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Jerry W Shay

Publications and source records attributed to Jerry W Shay.

At least 37 records · Page 2Linked to original sources
PubMed2005

IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression.

COX-2 overexpression and subsequent PGE(2) production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE(2) in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE(2) occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE(2) and inhibits angiogenesis.

Angiogenesis Inhibitors↗
PubMed2005

POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end.

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.

Alternative Splicing↗
PubMed2005

Meeting report: the role of telomeres and telomerase in cancer.

The role of telomeres and telomerase in cancer is an area of much recent interest. This conference sponsored by the American Association of Cancer Research provided a timely opportunity to bring together basic and clinical scientists interested in the field of telomeres and telomerase cancer biology. The meeting included over 250 attendees with 150 oral and poster presentations focused on understanding telomere and telomerase biology for the development of cancer therapeutics. The meeting chairpersons were Dr. Jerry W. Shay, University of Texas Southwestern Medical Center, Dallas, TX; Dr. Elizabeth H. Blackburn, University of California San Francisco, CA; and Dr. Maria A. Blasco, Spanish National Cancer Center, Madrid, Spain. The meeting provided an update on the field, pointing to areas in which our knowledge is deficient, and explored how the most promising areas may be advanced into translational research. This conference brought together cell and molecular biologists with clinicians interested in fundamental cancer mechanisms as they relate to telomeres and telomerase. The symposium consisted of formal presentations by prominent scientists working in these areas and by participants selected from submitted abstracts. In addition, there were two poster sessions. Whereas there were many basic research advances presented, the focus of this overview will be the areas of clinical advances.

Animals↗
PubMed2005

Telomere-end processing the terminal nucleotides of human chromosomes.

Mammalian telomeres end in single-stranded, G-rich 3' overhangs resulting from both the "end-replication problem" (the inability of DNA polymerase to replicate the very end of the telomeres) and postreplication processing. Telomeric G-rich overhangs are precisely defined in ciliates; the length and the terminal nucleotides are fixed. Human telomeres have very long overhangs that are heterogeneous in size (35-600 nt), indicating that their processing must differ in some respects from model organisms. We developed telomere-end ligation protocols that allowed us to identify the terminal nucleotides of both the C-rich and the G-rich telomere strands. Up to approximately 80% of the C-rich strands terminate in CCAATC-5', suggesting that after replication a nuclease with high specificity or constrained action acts on the C strand. In contrast, the G-terminal nucleotide was less precise than Tetrahymena and Euplotes but still had a bias that changed as a function of telomerase expression.

Animals↗
PubMed2005

Mechanism-based combination telomerase inhibition therapy.

Inhibition of telomerase is an exciting therapeutic target, since it is required for the long-term proliferation of most cancer cells but not present in most somatic cells. However, effective telomerase inhibitors have yet to be tested in clinical trials. In this issue of Cancer Cell, Seimiya and coworkers explore inhibiting tankyrase, an enzyme involved in making telomeres accessible to telomerase. Adding a partial inhibition of tankyrase to a partial inhibition of telomerase drove cancer cells into crisis and death. The combination of tankyrase and telomerase inhibitors may offer new opportunities for realizing the promise of telomerase inhibition therapy.

Humans↗
PubMed2005

Telomere biology in aging and cancer.

It is thought that a limited investment by the body in many types of maintenance and repair causes aging. Cell turnover is one mechanism of replacing damaged cells, and cell division thus contributes to good repair, but the number of times cells can divide is limited to form a barrier against cancer. Precancerous cells must divide many times to accumulate all of the mutations needed to become malignant. Limiting the number of times they can divide helps prevent cancer. The mechanism for counting cell divisions lies in structures at the ends of the chromosomes called telomeres that shorten with every division, eventually causing cell aging. This shortening can be prevented and cells immortalized using the enzyme telomerase, which can elongate telomeres. Immortalizing all of the cells in the body might increase repair but would remove the barrier to malignancy and would probably cause premature death from cancer in many cases, although the ability to immortalize cells opens up enormous opportunities for using normal cells for therapeutic purposes in localized areas. Eventually, once better controls and treatments for cancer are available, cellular rejuvenation by manipulating telomeres may reduce some of the physiological declines that accompany aging. Such treatments should increase health span, but because replicative aging represents only one of many processes that may contribute to overall human aging, modest increases in life span are expected at best.

