Michel M. Ouellette

Bio: Michel M. Ouellette is an academic researcher from University of Nebraska Medical Center. The author has contributed to research in topics: Pancreatic cancer & Telomerase. The author has an hindex of 31, co-authored 58 publications receiving 10133 citations. Previous affiliations of Michel M. Ouellette include University of Texas Southwestern Medical Center & University of Texas at Dallas.

Extension of life-span by introduction of telomerase into normal human cells

Abstract: Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for β-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT

Abstract: The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA poly-merases cannot fully replicate the ends of linear molecules1–4. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours5,6. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division7–9. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation10. In addition to the human telomerase RNA component (hTR; ref. 11), TP1/TLP1 (refs 12,13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme14, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (SpTrt!) telomerases15,17, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues17,18. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase

Abstract: The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Functional requirement of p23 and Hsp90 in telomerase complexes

Abstract: Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

Sonic hedgehog promotes desmoplasia in pancreatic cancer.

Abstract: Purpose: We investigated the contribution of Sonic hedgehog (SHH) to pancreatic cancer progression. Experimental Design: We expressed SHH in a transformed primary ductal-derived epithelial cell line from the human pancreas, transformed hTert-HPNE (T-HPNE), and evaluated the effects on tumor growth. We also directly inhibited the activity of SHH in vivo by administering a blocking antibody to mice challenged orthotopically with the Capan-2 pancreatic cancer cell line, which is known to express SHH and form moderately differentiated tumors in nude mice. Results: Our data provide evidence that expression of SHH influences tumor growth by contributing to the formation of desmoplasia in pancreatic cancer. We further show that SHH affects the differentiation and motility of human pancreatic stellate cells and fibroblasts. Conclusions: These data suggest that SHH contributes to the formation of desmoplasia in pancreatic cancer, an important component of the tumor microenvironment.

Embryonic Stem Cell Lines Derived from Human Blastocysts

Abstract: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Extension of life-span by introduction of telomerase into normal human cells

Abstract: Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for β-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.