Browsing by Subject "Telomerase"
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Item The Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus(2011-02-01) Wilson, Kimberly Anne; Porteus, Matthew H.The ability to direct hTERT expression through genetic control or tunable regulatory factors would advance our understanding of the transcriptional regulation of hTERT, and also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and zinc finger nucleases (ZFNs) to target sequences at the hTERT promoter. We first explored expansions to the repertoire of sites that can be targeted by ZFNs and modifications of ZFN architecture to accommodate such sites. A ZFN is made of a zinc-finger DNA binding domain (ZFP) linked to the FokI nuclease domain by a short amino acid “inter-domain linker”. The general sequence motif of a ZFN target is 5’-(ZFN site1)-(6 bp spacer)-(ZFN site2)-3’ and each half-site is 5’-GNNGNNGNN-3’. Variations of this motif come in the forms of variable spacer lengths, extra basepairs in-between triplets, and the inclusion of non-GNN triplets. To explore these types of target sites, we created ZFN variants that contained different inter-domain linkers, lengthened inter-finger linkers, and DNA binding domains created through hybridizing the modular assembly and OPEN methodologies. We show that through altering ZFN architecture, target sites with 5-7-bp spacers and those with ANN, CNN, and TNN triplets can be efficiently recognized and cut by ZFNs. We then generated new ZFPs to five ZFN target sites with 5- or 6-bp spacers in the hTERT locus based on those findings and made ZFTFs by linking the ZFPs to the VP16 transcriptional activation domain. We were able to identify several active ZFTFs that demonstrate a dose-dependent response. The same ZFPs were also converted into ZFNs and screened in combinatorial pairs in cell-based single-strand annealing assays and gene targeting assays. These screening strategies have pinpointed several ZFN pairs that may be useful in genomic editing of the hTERT locus. Our findings provide guidelines for modifying ZFP architecture to a wider array of potential target sites for use in developing ZFTFs and ZFNs at the hTERT promoter, which may be applicable towards inheritable, telomerase-based diseases and answering basic science questions about hTERT transcriptional regulation.Item Genetics of pulmonary fibrosis: telomerase & surfactant dysfunction(2009-04-24) Garcia, Christine KimItem Identifying, Characterizing and Inhibiting the Telomerase Regulatory Network(2015-04-07) Holohan, Brody Christopher; Corey, David R.; Brekken, Rolf A.; Cobb, Melanie H.; Shay, Jerry W.; Wright, Woodring E.Telomeres, which are structures that cap the ends of linear chromosomes are maintained by telomerase, a reverse transcriptase. Telomere length limits the self-renewal capacity for telomerase negative cells, and nearly all tumors circumvent this limitation through telomerase expression; as such, telomerase is an attractive target for cancer therapy. In order to identify new targets for anti-telomerase therapy, I demonstrate that a number of candidate genes are required for telomere maintenance in vitro through shRNA-mediated knockdown and telomere length analysis. Further, I show that Perifosine, a drug identified upstream of a number of the candidates can act as a telomerase inhibitor in a majority of cell lines evaluated in vitro as well as induce shortening of the shortest telomeres in tumors from human patients treated with Perifosine in a phase II clinical trial. Additionally, I identify a trans-generational trend in telomere length at birth in human populations that may bias estimates of telomere shortening rate that has public health implications. Lastly, using data from a large twin study, I have identified a network of genes that regulate the rate of telomere shortening in humans that may be used to clarify the association between telomere length, aging and age-related disease.Item Preclinical Studies of Telomerase Inhibitor Imetelstat in Non-small Cell Lung Cancer(2013-04-18) Frink, Robin Elizabeth; Shay, Jerry W.; Minna, John D.; Brekken, Rolf A.; White, Michael A.Telomerase is expressed in ~90% of all cancers but is not expressed in most somatic cells making it an attractive target for cancer therapy. Telomerase has two essential components, a reverse transcriptase (hTERT) and an RNA template (hTR or hTERC). The RNA template is used by the reverse transcriptase to add the TTAGGG hexameric repeats to elongate telomeres and compensate for the loss of telomeres each cell division caused by the end replication problem. Imetelstat is an oligonucleotide designed to bind the hTR telomerase