Telomerase is an enzyme that helps maintain the telomeric ends of chromosomes during DNA replication. Telomere lengths represent a balance between telomerase activity attempting to elongate their ends, and cell division that causes telomere shortening. As cells age, diminished telomerase activity allows a shortening of telomere lengths until they reach a target length that stimlulates apoptosis. Identifying a drug capable of upregulating telomerase activity may help increase cell (and even organismal) lifespan. The purpose of this thesis was to determine whether treatment of human primary foreskin fibroblast cultures with a 14 amino acid (aa) ependymin peptide mimetic upregulates (or at least maintains) telomerase activity and telomere lengths during cellular ageing. The 14aa peptide was previously shown to significantly increase the murine lifespan by 25%, so its activity was a logical candidate to test in this thesis. In a preliminary set of experiments, the human primary fibroblast cells were shown to respond to the 14aa drug by upregulating the antioxidative enzyme superoxide dismutase (SOD), thus human fibroblast cells likely contain the appropriate receptor for binding this drug. This same dose proved optimal for upregulating telomerase activity in the fibroblast cells an average of 57% relative to untreated cells (p value = 0.003). The upregulation appears to be specific for the sequence of aa in the 14aa drug since a“scrambled" peptide containing the same aa but in a different order showed no upregulation, even at doses 10-fold higher. Treatment of mice once per day or twice per day with the 14aa peptide was also found to upregulate telomerase activity in vivo in brain and heart. The activity was optimal at a 3.3 mg/kg dose for each aged organ, and was generally high in young organs. The activity observed in heart was a total surprise since heart cells are generally thought to be quiescent, and telomerase is usually associated with cell division, so perhaps telomerase has a function other than in cell division. The second part of the hypothesis tested whether treatment of fibroblast cells with the 14aa drug elongated (or prevented from shortening) telomere lengths in aged cells. A telomere length assay (TLA) based on a Southern hybridization approach using a telomere probe appeared to work well, since marker DNAs showed appropriate differences in their“telomere smears", and aged fibroblast cells showed shorter smears than young cells. However, no difference was observed between drug-treated versus vehicle-treated cells, even at the 10 ng/ml dose previously shown to strongly upregulate telomerase activity. So perhaps the upregulation of telomerase activity was not sufficient to provide a measurable increase in telomere lengths. Telomerase has been shown to extend the lifespan of virus-transformed human cells without showing any visible telomere lengthening (Blackburn et al, 1999), so perhaps telomerase can increase cell lifespan without increasing telomere lengths. To our knowledge, this is the only drug demonstrated to upregulate telomerase activity. Transforming cells with the viral T-antigen can upregulate telomerase, but T-antigen is not a therapeutic drug since it also causes cancer. Telomerase upregulation is known to occur during oncogenesis, but telomerase itself is not an oncogene since oncogenesis also requires the upregulation of oncogenes. Our lab previously showed this peptide does not upregulate the potent oncogene myc. If this proves to be the case for other oncogenes, using this 14aa drug to upregulate telomerase activity without activating oncogenes could prove extremely useful for helping prove telomerase is not an oncogene, and for extending cell lifespan.

Telomeres are specialized nucleoprotein structures found at the end of eukaryotic chromosomes. The telomere DNA in humans is composed of the sequence "5'-TTAGGG-3'" tandemly repeated in a stretch of 5-30kb of double stranded DNA. TTAGGG Repeat Factor 2 (TRF2) is a telomere DNA binding protein that has a critical role in telomere end protection. The current model for telomere protection by TRF2 is through its ability to remodel telomeres into looped higher-order structures, called the t-loop, which sequesters the end from DNA damage sensors. Since telomeres are known to be comprised of nucleosomal chromatin, it is important to determine how TRF2 binds to and affects the structure of nucleosomal arrays. The ability of TRF2 to bind to unusual DNA structures such as the t-loop and the single stranded/double (ss/ds) stranded telomere DNA junction may facilitate its binding to DNA in the form of nucleosomal arrays and promote higher-order chromatin structures. In this study, we have reconstituted a 2kb DNA fragment containing 550bp of telomere DNA into nucleosomal arrays and tested the binding of full-lengthTRF2 and four truncation mutants to telomeric nucleosomal arrays. Our data indicates that TRF2 and its truncation mutants bind to telomere nucleosomal arrays as well as it binds to telomere DNA. We used a novel electrophoretic technique, Analytical Agarose Gel Electrophoresis (AAGE), to measure changes in surface charge density, hydrodynamic radius, and conformational flexibility of DNA and nucleosomal arrays upon protein binding. Our results indicate that the C-terminal DNA binding Myb/SANT domain of TRF2 might be rearranging nucleosomal structure through either nucleosome sliding, unwrapping, or changing the arrangement of the linker DNA, while the N-terminal basic DNA binding region is causing nucleosomal arrays compaction. Instead of significant compaction, histone-free DNA undergoes DNA condensation and self-association. This activity is observed with the full-length protein and all regions of the protein, with the exception of TRF2-DBD, participate in the process. We speculate that the ability of TRF2-DBD to rearrange nucleosomal structure and N-terminal basic region to cause nucleosomal fiber compaction may allow TRF2 to promote t-loop formation in the context of chromatin. We propose that TRF2, possessing all the features, has a new role at telomeres as a chromatin architectural protein.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

