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Biomedical subjects

Jerry W Shay

Publications and source records attributed to Jerry W Shay.

At least 55 records · Page 3Linked to original sources
PubMed2004

Cell-type-specific responses to chemotherapeutics in breast cancer.

Recent microarray studies have identified distinct subtypes of breast tumors that arise from different cell types and that show statistically significant differences in patient outcome. To gain insight into these differences, we identified in vitro and in vivo changes in gene expression induced by chemotherapeutics. We treated two cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and two lines derived from luminal epithelium (MCF-7 and ZR-75-1) with chemotherapeutics used in the treatment of breast cancer and assayed for changes in gene expression using DNA microarrays. Treatment doses for doxorubicin and 5-fluorouracil were selected to cause comparable cytotoxicity across all four cell lines. The dominant expression response in each of the cell lines was a general stress response; however, distinct expression patterns were observed. Both cell types induced DNA damage-response genes such as p21(waf1), but the response in the luminal cells showed higher fold changes and included more p53-regulated genes. Luminal cell lines repressed a large number of cell cycle-regulated genes and other genes involved in cellular proliferation, whereas the basal cell lines did not. Instead, the basal cell lines repressed genes that were involved in differentiation. These in vitro responses were compared with expression responses in breast tumors sampled before and after treatment with doxorubicin or 5-fluorouracil/mitomycin C. The in vivo data corroborated the cell-type-specific responses to chemotherapeutics observed in vitro, including the induction of p21(waf1). Similarities between in vivo and in vitro responses help to identify important response mechanisms to chemotherapeutics.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Does a sentinel or a subset of short telomeres determine replicative senescence?

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

Cellular Senescence↗
PubMed2004

Telomeres are double-strand DNA breaks hidden from DNA damage responses.

A network of ATM/ATR-mediated events regulates cell cycle checkpoints and genomic integrity and contributes to the processing of DNA double-strand breaks in both genomic DNA and at telomeres. In yeast and in human cells, investigators, including, and Herbig et al., published in this issue of Molecular Cell, are beginning to decipher the signaling pathways involved at the telomeres.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Telomeric recombination in mismatch repair deficient human colon cancer cells after telomerase inhibition.

The majority of human malignancies use telomerase to maintain telomere homeostasis. Antitelomerase therapy is therefore a promising approach for a cancer-specific therapy. The alternative lengthening of telomeres pathway (ALT) is a recombination-based, telomerase-independent mechanism of telomere length control. It is widely believed that ALT could be engaged when cancer cells escape from telomerase inhibition. However, no reports exist that would support this concept of therapy resistance. We inhibited telomerase in a human cancer cell line with a mismatch repair defect and observed a telomerase-independent, ALT-like telomere elongation. This is the first report of inducing a telomerase-independent telomere elongation in human cancer cells when telomerase is inhibited, thus describing a novel mechanism of resistance to antitelomerase therapy.

Animals↗
PubMed2004

The frequency of homologous recombination in human ALT cells.

Instead of telomerase, some immortal cells use the alternative lengthening of telomeres pathway (ALT) to maintain their telomeres. There is good evidence that homologous recombination contributes to the ALT mechanism. Using an inducible GFP reporter system to measure the frequency of homologous recombination, we asked whether or not ALT cells exhibited a general change of the recombination machinery. Our results show that the frequency of homologous recombination for non-telomeric sequences in ALT cells is identical to that in telomerase positive cells, irrespective of whether the reporter was present at an intra-chromosomal location or next to a telomeric sequence. We conclude that the underlying recombination defect in ALT cells is restricted to telomeric sequences.

Base Sequence↗
PubMed2004

Hallmarks of senescence in carcinogenesis and cancer therapy.

