Browsing by Subject "Telomeres"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Genetic and Molecular Studies of Endometrial Cancer(2010-11-02) Akbay, Esra; Castrillon, Diego H.Endometrial cancer, which develops from the inner lining of the uterus, is the most common cancer of the female reproductive tract. Endometrial cancer is comprised of two epidemiologic, molecularly and genetically different subtypes known as type I and type II. Because of its clinical significance and aggressive behavior, my research focused on type II endometrial cancer. Type II endometrial cancer is associated with advanced age and TP53 mutations, which suggests a link between telomeres and the development of type II tumors. Telomeres and other markers of telomere status in type I and type II tumors were analyzed, short telomeres are observed in both tumor types. However, only type II tumors are associated with critical telomere shortening in the adjacent, morphologically normal epithelium. This suggests that telomere shortening contributes to the initiation of type II, but not of type I tumors. To further explore this hypothesis, 5th generation telomerase knockout mice with critically short telomeres were analyzed and a distinctive endometrial lesion that resembles the in situ precursor of type II carcinomas, endometrial intraepithelial carcinomas were observed. Subsequently, the role of dysfunctional telomeres in endometrial cancer development was investigated in the mouse by conditionally inactivating Pot1a, a key component of the shelterin complex that stabilizes telomeres in endometrial epithelium. Inactivation of Pot1a by itself does not stimulate endometrial carcinogenesis. However, simultaneous inactivation of Pot1a and p53 results in endometrial intraepithelial carcinoma-like lesions by 9 months and metastatic tumors in 100% of the animals by 15 months. These tumors are poorly differentiated endometrial carcinomas with prominent nuclear atypia, DNA damage, and aneuploidy, resembling human type II tumors. These studies thus lend support to the hypothesis that dysfunctional telomeres play a critical role in type II endometrial carcinogenesis.Item Human shRNA Library Screening to Dissect Pathways Involved in Telomerase Actions(2011-12-12) Hoshiyama, Hirotoshi; Shay, Jerry W.The minimal components of human telomerase are the human telomerase reverse transcriptase (hTERT) and the human telomerase template RNA (hTR). Although it is known that both components are minimally sufficient to reconstitute telomerase activity, the factors involved in any of the multiple steps of telomerase action such as telomerase assembly, telomerase recruitment to telomeres, and telomere extension/regulation are not well understood. There are a large numbers of proteins that have associations with telomerase, yet the functional roles of those in telomere maintenance and telomerase regulation are not well understood. Identifying novel proteins and pathways involved in any of these important telomerase-associated functions will be useful for identifying new targets for the development of novel inhibitors that block telomerase function in cancer cells. Therefore, my goal has been to develop methods to dissect these molecular pathways and identify functional factors involved in any step of telomerase actions. To accomplish this, I designed a selective screening system by exploiting lentiviral shRNA libraries and tetracycline inducible-hTERT cell lines that is hTR deficient but expressing mutant hTR. Thus, the overall strategy of the screening system may be considered a “synthetic rescue screen”. In brief this screen was set up to rescue cells from apoptotic death due to mutant sequence incorporation at telomeres by reducing the gene expressions with lentiviral shRNA libraries. This allows us to look a set of genes involved in pathways involved in functional aspects of telomerase actions, not based on structural association with telomerase. During the work, I have established multiple lines of inducible-hTERT cells to use in selective screening systems. I have also developed a method for rapid construction of high-complexity custom shRNA libraries for targeted screening and re-evaluate hundreds of primary candidate genes to identify smaller numbers of secondary candidate genes by removing false positives. In order to analyze the pooled shRNA screening result, I have developed a method for quantitative identification of half-hairpins from a pooled shRNA library based on the pGIPZ vector. Introducing multiplexing codes and refining sample preparation schemes resulted in the predicted ability to detect two-fold enrichments followed by massive parallel sequencing. Development of those methods allowed me to identify several candidate proteins, which may be involved in telomerase actions.Item Telomere Dynamics and End Processing in Mammalian Cells(2006-05-15) Sfeir, Agnel J.; Shay, Jerry W.Telomeres are repetitive DNA sequences that end in single-stranded 3' overhangs. With each cell division, normal human cells lose a small amount of telomeric DNA due to the end-replication problem and the action of an unidentified nuclease. In order for tumor cells to divide indefinitely, they maintain telomere length by expressing the enzyme telomerase. The end structure of mammalian telomeres is not very well understood. Two assays were developed using ligation and PCR amplification to identify the terminal nucleotides of both the C-rich and G-rich telomeric strands in human cells. The results showed that ~ 80 % of the C-strands terminate precisely in ATC-5', demonstrating that the nuclease resection of the C-strand post replication is specific for a single nucleotide. In contrast, the last base of the G-strand in normal human cells was less precise with 70% of the ends being TAG-3', TTA-3' or GTT-3'. An enrichment for the TAG-3' end was noted in cells that express telomerase. A series of nucleases were tested for their involvement in specifying the last base of C-strands and the results indicated that none of those nucleases were responsible for telomere-end resection. Inhibiting the normal function of most telomere binding proteins altered normal telomere function, however only one protein (POT-1) influenced last base specificity. Knocking down POT-1 in normal and tumor cells randomized the last base of the C-strand. These finding have important implications for the processing events that act on the telomere ends and they will help identify the nuclease that resects the chromosome ends. In the second part of this study, the dynamics of telomerase action in mammalian cells was examined. Using a PCR-based, single telomere-length measurement assay (STELA) we showed that telomerase adds an average of 250-nucleotides per end in one replication cycle. Cell cycle studies showed that while the telomeres on the Xp chromosome replicated in early S-phase their elongation by telomerase took place during late S/early G2 phase. Therefore, in mammalian cells telomerase action is not coupled to DNA replication. These studies will provide much needed information for exploiting our knowledge of telomere biology for telomerase-based therapeutic purposes.Item Telomere Position Effect in Human Cells(2003-04-01) Baur, Joseph Anthony; Shay, Jerry W.Telomeres are tracts of repetitive DNA that cap the ends of linear chromosomes. Each time the chromosome is duplicated, a small amount of telomeric DNA is lost from the end due to factors inherent in the mechanism of DNA replication. The result is a net shortening of telomeres with each cell division, unless new repeats are synthesized through the action of the enzyme telomerase. Most human somatic cells lack telomerase activity and so continued cell division leads to telomere shortening. After a limited number of divisions (the "Hayflick limit"), it is believed that a few critically shortened telomeres trigger a state of growth arrest termed replicative senescence.