template component and inhibit telomerase leading to progressive telomere shortening associated senescence or cell death. The work described here examined the efficacy of imetelstat in a panel of non-small cell lung cancer (NSCLC) cell lines. Imetelstat was tested in a short-term liquid colony formation assay, a 5-day drug response assay, and long-term continuous treatment in vitro and in vivo. The panel of over 70 NSCLC cell lines used for this study ranged from 1.5 kb to 20 kb in average telomere length as well as a wide range in telomerase activity, growth rate, NSCLC subtype, and oncogenotype providing a broad basis for comparison of response to imetelstat. All cell lines tested showed inhibition to telomerase with imetelstat treatment. In liquid colony formation, a wide range of response to 3 uM imetelstat was seen. Colony formation inhibition ranged from 96% inhibition in HCC44 to H441 which shows a greater than 2-fold increase in colony forming ability in the presence of imetelstat, though not statistically significant. 1 uM imetelstat was given long-term in 8 different cell lines and telomerase inhibition and telomere shortening was observed in all cases. Continuous treatment led to a reduction in growth rate and eventual cell death in all but two cell lines and imetelstat response time varied among the cell lines based on initial telomere length and growth rate. Calu-3 had the fastest response time (11 days or as few as 2 population doublings) to see a change in growth rate and 32 population doublings for cell death in all cells. Calu-3, H1648 and HCC827 all showed reduced growth rate in the presence of imetelstat in vivo as well. Imetelstat inhibits telomerase, shortens telomeres and leads to cell death in many NSCLC cell lines both in vitro and in vivo supporting the idea of telomerase inhibition for the treatment of lung cancers.Item The Regulation of hTERT by Alternative Splicing(2017-07-27) Yuan, Laura Yu; Yu, Hongtao; Shay, Jerry W.; Wright, Woodring E.; Fontoura, Beatriz; Cobb, Melanie H.Telomeres are non-coding DNA hexameric repeats (TTAGGG in mammals) located at the ends of linear chromosomes that, along with their associated proteins, protect against the loss of genomic material during cell division and prevent the recognition of chromosome ends as double-strand breaks. Human telomeres shorten with continued cell proliferation but are maintained by human telomerase reverse transcriptase (hTERT), an enzyme that synthesizes telomeric repeats using an RNA template. The regulation of telomerase has been studied at many levels--from epigenetic and transcriptional regulation to the alternative splicing of hTERT pre-mRNA into catalytically inactive splice variants. Our hypothesis is that if the regulation of telomerase reverse transcriptase splicing is necessary for telomere length homeostasis, altering telomerase splicing to decrease the production of full-length hTERT and will result in decreased telomerase activity and subsequently telomere shortening. We focused our efforts on identifying splicing factors are involved in hTERT splicing and characterized the role of two splicing factors, NOVA1 and PTBP1, in regulation of hTERT splicing in non-small cell lung cancer cells. We show that these splicing factors are important for full-length hTERT, telomerase activity and telomere length maintenance in vitro. Xenograft studies suggest that NOVA1 is also important for tumor growth in vivo. We found that these splicing factors are able to directly interact with hTERT in a region our group previously identified to be important for hTERT splicing. Altogether, our work suggests that splicing factors are important for hTERT regulation and telomerase activity in cancer. Since telomerase activity is undetectable in most somatic tissues but is increased in the vast majority of human cancers, dependence on telomerase represents a key vulnerability in cancer tissues which could be therapeutically targetable.Item Regulation of Human Telomerase Alternative Splicing(2014-06-10) Wong, Sze; Fontoura, Beatriz; Wright, Woodring E.; Shay, Jerry W.; Conrad, Nicholas; Martinez, ElisabethTelomerase adds TTAGGG repeats onto chromosome ends (telomeres). Since telomerase is expressed in ~90% of all human cancer cells while being absent in most somatic tissues, telomerase is an almost universal cancer therapeutic target. Yet, the clinical application of an effective telomerase inhibitor is still lacking. The pre-mRNA of the catalytic subunit of human telomerase (hTERT) may be alternatively spliced into 22 different isoforms. Only a small fraction of hTERT transcripts are spliced into the full length isoform, the form capable of being