Woodring Wright - US grants

DESCRIPTION (provided by applicant): Normal human cells have a limited capacity to proliferate, a process termed replicative aging. Increasing evidence over the last decade has implicated telomeres, the structures that cap the ends of the chromosomes, as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 5' end of a linear chromosome, leaving a 3' overhang and causing the chromosomes to shorten every time a cell divides. Human telomeres are composed of many kilobases of the repetitive sequence TTAGGG that together with telomere binding proteins prevent the cell from recognizing the end of the chromosome as a DNA break needing repair. Cellular senescence may occur when some of the telomeres have shortened sufficiently to induce a DNA damage signal. Cancer cells escape the proliferative limits of replicative aging by up-regulating the expression of telomerase, an enzyme capable of adding telomere repeats to the ends of the chromosomes and maintaining their length. We have developed methods for identifying the presence of modified nucleotides in the subtelomeric DNA, for purifying telomeres (based on the presence of the 3' G-rich overhang) that yields a greater than 1,000-fold enrichment in a single step, for determining the size of the overhangs, and for measuring telomere sizes in interphase nuclei. These advances will permit us to address the following Specific Aims: 1) To understand the structure and function of base modifications in subtelomeric/telomeric DNA; 2) To determine what regulates the rate of telomere shortening; and 3) To define when and where cells with short telomeres accumulate in vivo in humans. Knowledge gained from these studies may lead to the ability to manipulate rates of telomere shortening, with consequences both for slowing cellular senescence and enhancing the efficacy of anti-telomerase cancer therapeutics. In addition, these investigations should identify the appropriate tissues and pathologies in which a causal relationship between replicative aging and organismal aging/disease can be tested experimentally, ultimately providing the basis for the development of direct therapeutic applications.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Normal human cells have a limited capacity to proliferate, a process termed replicative aging. Increasing evidence has implicated telomeres, the structures that cap the ends of the chromosomes, as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging-strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 5''''end of a linear chromosome, leaving a 3''''overhang and causing the chromosomes to shorten every time a cell divides. Human telomeres are composed of many kilobases of the repetitive sequence TTAGGG that, together with telomere-binding proteins, prevent the cell from recognizing the end of the chromosome as a DNA break needing repair. Cellular senescence may occur when some of the telomeres have shortened sufficiently to induce a DNA damage signal. Cancer cells escape the proliferative limits of replicative aging by up-regulating the expression of telomerase, an enzyme capable of adding telomere repeats to the ends of the chreomsomes and maintaining their length. Using methods for identifying the presence of modified nucleotides in subtelomeric DNA, for purifying telomeres (based on the presence of the 3''''G-rich overhang) that yields a greater than 1000-fold enrichment in a single step, for determining the size of the overhangs, and for measuring telomere sizes in interphase nuclei. Chromatographic separation for the telomeric nucleobases has been developed. It is a HILIC based separation appropriate for these very hydrophilic compounds and it also affords high mass spectrometric sensitivity. The method has been adapted to be used on nano-LC columns and nano electro spray ionization to increase the sensitivity

DESCRIPTION (provided by applicant): Telomerase prevents the shortening of the ends of each chromosome that normally occurs with every cell division. Telomerase is repressed in most normal cells, and serves as a brake against the progression of premalignant cells by limiting the number of divisions they can undergo while accumulating mutations. Almost all cancers have had to upregulate/reactivate telomerase in order to overcome this limit. Telomerase is regarded as an ideal cancer therapeutic target because of its required expression in ~85% of all tumors and its absence in most normal cells. However, progress towards identifying good inhibitors has been slow and only one, an oligonucleotide directed at the catalytic site of the telomerase template RNA, is in Phase I/II clinical trials. This application proposes to investigate alternative splicing of the telomerase mRNA as a potential additional target for increasing the effectiveness of telomerase inhibition. Less than 5% of the total telomerase (hTERT) mRNA is spliced to give a full length mRNA capable of being translated into active protein. Three approaches for manipulating this splicing to reduce the amount of functional protein produced will be used: 1) Oligonucleotides directed against splicing factor binding sites have been shown to be able to cause exon-skipping, and are in clinical trials for a number of genetic diseases. We have already identified a variety of oligonucleotides that can increase non-functional alternative spliced telomerase mRNAs. These and others will be characterized and refined in order to optimize their effectiveness; 2) We have created a minigene that recapitulates the important alternatively spliced variants of telomerase. A deletion analysis will be used to define the important regulatory sequences that control telomerase alternative splicing. Using that knowledge, the important RNA sequences will be used to affinity purify and identify splicing factors important for telomerase alternative splicing; and 3) The minigene characterized above will be used to create a screen that will allow the preferential survival of cells with decreased full-length mRNA. We will infect shRNA or cDNA libraries to identify splicing factors important for the regulation of telomerase splicing. The knowledge and reagents created using the approaches above will permit chemical screens to be developed in order to identify small molecules capable of reducing full-length telomerase splice forms by increasing the fraction of non-functional alternatively spliced messages. Telomerase inhibition has the potential to be an almost universal treatment for cancer. This project will expand the molecular targets capable of inhibiting telomerase and may finally lead to the development of highly effective telomerase inhibitors. PUBLIC HEALTH RELEVANCE: Most cancers require telomerase for their long-term proliferation, and telomerase inhibition has the potential to be an almost universal cancer treatment. This application explores the ability to manipulate the alternative splicing of telomerase in order to develop more effective cancer treatments.