Biomedical subjects

Sheila A Stewart

Publications and source records attributed to Sheila A Stewart.

12 recordsLinked to original sources

PubMed2006

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Animals↗

PubMed2006

Telomeres: cancer to human aging.

The cell phenotypes of senescence and crisis operate to circumscribe the proliferative potential of mammalian cells, suggesting that both are capable of operating in vivo to suppress the formation of tumors. The key regulators of these phenotypes are the telomeres, which are located at the ends of chromosomes and operate to protect the chromosomes from end-to-end fusions. Telomere erosion below a certain length can trigger crisis. The relationship between senescence and telomere function is more complex, however: Cell-physiological stresses as well as dysfunction of the complex molecular structures at the ends of telomeric DNA can trigger senescence. Cells can escape senescence by inactivating the Rb and p53 tumor suppressor proteins and can surmount crisis by activating a telomere maintenance mechanism. The resulting cell immortalization is an essential component of the tumorigenic phenotype of human cancer cells. Here we discuss how telomeres are monitored and maintained and how loss of a functional telomere influences biological functions as diverse as aging and carcinogenesis.

Aging↗

PubMed2005

High-efficiency RNA interference in human embryonic stem cells.

RNA interference methodology suppresses gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. In this study, we used retroviral and lentiviral vectors to deliver small interfering RNAs and report high-efficiency silencing of a green fluorescent protein (GFP) trans gene and the stem cell-specific transcription factors Oct4/POU5F1 and Nanog in human embryonic stem cells. Gene knockdown of Oct4 and Nanog promotes differentiation, thereby demonstrating a role for these factors in human embryonic stem cell self-renewal.

Antigens, Surface↗

PubMed2005

Telomere maintenance and tumorigenesis: an "ALT"ernative road.

The acquisition of cellular immortality is a critical step in human tumorigenesis. While the vast majority of human tumors activate the catalytic component of telomerase (hTERT) to stabilize their telomeres and attain immortality, a significant portion (7-10%) utilize a poorly defined alternative form of telomere maintenance referred to as ALT. Interestingly, telomerase activation is often favored in tumors arising from the epithelial compartment whereas ALT occurs in a more significant portion of tumors that arise from tissues of mesenchymal origin. This observation raises the possibility that cell type specific mechanisms favor the activation of telomerase versus ALT in human tumorigenesis. Because cellular immortality is critical to tumorigenesis it may represent an important anti-neoplastic target. Indeed, several approaches have successfully eliminated telomerase activity in human tumor models and some of these approaches are now moving into clinical trials. While these results are encouraging, it is clear that these approaches will have no impact on cells that utilize the ALT mechanism for telomere maintenance. Furthermore, the existence of ALT raises the possibility that telomerase-positive tumors undergoing anti-telomerase therapies may escape by activating the ALT pathway. For these reasons a detailed understanding of the ALT pathway is critical to the future design of anti-neoplastic therapies.

Cell Transformation, Neoplastic↗

PubMed2003

Telomerase maintains telomere structure in normal human cells.

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Antibodies, Monoclonal↗

PubMed2003

Erosion of the telomeric single-strand overhang at replicative senescence.

Cultured primary human cells inevitably enter a state of replicative senescence for which the specific molecular trigger is unknown. We show that the single-strand telomeric overhang, a key component of telomere structure, is eroded at senescence. Expression of telomerase prevents overhang loss, suggesting that this enzyme prevents senescence by maintaining proper telomere structure. In contrast, progressive overhang loss occurs in cells that avoid senescence through the inactivation of p53 and Rb, indicating that overhang erosion is the result of continuous cell division and not a consequence of senescence. We thus provide evidence for a specific molecular alteration in telomere structure at senescence and suggest that this change, rather than overall telomere length, serves to trigger this state.

Antigens, Viral, Tumor↗

PubMed2003

Lentivirus-delivered stable gene silencing by RNAi in primary cells.

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Dendritic Cells↗

PubMed2002

Telomerase contributes to tumorigenesis by a telomere length-independent mechanism.

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.

Blotting, Western↗

PubMed2002

Immortalization and transformation of primary human airway epithelial cells by gene transfer.

One critical step in the development of a cancerous cell is its acquisition of an unlimited replicative lifespan, the process termed immortalization. Experimental model systems designed to study cellular transformation ex vivo have relied to date on the in vitro selection of a subpopulation of cells that have become immortalized through treatment with chemical or physical mutagens and the selection of rare clonal variants. In this study, we describe the direct immortalization of primary human airway epithelial cells through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT. Cells immortalized in this way are now responsive to malignant transformation by an introduced H-ras or K-ras oncogene. These immortalized human airway epithelial cells, which have been created through the stepwise introduction of genetic alterations, provide a novel experimental model system with which to study further the biology of the airway epithelial cell and to dissect the molecular basis of lung cancer pathogenesis.

Blotting, Western↗

PubMed2002

Senescence: does it all happen at the ends?

Over 60 years ago Barbara McClintock described the telomere and suggested that it protected the chromosome from illegitimate or end-to-end fusion, thus functioning to protect the genome. Since that time we have discovered that the telomere is a complex structure composed of both DNA and a growing list of associated proteins that together serve to regulate the length of the telomere and, as predicted by McClintock, protect genomic integrity. In addition to its protective role, the telomere has also been hypothesized to serve as a molecular clock that tallies the number of cell divisions and limits further divisions at a predetermined point. However, the precise role of telomeres in predicting and limiting cellular lifespan remains a matter of much debate. In this review, we highlight some of the salient points of basic telomere biology and relate them to the current controversies surrounding the role of telomeres and telomerase in cellular senescence.

Cell Division↗

PubMed2002

Multiple levels of telomerase regulation.

Normally, cell division leads to shortening of telomeres, the nucleoprotein complexes located at the ends of linear chromosomes. When telomeres reach a critically short length, cells cease to divide. However, immortal tumor cells display stable telomere lengths and are able to maintain their proliferative state. Wong and colleagues have found that telomerase is sequestered by nucleoli during certain stages of the cell cycle, decreasing the likelihood of telomerase access to chromatin until the late S phase. Additionally, they demonstrate that ionizing radiation tends to keep telomerase sequestered in nucleoli, whereas cell transformation leads to telomerase translocation into the nucleoplasm, where, presumably, it can catalyze the lengthening of telomeres at appropriate and inappropriate sites. The sequestration of telomerase thus imposes a newly identified level of regulation on telomerase activity, implicating telomerase localization as a potentially useful target for pharmacotherapy.

Cell Cycle↗

PubMed2002

Prospects for anti-neoplastic therapies based on telomere biology.

The maintenance of specialized nucleoprotein structures at the ends of human chromosomes called telomeres is essential for chromosome stability, and plays a fundamental role in the regulation of cellular lifespan. Without new synthesis of telomeres, chromosome ends shorten with progressive cell division, eventually triggering either replicative senescence or apoptosis when telomere length becomes critically short. The regulation of telomerase activity in human cells plays a significant role in the development of cancer. Telomerase is tightly repressed in the vast majority of normal human somatic cells but becomes activated during cell immortalization and in cancers. Recent work has demonstrated that inhibiting or targeting telomerase shows promise as a novel anti-neoplastic strategy; however, the biology of telomeres and telomerase predict that such approaches will differ in important ways from traditional cytotoxic drug therapies. Understanding telomerase biology may eventually lead to several types of clinically effective, telomerase-based therapies for neoplastic disease.

Animals↗