translated into function hTERT with reverse transcriptase activity. If telomerase activity is partially regulated at the level of RNA splicing, then telomerase activity may be modulated to increase the amount of nonfunctional transcripts and decrease the amount of full-length hTERT and this would be a novel anti-cancer therapeutic approach. A hTERT minigene was created to understand the cis-regulatory elements governing hTERT splicing. A 1.1kb region of 38 bp repeats (block 6) ~2 kb from the exon/intron junctions is essential for the exclusion of exons 7 and 8. Block 6 repeats suggested that RNA:RNA pairing may regulate splicing of hTERT. Mutations within the repeat sequence that abolish exon skipping were corrected by compensatory mutations in the pre-mRNA. To identify trans-acting regulators of hTERT alternative splicing, the minigene was modified into a dual-luciferase reporter for a selected 528 RNA-binding proteins/splicing factors siRNA screen. Our initial validation focused on 45 factors with enzymatic activity and resulted in CDC-Like Kinase 1 (CLK-1) as a potential hTERT splicing modulator. Knock-down of CLK-1 altered hTERT splicing, resulting in reduction in telomerase activity and telomere shortening. CLK-1 belongs to the Clk family of dual-specificity nuclear kinases that can auto-phosphorylate SR proteins. TG003, a chemical CLK-1/4 inhibitor, results in less full length hTERT splicing and a decrease in cancer cell telomerase activity. This approach provides a platform to identify additional hTERT splicing regulators that may be suitable drug targets to increase or decrease telomerase activity. Altogether, these results demonstrate the potential of manipulating hTERT splicing as a target for both chemotherapy and regenerative medicine.Item Screening and surveillance of pancreas neoplasia: Can we and should we?(2008-07-11) Lara, Luis F.Item [Southwestern News](1995-02-28) Lyons, MorganItem [Southwestern News](2001-01-03) Cofer, BrianItem [Southwestern News](1999-12-07) Stieglitz, HeatherItem [Southwestern News](1994-12-23) Lyons, MorganItem [Southwestern News](1999-05-13) Steeves, Susan A.Item [Southwestern News](2005-09-01) McKenzie, AlineItem [Southwestern News](2003-09-15) Maier, ScottItem [Southwestern News](1998-12-29) Stieglitz, HeatherItem [Southwestern News](1996-05-01) Lyons, MorganItem Telomere and Telomerase Dynamics in Human T Lymphocytes(2017-06-28) Huang, Ejun; Zhu, Hao; Shay, Jerry W.; Wright, Woodring E.; Burma, Sandeep; Brekken, Rolf A.Advanced cancer is characterized by a phenotype that permits cells to divide indefinitely while cellular aging is characterized by cells ceasing to divide. It is believed that senescence may have evolved as an anti-cancer protection mechanism in long-lived species such as humans. Therefore, cancer and aging are essentially opposite ends of the same problem. Telomeres are the cellular aging-clock that determines a cell's capability of proliferation. When normal cells divide, telomeres gets progressively shorter. When telomeres reach a critical short length, cells stop dividing. To compensate for the loss of telomeres, telomerase is a ribonucleoprotein enzyme complex that elongates telomeres, but its expression is restricted to certain subsets of cell types. Investigating how telomerase is regulated in normal cells may provide insights and new approaches to block telomerase activity in cancer cells or to re-activate telomerase in aging cells. Immune cells play central roles in defending humans from pathogens, infections, and malignant cells. Immunotherapy has shown great potential in cancer treatment and has gained increasing public attention. Thus, a better understanding of how to increase the proliferation efficiency of normal immune cells, especially in older individuals whose immune stem-like cells may becoming less efficient is important. Due to the accessibility of blood from volunteers, immune cells are a model system to investigate healthy aging. For my doctoral research, I decided to study telomeres and telomerase dynamics in immune cells. Using novel techniques in telomere length analysis and telomerase activity measurements developed in our lab, I discovered that a subset of CD28+ T cells show robust telomerase activity and the ability to maintain telomere lengths during stimulated cell proliferation. In a centenarian study, T cells from a subset of centenarians showed stronger telomerase activation compared with many younger individuals. RNA-seq analysis revealed distinct differences between high performance centenarians and other age groups. Finally, we developed novel techniques using ddPCR for quantitating mitochondrial DNA numbers per cell and