Biomedical subjects

Jerry W Shay

Publications and source records attributed to Jerry W Shay.

At least 73 records · Page 4Linked to original sources

PubMed2003

BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination.

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.

Active Transport, Cell Nucleus↗

PubMed2003

Adenoviral-mediated retinoblastoma 94 produces rapid telomere erosion, chromosomal crisis, and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells.

Retinoblastoma (RB)94, which lacks the NH(2)-terminal 112 amino acid residues of the full-length RB protein (RB110), is a more potent tumor and growth suppressor than RB110. In this study, Ad-RB94, but not Ad-RB110, produced marked growth inhibition, cytotoxicity, caspase-dependent apoptosis, and G(2)-M block in the human RB-negative, telomerase-positive bladder cancer cell line UM-UC14. This effect was completely inhibited by pretreatment with caspase inhibitors (P < 0.0001). Similar results were seen in RB-positive and other RB-negative bladder cancer cell lines. Ad-RB94 produced rapid telomere length shortening and loss of telomere signal, which was associated with polyploidy and chromosomal aberrations (P < 0.001). Ad-RB94, however, showed no cytotoxicity to telomerase-negative human normal urothelium cells but was highly cytotoxic to telomerase-positive human E6 and E7 immortalized urothelial cells (P < 0.0001). In addition, telomerase-negative cells, which maintain their telomere length through an alternative lengthening of telomeres DNA recombination pathway, showed no cytotoxicity to RB94. These results suggest that the induction of rapid telomere erosion and chromosomal crisis by RB94 in telomerase-positive cancer and in telomerase-expressing immortalized human cells is a major factor in its selective and potent tumor suppression and cytotoxic activity. The lack of cytotoxicity to normal cells should also provide a high therapeutic index when used in gene therapy protocols for the treatment of bladder and other cancers.

Adenoviridae↗

PubMed2003

Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells.

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16(INK4a)-associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16(INK4a) and milder upregulation of p53 and p21(WAF1), which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.

Adult↗

PubMed2003

hTERT associates with human telomeres and enhances genomic stability and DNA repair.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

DNA Damage↗

PubMed2003

Differential gene expression profiles between neuroblastomas with high telomerase activity and low telomerase activity.

PURPOSE: Neuroblastoma shows remarkable heterogeneity, resulting in favorable or unfavorable outcomes. The authors previously reported that high levels of telomerase activity correlated with unfavorable tumors, and telomere shortening without telomerase activity correlated with tumor regression. To identify the genes responsible for the biological characteristics of neuroblastoma, the authors applied microarray techniques. METHODS: Mixtures of total RNAs extracted from 10 neuroblastoma tissues with high and 10 with low telomerase activity were labeled with Cy3 or Cy5 by reverse transcriptase reaction, respectively, and hybridized with our original microarrays prepared with a cDNA library of human fetal brain. RESULTS: Expression of 63 genes including MYCN, hTERT, HSPCA, and cell cycle-related proteins was found to be increased in neuroblastomas with high telomerase activity, whereas another 46 genes, including neural differentiating genes, were detected as highly expressed in tumors with low telomerase activity. CONCLUSIONS: The expression profiling data indicated clusters of upregulated and downregulated genes tumors with high or low telomerase activity. The genes involved in differentiation/growth arrest of tumor cells were closely related to low telomerase activity in neuroblastoma. The genes overexpressed in tumors with high telomerase activity, including cell-cycle-related genes and transcriptional factors, would be candidates for novel prognosis-predicting factors as well as new therapeutic targets in aggressive neuroblastomas.

DNA, Neoplasm↗

PubMed2003

Developmental differences in the immortalization of lung fibroblasts by telomerase.

The role of ambient (21%) and physiological oxygen (2-5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan-shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long-term culture requirements for the immortalization of WI-38 fetal lung fibroblasts included supplementation with N-(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end-replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes.

Adult↗

PubMed2003

Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts.

PURPOSE: The purpose of this study was to determine whether TGFbeta induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines. METHODS: Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFbeta in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of alpha-smooth muscle actin (alpha-SMA). RESULTS: Treatment of cultured keratocytes with TGFbeta (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of alpha-SMA, which was blocked by the addition of RGD-containing peptides (100 microM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in alpha-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor. CONCLUSIONS: Induction of myofibroblast differentiation by TGFbeta in cultured human keratocytes and hTERT corneal fibroblasts occurs through a similar signal transduction pathway to that previously identified in the rabbit, which involves an autocrine PDGF feedback loop.

Actins↗

PubMed2002

Human telomerase can immortalize Indian muntjac cells.