Cellular senescence is a signal transduction program leading to irreversible cell cycle arrest. This growth arrest can be triggered by many different mechanisms including recognition by cellular sensors of DNA double-strand breaks leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. Senescence is initiated by the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals (STASIS: stress or aberrant signaling-induced senescence). The process of carcinogenesis involves a series of changes that allow tumor cells to bypass the senescence program. Nevertheless, tumor cells retain the capacity to undergo senescence. Treatment of tumor cells with many conventional anticancer therapies activates DNA damage signaling pathways, which induce apoptosis in some cells and senescence in others. Overexpression of tumor suppressors or inhibition of oncogenes can also induce rapid senescence in tumor cells. Senescent cells, while not dividing, remain metabolically active and produce many secreted factors, some of which stimulate and others inhibit the growth of tumors. The emerging knowledge about the pathways that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.

Animals↗
PubMed2004

The establishment of telomerase-immortalized Tangier disease cell lines indicates the existence of an apolipoprotein A-I-inducible but ABCA1-independent cholesterol efflux pathway.

Tangier disease (TD) is a human genetic disorder associated with defective apolipoprotein-I-induced lipid efflux and increased atherosclerotic susceptibility. It has been linked to mutations in the ATP-binding cassette protein A1 (ABCA1). Here we describe the establishment of permanent Tangier cell lines using telomerase. Ectopic expression of the catalytic subunit of human telomerase extended the life span of control and TD skin fibroblasts, and (in contrast to immortalization procedures using viral oncogenes) did not impair apolipoprotein A-I-induced lipid efflux. The key characteristics of TD fibroblasts (reduced cholesterol and phospholipid efflux) were observed both in primary and telomerase-immortalized fibroblasts from two unrelated homozygous patients. Surprisingly, the apolipoprotein-inducible cholesterol efflux in TD cells was significantly improved after immortalization (up to 40% of normal values). In contrast to ABCA1-dependent cholesterol efflux, this efflux was not inhibited by brefeldin A, glybenclamide, or intracellular ATP depletion but was inhibited in the presence of cytochalasin D. Apolipoprotein A-I-dependent cholesterol efflux was inversely correlated with the population doubling number in cell culture and was inhibited up to 40% in near-senescent normal diploid fibroblasts. This inhibition was completely reversed by telomerase. Thus ectopic expression of telomerase is a way to circumvent the lack of critical experimental material and represents a major improvement for studying cholesterol efflux pathways in lipid disorders. Our findings indicate the existence of an ABCA1-independent but cytoskeleton-dependent cholesterol removal pathway that may help to prevent early atherosclerosis in Tangier disease but may also be sensitive to aging phenomena ex vivo and possibly in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

Spontaneous reactivation of a silent telomeric transgene in a human cell line.

Subtelomeric reporter genes in human cells are silenced in a telomere length-dependent manner. Here we show that a subtelomeric reporter gene is expressed in only a subpopulation of cells within a clone and that this heterogeneity is generated by switching between expression states. We observed frequent reversion from the silenced state back to active expression. This process was more prominent for subtelomeric transgenes; however, we also observed cases of spontaneous reversion in control clones bearing the reporter at an internal site. We additionally show that treatment of these cells with 5-bromodeoxyuridine results in strong activation of the transgene. Although similar findings have been reported previously in mouse cells, this is, to our knowledge, the first direct observation of ongoing fluctuations in transcription in clonal populations of human cells. Our results suggest that this mechanism, as opposed to progressive silencing or a delayed fixing of expression states, accounts for the variegation in expression observed for subtelomeric transgenes in human cells. These data imply that telomere shortening during human aging could lead to stochastic activation of subtelomeric genes.

Aging↗
PubMed2004

A reproducible laser-wounded skin equivalent model to study the effects of aging in vitro.