Aging↗
PubMed2005

Function of AP-1 in transcription of the telomerase reverse transcriptase gene (TERT) in human and mouse cells.

The transcriptional regulation of the human telomerase catalytic subunit (hTERT) plays a critical role in telomerase activity. Approximately 200 bp of the proximal core promoter is responsible for basic hTERT expression; however, the function of the distal regulatory elements remains unclear. The transcription factor activator protein 1 (AP-1) is involved in cellular proliferation, differentiation, carcinogenesis, and apoptosis and is expressed broadly in both cancer and normal cells. There are several putative AP-1 sites in the hTERT promoter, but their functions are unknown. The present study examined the regulatory role of AP-1 in hTERT gene transcription. Overexpression of AP-1 leads to transcriptional suppression of hTERT in cancer cells. The combination of c-Fos and c-Jun or c-Fos and JunD strongly suppresses hTERT promoter activity in transient-expression analyses. The hTERT promoter region between -2000 and -378 is responsible for this function. Gel shift and supershift analyses, as well as ChIP, show binding of JunD and c-Jun on two putative AP-1 sites within this region. Mutations in the AP-1 binding sites rescued suppressions caused by AP-1, suggesting this is a direct regulation of the hTERT promoter. In contrast, there was no effect on mTERT expression or mTERT promoter activity by AP-1 overexpression in mouse fibroblasts. The species-specific function of AP-1 in TERT expression may in part help explain the difference in telomerase activity between normal human and mouse cells.

Animals↗
PubMed2005

Human telomeres maintain their overhang length at senescence.

Normal human cells in culture enter replicative senescence after a finite number of population doublings. The exact molecular mechanisms triggering the growth arrest are poorly understood. A recent report on the disappearance of the G-rich 3' telomeric overhang in senescent cells led to the hypothesis that loss of the 3' G-rich overhang is the molecular signal that triggers senescence. Here, we describe a quantitative assay to measure the length of the G-rich 3' telomeric overhangs from cultured cells. Using both this assay and the conventional nondenaturing hybridization assay for measuring G-rich overhangs, we show that normal human fibroblasts can maintain their overhangs at senescence. Furthermore, cells do not lose their overhangs when they bypass senescence after the inactivation of p53 and Rb. We thus conclude that a global reduction in overhang length is not the molecular signal that triggers replicative senescence.

Antigens, Polyomavirus Transforming↗
PubMed2005

Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.

PURPOSE: To develop and characterize a telomerase-immortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms involved in regulating human corneal epithelial cell differentiation. METHODS: Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air-lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti-Ki-67, anti-p16), stratification (phalloidin and anti-ZO-1), and differentiation (anti-K3, anti-BCL-2 and TUNEL labeling). RESULTS: hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 10. Air-lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO-1-stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5/K14) with K3-corneal keratin marker expression in long-term, air-lifted culture. Anti-BCL-2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL-2 expression in TUNEL-labeled surface epithelial cells. CONCLUSIONS: hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecular mechanisms regulating corneal epithelial cell differentiation and desquamation.

Blotting, Western↗
PubMed2005

Use of telomerase to create bioengineered tissues.