The mechanisms of replicative senescence by telomere shortening are not fully understood. The Indian muntjac has the fewest chromosomes of all mammals, greatly simplifying the analysis of each telomere over time. In this study, telomere shortening was observed throughout the life span of cultured normal muntjac cells by quantitative fluorescence in situ hybridization and terminal restriction fragment analysis. Ectopic expression of the human telomerase catalytic subunit in these cells reconstituted telomerase activity, extended telomere lengths, and immortalized the cells, demonstrating that the Indian muntjac cells can serve as a telomere-based replicative senescence model for human cells. In one strain, two chromosome ends had significantly shorter telomeres than the other ends, which led to a variety of chromosome abnormalities. Near senescence, additional ends became telomere signal free, and chromosome aberrancies increased dramatically. Interstitial telomere sequences coincided with fragile sites, suggesting that these remnants of chromosome fusion events might contribute to genome instability. One SV40-immortalized cell line lacked telomerase, and its genetic instability was corrected by the ectopic expression of telomerase, confirming that too-short telomeres were the source of abnormalities. Indian muntjac cells provide an excellent system for understanding the mechanism of replicative senescence and the role of telomerase in the elongation of individual telomeres.

Animals↗

PubMed2002

p16(INK4a) inactivation is not required to immortalize human mammary epithelial cells.

Using standard culture conditions, primary human mammary epithelial cells (HMECs) undergo a premature, transient growth arrest termed M0 (mortality stage 0) after 10-15 population doublings in vitro. It has been reported that emergence from this growth arrest by the abrogation of p16(INK4a), a cyclin-dependent kinase inhibitor, and expression of the catalytic component of human telomerase (hTERT) are necessary for HMEC immortalization. Here we show that primary HMECs, grown on feeder layers, do not undergo this growth arrest and can be immortalized without abrogating p16. These findings support the concept that the so-called M0 stage represents a cell culture stress-induced growth arrest and that hTERT is sufficient to immortalize HMECs when cultured under adequate conditions.

Breast↗

PubMed2002

Human Ku70/80 associates physically with telomerase through interaction with hTERT.

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.

Antigens, Nuclear↗

PubMed2002

Oligonucleotide N3'-->P5' phosphoramidates as efficient telomerase inhibitors.

Human telomerase is a unique reverse transcriptase that is expressed in multiple cancers, but not in the vast majority of normal cells. The enzyme is responsible for telomere protection and maintenance, and supports the proliferative immortality of cancer cells. Thus, it has been proposed that the specific inhibition of telomerase activity in tumors might have significant and beneficial therapeutic effects. To this goal we have designed, synthesized, and evaluated several oligonucleotide N3'-->P5' phosphoramidates as telomerase inhibitors. These oligonucleotides are complementary to the template region of the RNA domain of telomerase (hTR). The prepared compounds were evaluated in HME50-5E breast epithelial cells, where their effects on telomerase activity were determined using a cell-based telomerase (TRAP) assay at 24 as well as 72 h after exposure to compounds. The oligo-N3'-->P5' phosphoramidate inhibited telomerase activity in cells in the presence of the cellular up-take enhancer (FuGENE6) in a dose- and sequence-dependent manner, with IC(50) values of approximately 1 nM. Inhibition of telomerase activity by this compound without the lipid carrier was not efficient. However, the isosequential oligonucleotide N3'-->P5' thio-phosphoramidate was able to inhibit telomerase activity with or without lipid carriers at nM, or low-microM concentrations, respectively. This inhibition of telomerase activity in HME50-5E cells by the oligonucleotide thio-phosphoramidates was also sequence specific. Long-term treatment of the cells with 0.5 microM of FuGENE6 formulated 13-mer thio-phosphoramidates, fully complementary to hTR, resulted in gradual telomere shortening, followed by cellular senescence and apoptosis, as would be predicted for a telomerase inhibitor. The mismatched control compound had no effect on cell proliferation. The results suggest that the oligonucleotide N3'-->P5' phosphoramidates, and particularly thio-phosphoramidates, might be further developed as selective anti-telomerase reagents.

Amides↗

PubMed2002

A model for heterogeneous nuclear ribonucleoproteins in telomere and telomerase regulation.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of nucleic acid binding proteins that are often found in, but not restricted to, the 40S-ribonucleoprotein particle. Subsets of hnRNPs are strictly nuclear while others shuttle between the nucleus and cytoplasm. Members of the hnRNP family have been implicated to have roles in many aspects of mRNA maturation/turnover and in telomere and telomerase regulation. Telomeres are repetitive DNA elements mainly found at the ends of human chromosomes. In most normal cells, telomeres shorten with each cell division. Telomere shortening can be compensated for by a ribonucleoprotein complex, called telomerase. Telomerase, consisting of an integral RNA and catalytic protein component as well as several auxiliary factors, extends the 3'-G-rich strand of the ends of the telomeres. Here we present new data and describe a model that implicates the telomerase bound hnRNPs in promoting telomere access by interacting with telomeres. Telomere bound hnRNPs include hnRNP A1, A2-B1, D and E and telomerase bound hnRNPs including hnRNPA1 C1/C2 and D. The telomere and telomerase bound hnRNPs may prove to be good targets for regulating telomere length.