Skin aging involves both chronological and photoaging processes. The effects of these processes are often overlapping and include changes in both the stratified epithelium and the fibroblast-rich dermis. Wound healing is frequently delayed with aging and can result in scarring. A skin equivalent model can be used to study the role of cells and the extracellular matrix in the process of wound healing. Current studies using this model employ a full-thickness wound placed atop a nonwounded dermis to mimic a partial-thickness wound. However, a true reproducible partial-thickness wound model has yet to be described. In this study, we investigated whether a laser-wounded skin equivalent would be a useful partial-thickness wound healing model. Three lasers were compared for the ability to generate a reproducible wound: an erbium-YAG, a high-powered excimer, and a low-powered excimer laser. Reepithelialization ability was tested using newborn and adult skin keratinocytes, adult esophageal keratinocytes, and cdk4-overexpressing newborn keratinocytes. Keratinocyte compartmentalization and basement membrane formation were assessed by immunofluorescence. The erbium-YAG and high-powered excimer laser cut reproducible wounds but left the remaining surface either discolored due to thermal damage and/or ragged; keratinocytes were unable to migrate into the wound area. The low-powered excimer laser cut reproducible wounds, leaving the cut surface intact and visibly unaltered; keratinocytes reepithelialized the wound in a collagenase-dependent manner within 3 days; and return of compartmentalization and basement membrane occurred within 14 days. The laser-wounded skin equivalent is an adjustable, reproducible partial-thickness wound model where keratinocyte biology akin to in vivo can be studied, and will be useful to study the effects of aging on wound healing.

Basement Membrane↗
PubMed2004

Modification of subtelomeric DNA.

There is a discrepancy in telomere length as measured by signal intensity of telomere restriction fragments on gels and fluorescence in situ hybridization analysis. This difference has been ascribed to the X-region, a segment of subtelomeric DNA that is resistant to being cut by restriction enzymes. To explore the nature of this region, we analyzed the digestibility of an artificial seeded telomere in HeLa cells as well as the Xp/Yp autosomal telomere in human BJ fibroblasts. We found that there is a substantial fraction of subtelomeric DNA containing restriction sites that is not digested with enzymes such as EcoRI, NlaIII, and SphI. Comparison of methylation-sensitive and -resistant enzymes excluded the possibility of the X-region being maintained by DNA methylation. We show that the X-region represents a variable domain whose size changes with telomere length, and neither non-TTAGGG sequences nor cytidine methylation can adequately explain the size of the X-region.

Base Sequence↗
PubMed2004

Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT).

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.

Cell Division↗
PubMed2004

Analysis of mammalian telomere position effect.

Methods relating to the positioning of a transgene next to a newly formed telomere in human (HeLa) cells and the subsequent analysis of the resulting clones are described. These include vector design, analysis of integration sites by Southern blotting, pharmacological relief of silencing, and enhancement of silencing by telomere elongation. Several potential pitfalls of applying these techniques to other cell lines are discussed. In addition, detailed instructions are provided for several more general methods related to human telomeres including terminal restriction fragment analysis and purification of telomeres from digested genomic DNA. This chapter summarizes the techniques currently in use that relate to human telomere position effect.

Animals↗
PubMed2003

Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency.

Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our reporter construct to investigate if telomeric sequences near an induced double-strand break alter the frequency of recombination between nontelomeric sequences, and found no differences among the different cell lines analysed. Our results indicate that the underlying defect in homologous recombination in ALT cells does not affect sequences independent of their chromosomal location but is likely to be primarily a specific telomeric defect.

Cell Division↗
PubMed2003

Aneuploidy and telomere attrition are independent determinants of crisis in SV40-transformed epithelial cells.

Replicative immortality is achieved in vitro by overcoming two mortality checkpoints, M1 (senescence) and M2 (crisis). Cancer cells are thought to overcome M2 by activating telomerase, an enzyme believed to confer genomic stability in addition to maintaining telomeric sequences above a critical length. Here we show that a subset of cultured ovarian cystadenoma cells expressing SV40 large T-antigen, which allows bypassing of M1, develop a specific type of genomic instability, characterized by numerical (as opposed to structural) chromosomal alterations, that leads to non-telomere-based premature growth arrest/crisis. Cells recover from this type of growth arrest and stabilize their ploidy status without telomerase expression. In these cases, telomeres continue to shorten until a second, telomere-based growth arrest/crisis event is reached. Transfection of the catalytic subunit of telomerase does not immortalize cells harboring severe abnormalities in their DNA ploidy but results in immortalization of diploid cells. We conclude that changes in DNA ploidy constitute an important determinant of growth arrest that is independent of telomere attrition in a subset of SV40 large T-antigen-expressing cystadenoma cells. Reestablishment or emergence of ploidy stability, which is not always dependent on telomerase activation, is necessary for acquisition of the potential to achieve replicative immortality.