Telomeres are repetitive DNA (TTAGGG) elements at the ends of chromosomes. Telomerase is a ribonucleoprotein complex that catalyzes the addition of telomeric sequences to the ends of chromosomes. The catalytic protein component of telomerase (hTERT) is expressed only in specific germ line cells, proliferative stem cells of renewal tissues, and cancer cells. The expression of hTERT in normal cells reconstitutes telomerase activity and circumvents the induction of senescence. Telomeres shorten with each cell division, eventually leading to senescence (aging), due to incomplete lagging DNA strand synthesis and end-processing events, and because telomerase activity is not detected in most somatic tissues. There are specific tissues and locations in which replicative senescence likely contributes to the decline in human physiological function with increased age and with chronic illnesses. While expressing hTERT in cells results in the maintenance of telomere length and greatly extended life span, blocking replicative aging systemically would be predicted to increase the potential for tumor formation. However, there are many situations in which the transient rejuvenation of cells could be beneficial. Ectopic expression of hTERT has been shown to immortalize human skin keratinocytes, dermal fibroblasts, muscle satellite (stem), and vascular endothelial, myometrial, retinal-pigmented, and breast epithelial cells. In addition, human bronchial, corneal and skin cells expressing hTERT can be used to form organotypic (3D) cultures (bioengineered tissues) that express differentiation-specific proteins, demonstrating that hTERT by itself does not alter normal physiology. The production of hTERT-engineered tissues offers the possibility of producing tissues to treat a variety of chronic diseases and age-related medical conditions that are due to telomere-based replicative senescence.

Aging↗
PubMed2005

Lack of mutations of the human telomerase RNA gene (hTERC) in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS), considered a pre-leukemic state, has recently been categorized as a subset of bone marrow failure syndromes. Unlike other subtypes of bone marrow failure syndromes, such as aplastic anemia or dyskeratosis congenita, little is known about genetic alterations of human telomerase in MDS, despite the fact that immune cells from patients with MDS frequently exhibit telomere attrition.

Adult↗
PubMed2005

Marked telomere fluctuation of leukocytes during graft-versus-host disease in allogeneic stem cell transplantation.

Immune dysfunction after allogeneic stem cell transplantation (SCT) is closely associated with cell turnover of lymphocytes and homeostasis of hematopoietic stem cells. Telomeres, repetitive sequences (TTAGGG)n on the end of linear chromosomes, reflect the mitotic history of stem cells. Using telomere fluorescence in situ hybridization (FISH) and flow cytometry (flow-FISH), we measured telomere length in lymphocytes and neutrophils at various intervals to analyze the relationship between telomere length change and clinical features in 5 patients who underwent allogeneic bone marrow transplantation. During the first year after allogeneic stem cell transplantation, a marked fluctuation of telomere length in peripheral blood leukocytes was observed in all recipients, and in 3 patients there was a reduction of telomere length during chronic graft-versus-host disease (GvHD) or during post-transplant lymphoproliferative disorder. The reduction of telomere length during GvHD was evident in lymphocytes and neutrophils, but telomere length in neutrophils tended to recover earlier than that observed in lymphocytes. The rapid reduction of telomere length in leukocytes during GvHD was too extensive to be explained by the end-replication problem, suggesting the presence of a telomerically unstable hematopoietic condition after transplant in vivo.

Bone Marrow Transplantation↗
PubMed2004

Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins.