published_or_final_version / Pathology / Master / Master of Philosophy

The overall survival for patients with glioblastoma multiforme (GBM) has not improved in two decades. A better understanding of the molecular basis for gliomagenesis would aid therapeutic advances. Recombinational based alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) distinct from telomerase, which serves as a prognostic factor in GBM. In this thesis, I have compared components of the p53 axis, namely p53, p21[WAF-1] and the paired box-containing transcription factors (PAX) PAX2, 5, and 8, with TMM in gliomas. Analysis of TP53 status in relation to TMM in 110 gliomas revealed that activation of ALT during tumorigenesis possibly requires loss of normal TP53 function (P < 0.0001). Overexpression of p21[WAF-1] was also found to correlate with telomerase-positive gliomas (P = 0.0002). Moreover, high p21[WAF-1] expression is a poor prognostic factor in patients under 56 years (P = 0.015). Telomere length (TL) was also found as a prognostic factor, such that short TL (< 5 kb) is a poor prognostic factor in the group without defined TMM ("None")(P = 0.0160). Pax2,5 and 8 belong to Group II of the PAX family. They are expressed at the midbrain-hindbrain boundary (MHB) and in the neural tube of the vertebrate embryo, whereas their expression levels are low in the adult brain. To explore their roles in glioma pathology, I analyzed mRNA levels in 54 gliomas and 16 established glioma cell lines. Increased levels of PAX8 mRNA were detected in 74.1% of gliomas and 62.5% of established glioma cell lines by real time PCR. Sixty-six percent of glioma specimens expressed high levels of active PAX2, 5, and 8 by immunohistochemistry. There were more males than females having high PAX2 expression (P = 0.0408). Suppression of PAX8 by small interfering RNA induced glioma cell death, independent of TP53 status. These findings identify PAX8 as a survival factor for GBM, and PAX2, 5, and 8 expression as contributing to the aggressive behavior of gliomas. The mRNA level of PAX8 showed a positive correlation with telomerase activity in glioma biopsies (r� = 0.75, P < 0.001). The relationship was explored and I found that PAX2 and PAX8 are able to activate the reporter constructs of both the catalytic subunit (hTERT) and the RNA component (hTR) of telomerase. PAX8 had a stronger effect than PAX2 on the activation of the hTERT and hTR promoters. By electrophoretic mobility shift assay, Western blotting and telomerase activity assay, I showed that PAX8 bound directly to hTERT and hTR promoters, and upregulated hTERT protein and telomerase activity. Moreover, gliomas carrying wild type TP53 had higher levels of PAX8 expression compared to those with mutant TP53 (P = 0.0075) suggesting that PAX8 is significant only in some GBMs during gliomagenesis. These results show that the oncofetal proteins, PAX2 and PAX8, may have roles in telomerase regulation. Taken together, molecular markers examined in this thesis suggest gliomas with different TMMs are derived from different pathways.

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3' assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. We show that K+ and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells. The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, a protein essential for telomere maintenance, is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3'-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5'-d(TTAGGG)4-3', to 5'-dTTAGGG(TTAGAG)2TTAGGG-3', reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexes. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2 G-quadruplex interactions, suggesting a novel mechanism for TRF2 recognition of the ds/ss junction of telomeres.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Hematopoietic stem cells; Umbilical cord blood. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 134-150).

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Bibliography: leaves 102-114.

The overall survival for patients with glioblastoma multiforme (GBM) has not improved in two decades. A better understanding of the molecular basis for gliomagenesis would aid therapeutic advances. Recombinational based alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) distinct from telomerase, which serves as a prognostic factor in GBM. In this thesis, I have compared components of the p53 axis, namely p53, p21[WAF-1] and the paired box-containing transcription factors (PAX) PAX2, 5, and 8, with TMM in gliomas. Analysis of TP53 status in relation to TMM in 110 gliomas revealed that activation of ALT during tumorigenesis possibly requires loss of normal TP53 function (P < 0.0001). Overexpression of p21[WAF-1] was also found to correlate with telomerase-positive gliomas (P = 0.0002). Moreover, high p21[WAF-1] expression is a poor prognostic factor in patients under 56 years (P = 0.015). Telomere length (TL) was also found as a prognostic factor, such that short TL (< 5 kb) is a poor prognostic factor in the group without defined TMM ("None")(P = 0.0160). Pax2,5 and 8 belong to Group II of the PAX family. They are expressed at the midbrain-hindbrain boundary (MHB) and in the neural tube of the vertebrate embryo, whereas their expression levels are low in the adult brain. To explore their roles in glioma pathology, I analyzed mRNA levels in 54 gliomas and 16 established glioma cell lines. Increased levels of PAX8 mRNA were detected in 74.1% of gliomas and 62.5% of established glioma cell lines by real time PCR. Sixty-six percent of glioma specimens expressed high levels of active PAX2, 5, and 8 by immunohistochemistry. There were more males than females having high PAX2 expression (P = 0.0408). Suppression of PAX8 by small interfering RNA induced glioma cell death, independent of TP53 status. These findings identify PAX8 as a survival factor for GBM, and PAX2, 5, and 8 expression as contributing to the aggressive behavior of gliomas. The mRNA level of PAX8 showed a positive correlation with telomerase activity in glioma biopsies (r� = 0.75, P < 0.001). The relationship was explored and I found that PAX2 and PAX8 are able to activate the reporter constructs of both the catalytic subunit (hTERT) and the RNA component (hTR) of telomerase. PAX8 had a stronger effect than PAX2 on the activation of the hTERT and hTR promoters. By electrophoretic mobility shift assay, Western blotting and telomerase activity assay, I showed that PAX8 bound directly to hTERT and hTR promoters, and upregulated hTERT protein and telomerase activity. Moreover, gliomas carrying wild type TP53 had higher levels of PAX8 expression compared to those with mutant TP53 (P = 0.0075) suggesting that PAX8 is significant only in some GBMs during gliomagenesis. These results show that the oncofetal proteins, PAX2 and PAX8, may have roles in telomerase regulation. Taken together, molecular markers examined in this thesis suggest gliomas with different TMMs are derived from different pathways.