Aneuploidy↗
PubMed2003

Telomere dysfunction: a potential cancer predisposition factor.

BACKGROUND: Genetic instability associated with telomere dysfunction (i.e., short telomeres) is an early event in tumorigenesis. We investigated the association between telomere length and cancer risk in four ongoing case-control studies. METHODS: All studies had equal numbers of case patients and matched control subjects (92 for head and neck cancer, 135 for bladder cancer, 54 for lung cancer, and 32 for renal cell carcinoma). Telomere length was measured in peripheral blood lymphocytes from study participants. Genetic instability was assessed with the comet assay. Patient and disease characteristics were collected and analyzed for associations with risk for these cancers. All statistical tests were two-sided. RESULTS: Telomeres were statistically significantly shorter in patients with head and neck cancer (6.5 kilobases [kb]) than in control subjects (7.4 kb) (difference = 0.9 kb, 95% confidence interval [CI] = 0.5 to 1.2 kb; P<.001). Nine percent of patients with head and neck cancer were in the longest quartile of telomere length, whereas 59% were in the shortest quartile. Similar patterns were observed for lung, renal cell, and bladder cancer. When subjects were categorized into telomere length quartiles defined by the distribution in control subjects, the following inverse relationship between telomere length and cancer risk was observed: adjusted odds ratios [ORs] for decreasing quartiles = 0.84 (95% CI = 0.36 to 1.97), 1.77 (95% CI = 0.72 to 4.36), and 5.11 (95% CI = 1.90 to 13.77). In stratified analysis, we found a suggestive greater-than-additive interaction between smoking status and telomere length: for ever smokers with short telomeres, OR = 25.05 (95% CI = 6.91 to 90.73); for never smokers with short telomeres, OR = 6.18 (95% CI = 1.72 to 22.13); and for ever smokers with long telomeres, OR = 6.49 (95% CI = 1.54 to 27.38). Telomere length was statistically significantly and inversely associated with baseline and mutagen-induced genetic instability. CONCLUSION: Short telomeres appear to be associated with increased risks for human bladder, head and neck, lung, and renal cell cancers.

Adult↗
PubMed2003

A peroxisome proliferator-activated receptor-gamma agonist and the p53 rescue drug CP-31398 inhibit the spontaneous immortalization of breast epithelial cells.

Cell immortalization is a critical and rate-limiting step in cancer progression. Agents that inhibit cell immortalization may have utility for novel molecular chemopreventive strategies. Preimmortal breast epithelial cells derived from a patient with the Li-Fraumeni Syndrome (LFS) can spontaneously immortalize in vitro at a measurable and reproducible frequency. In the present study, these cells were treated in vitro with low (nM) concentrations of potential and otherwise clinically validated chemopreventive agents, including several nonsteroidal anti-inflammatory drugs, rosiglitazone maleate, and the p53 rescue drug CP-31398. Rosiglitazone maleate (P < 0.05) and CP-31398 (P < 0.05) significantly inhibited the frequency of spontaneous immortalization of LFS breast epithelial cells compared with untreated controls. Nonsteroidal anti-inflammatory drugs, including specific cyclooxengenase-2 inhibitors, only moderately inhibited the spontaneous immortalization of preimmortal LFS breast epithelial cells. The significant effects of the p53 rescue drug CP-31398 correlated with the increase in cellular death induced by telomere shortening-induced DNA damage signals, including increases in p53 and p21 protein levels. Because immortalization is one step in cancer progression, these studies show the potential usefulness of a cell-based model system to screen the effects of known and potentially novel chemopreventive agents, using cell immortalization as an end point.

Anti-Inflammatory Agents, Non-Steroidal↗