validated the differences we found in centenarian samples. Altogether, my doctoral project has added value to our current knowledge of how telomerase is regulated during immune responses and revealed its importance in longevity studies.Item Telomere Length Studies in Human Cancer Cells(2011-08-10) Buseman, Christen Marie; Shay, Jerry W.Telomeres consist of repetitive DNA sequences and their associated binding proteins, and serve to protect linear chromosome ends from being recognized as double stranded breaks in need of repair. The telomeres of most normal diploid cells shorten with every cell division until they reach a critically short length, at which time the cells undergo senescence or apoptosis. Cancer cells which have the ability to divide indefinitely must prevent their telomeres from becoming critically short, and the majority of cancer cells achieve this by upregulating telomerase. Maintaining telomere length involves regulating the dynamic between telomere shortening and telomere elongation. However, there are still many aspects of this dynamic regulatory process that are unknown. Many methods of telomere length assessment have been developed that utilize a variety of molecular techniques, but a major shortcoming of these methods is that they lack the ability to detect single short telomeres that are thought to trigger replicative senescence. Thus, the objective of this work was to develop an assay, named Universal STELA, which can generate an accurate distribution of telomere lengths on all chromosomes and allow for the study of the shortest telomeres in experimental settings. Universal STELA was first used to determine if cancer stem cells are susceptible to telomerase inhibition therapy because they have a larger fraction of shorter telomeres than non cancer stem cells. Cancer stem cells are thought to contribute to cancer metastasis and recurrence, and therapies like telomerase inhibition that target cancer stem cells may lead to more durable treatment outcomes. Universal STELA was next used to investigate regulation of telomerase action. C- and G-STELA were used to determine that telomerase activity is coupled to telomere replication, while C-strand fill-in is delayed until S/G2. Universal STELA was used to compare the rate of elongation of short, average and long telomeres when telomeres are shorter than their maintenance length.Item Telomere Specific Homologous Recombination in the Alternative Lengthening of Telomeres(2013-01-16) Whitington, Aric J.; Shay, Jerry W.; Wright, Woodring E.Over twenty years have passed since the discovery of telomerase-independent telomere maintenance, yet the precise details of the ALT mechanism remain a mystery. A growing body of evidence suggests that ALT cells maintain telomeres by homologous recombination (Reddel 2003 for Review). Groundbreaking work by Oliver Bechter demonstrated that ALT cells and telomerase-positive cells show no difference in the rate of general HR. This study fundamentally shaped our current concept of the ALT mechanism, implying that it involves preferential recombination of telomeric repeats. Since ALT seems to require proteins involved in normal HR, it follows that this telomeric recombination must be suppressed in telomerase positive or normal cells. However, to date there has been no direct evidence to support this hypothesis. Seeking to investigate the rates of telomere specific recombination, previous work in the Shay and Wright lab utilized the Tel-Tel vector. However, due to the method of integration only a limited number of clones could be analyzed and no statistically significant conclusions could be made. My work has focused on remedying this limitation. I have developed a strategy for integrating the Tel-Tel vector into a variety of host cell lines and generating a large number of distinct clones for each line. Using this strategy I was able to measure the average rates of telomeric HR for each cell line and provide the first direct evidence that ALT cells show distinctly elevated levels of increased telomere-specific HR. Additionally, I have constructed a control vector which functions in the same manner as Tel-Tel, differing only in that the telomeric repeats are replaced by non-telomeric repeat sequence. Using this vector (referred to as Mut-Mut) and the same incorporation method, I have demonstrated that there is no significant difference in the rates of general HR in ALT and telomerase positive cells. Finally, I used this novel ALT reporter as well as previously established methods to investigate some proteins that may play a central role in the ALT mechanism.Item [UT Southwestern Medical Center News](2007-04-24) McKenzie, Aline