Animals↗

PubMed2002

Telomere biology and cellular aging in nonhuman primate cells.

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.

Animals↗

PubMed2002

Telomerase regulation: not just flipping the switch.

Significant research efforts have focused on the transcriptional regulation of the catalytic protein component of telomerase, hTERT (human telomerase reverse transcriptase). However, increasing evidence indicates that regulation of hTERT is complex and not as simple as just flipping a switch. Although much remains to be learned about the regulation of telomerase, generation of full-length hTERT protein is likely to be the limiting step in the formation of active telomerase. Regulation of hTERT transcription, transport to the nucleus, assembly of the telomerase holoenzyme, recruitment of telomerase to the telomere, and the role of post-translational modifications of hTERT protein are areas of intense investigation. It is hoped that shedding light on these diverse areas of telomerase regulation will allow us to manipulate telomerase activity for therapeutic purposes.

Active Transport, Cell Nucleus↗

PubMed2002

Telomerase in cancer and aging.

The telomere-telomerase hypothesis is the science of cellular aging (senescence) and cancer. The ends of chromosomes, telomeres, count the number of divisions a cell can undergo before entering permanent growth arrest. As divisions are being counted, events occur on the cellular and molecular level, which may either delay or hasten this arrest. As humans age, a particular concern is the accumulation of events that lead to the progression of cancer. Telomerase is a mechanism that most normal cells do not possess, but almost all cancer cells acquire, to overcome their mortality and extend their lifespan. This review aims to provide a comprehensive understanding of the role of telomerase in cancer development, progression, diagnosis, and in the future, treatment. The ultimate goal of telomerase research is to use our understanding to develop anti-telomerase therapies, an almost universal tumor target.

Aging↗

PubMed2002

Telomerase: a target for cancer therapeutics.

The continuous growth of advanced malignancies almost universally correlates with the reactivation of telomerase. While there is still a great deal of basic and applied research to be done, telomerase remains a very attractive novel target for cancer therapeutics. In this review, we will discuss the challenges and the pros and cons of the most promising antitelomerase approaches currently being investigated.

Animals↗

PubMed2002

Historical claims and current interpretations of replicative aging.

Replicative aging is the process by which most normal human cells "count" the number of times they have divided, eventually undergoing a growth arrest termed cellular senescence. This process is dependent on the shortening of telomeres, repeated sequences at the ends of the chromosomes. The loss of telomeric sequences with each cell division eventually induces a growth arrest that has a similar phenotype to that of cells stressed by inadequate culture or other conditions. Experiments over the past several years have identified species in which replicative aging does not occur and many examples in which a failure to proliferate has been misinterpreted as replicative senescence. Insights from these studies now permit a reevaluation of much of the seemingly contradictory data concerning replicative aging. There are good theoretical reasons for believing a limited proliferative capacity contributes to declining tissue homeostasis with increasing age. Although the presence of telomere shortening provides strong circumstantial evidence that replicative aging is occurring in vivo, thus far there is only very limited direct evidence for actual physiological effects of replicative aging.

Cell Differentiation↗

PubMed2002

Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again.

Telomerase is a ribonucleoprotein complex that catalyses the addition of TTAGGG repeats onto telomeres, repetitive DNA structures found at the ends of linear chromosomes. The majority of human somatic tissues do not display telomerase activity and undergo telomeric shortening with consecutive divisions. This telomeric shortening results in replicative senescence in vitro and likely in vivo. Telomerase activity is present in the vast majority of tumors, preventing telomeric shortening and thereby enabling indefinite cell divisions. Telomerase activity is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards. However, regulated telomerase activity is seen in basal/stem cell compartments of highly regenerative tissues, such as those of the immune system, skin, and intestine. Avian species display telomerase repression and telomeric shortening similar to that seen in humans. However, rodents retain telomerase-competency throughout their lifespan and have not been shown to display division-dependent telomere shortening. The regulation of telomerase activity in plants is less well understood, although early indications suggest ubiquitous competency. The aim of this review is to present current data regarding developmental regulation of telomerase in humans, mice, chickens and flowering plants. Differentiation, quiescence and telomerase activity regulation will then be addressed in three human representative tissue systems; blood, skin, and intestine. We will also highlight similarities, differences and misconceptions in the developing field of telomere and telomerase biology.

Animals↗