By expressing two genes (hTERT and Cdk4), we have developed a method to reproducibly generate continuously replicating human bronchial epithelial cell (HBEC) lines that provide a novel resource to study the molecular pathogenesis of lung cancer and the differentiation of bronchial epithelial cells. Twelve human bronchial epithelial biopsy specimens obtained from persons with and without lung cancer were placed into short-term culture and serially transfected with retroviral constructs containing cyclin-dependent kinase (Cdk) 4 and human telomerase reverse transcriptase (hTERT), resulting in continuously growing cultures. The order of introduction of Cdk4 and hTERT did not appear to be important; however, transfection of either gene alone did not result in immortalization. Although they could be cloned, the immortalized bronchial cells did not form colonies in soft agar or tumors in nude mice. The immortalized HBECs have epithelial morphology; express epithelial markers cytokeratins 7, 14, 17, and 19, the stem cell marker p63, and high levels of p16(INK4a); and have an intact p53 checkpoint pathway. Cytogenetic analysis and array comparative genomic hybridization profiling show immortalized HBECs to have duplication of parts of chromosomes 5 and 20. Microarray gene expression profiling demonstrates that the Cdk4/hTERT-immortalized bronchial cell lines clustered together and with nonimmortalized bronchial cells, distinct from lung cancer cell lines. We also immortalized several parental cultures with viral oncoproteins human papilloma virus type 16 E6/E7 with and without hTERT, and these cells exhibited loss of the p53 checkpoint and significantly different gene expression profiles compared with Cdk4/hTERT-immortalized HBECs. These HBEC lines are a valuable new tool for studying of the pathogenesis of lung cancer.

Cell Growth Processes↗
PubMed2004

Senescence and immortalization: role of telomeres and telomerase.

Telomere dynamics are a critical component of both aging and cancer. Telomeres progressively shorten in almost all dividing cells and most human cells do not express or maintain sufficient telomerase activity to fully maintain telomeres. There is accumulating evidence that when only a few telomeres are short, they form end-associations, leading to a DNA damage signal resulting in replicative senescence (a cellular growth arrest, also called the M1 stage). In the absence of cell-cycle checkpoint pathways (e.g. p53 and or p16/Rb), cells bypass M1 senescence and telomeres continue to shorten eventually resulting in crisis (also called the M2 stage). M2 is characterized by many 'uncapped' chromosome ends, end-fusions, chromosome breakage fusion-bridge cycles, mitotic catastrophe and a high fraction of apoptotic cells. In a rare M2 cell, telomerase (a cellular reverse transcriptase) can be reactivated or up-regulated, resulting in indefinite cell proliferation. This cellular immortalization is a potentially rate-limiting step in carcinogenesis that is important for the continuing evolution of most advanced cancers. In this perspective we will present our views on the evidence for telomere dysfunction in aging and in cancer progression. We will argue that telomere shortening in the absence of other alterations may be a potent tumor suppressor mechanism and we will discuss the evidence for and against the major molecular mechanisms proposed to initiate replicative senescence.

Aging↗
PubMed2004

Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes.

Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited. We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes. In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome. This finding contrasts with the coordinated timing of both ends of each chromosome in yeast. Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later. Finally, replication timing of some but not all telomeres was influenced by telomere length. Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action. The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci.

Animals↗
PubMed2004

Disparate effects of telomere attrition on gene expression during replicative senescence of human mammary epithelial cells cultured under different conditions.

Telomere shortening in populations of human mammary epithelial cells (HMECs) that survive early replicative arrest (M0) by the inactivation of p16(INK4A) during cell culture on plastic dishes leads to a state of permanent replicative arrest termed senescence. While culture of HMECs on feeder layers abrogates M0 and p16(INK4A) inactivation, progressive telomere attrition in these cells also eventually results in permanent replicative arrest. Expression of telomerase prevents both senescence on plastic (S-P) and senescence on feeder layers (S-FL) in HMECs, as it does also in cultured primary human fibroblasts. We report here that the gene expression profiles of senescence in HMECs of the same lineage maintained under different culture conditions showed surprisingly little commonality. Moreover, neither of these senescence-associated profiles in HMECs resembles the profile for senescence in human fibroblasts. These results indicate that senescence-associated alterations in gene expression resulting from telomere attrition are affected by culture conditions as well as by cell origins, and argue that replicative senescence at the molecular level is a diverse rather than unique cellular process.

Adult↗
PubMed2004

Different roles for caveolin-1 in the development of non-small cell lung cancer versus small cell lung cancer.

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n = 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n = 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n = 15) and 9% of NSCLCs (n = 11), whereas 5-aza-2'deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.

Carcinoma, Non-Small-Cell Lung↗