. An enzyme called telomerase replenishes the DNA at telomeres. Telomerase consists of both a protein resembling the reverse transcriptase subunit of HIV, and RNA. While RNA is usually produced as an intermediate during protein synthesis, the RNA component of telomerase instead acts as a template to be copied by the reverse transcriptase of telomerase to enable the telomeric DNA to be extended. Many proteins bind to telomeres and protect them and prevent them from fusing with other telomeres. In most cells, once the cell is fully developed, the enzyme telomerase is switched off. This means that the telomeres cannot be extended; instead, they gradually shorten with each cell division. This shortening of the telomeres acts as a signal to the cell that it has reached a certain age, and needs to stop growing and dividing. It is this mechanism that determines cellular aging, so that cells do not live forever. However, in some cells such as germ cells and stem cells which continually give rise to new cells, the telomerase enzyme is never switched off, and the telomeres do not shorten. These cells, therefore, have stabilised telomere lengths and do not stop dividing. Telomerase is reactivated in most cancer cells, conferring immortality on these cells. Since telomerase is highly active in most forms of breast human cancer, it provides a potential anti-neoplastic therapeutic target. We plan to study signalling mechanisms from the telomere to the rest of the cell. We will induce mutations in the RNA component (template) of telomerase which will cause the telomere itself to become mutated (altered). This is known to cause the cell to die in a matter of days but what it is that happens to the telomere structure itself to signal cell death is unknown. Therefore, using a microscope we will watch different telomere-associated proteins come off or go onto telomeres in response to the altered telomere. We will also use a variety of reagents and chemicals to study the signalling pathways from the telomere that cause the cell to die. These studies will increase our understanding on how telomeres and telomerase signal to the cell to either stop or continue dividing and illuminate how the reactivation of telomerase contributes to an increased risk of breast cancer.