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1.  Improving cell therapy – experiments using transplanted telomerase-immortalized cells in immunodeficient mice 
Cell therapy is the use of stem cells and other types of cells in various therapies for age-related diseases. Two issues that must be addressed before cell therapy could be used routinely in medicine are improved efficacy of the transplanted cells and demonstrated long-term safety. Desirable genetic modifications that could be made to cells to be used for cell therapy include immortalization with hTERT (human telomerase reverse transcriptase). We have used a model for cell therapy in which transplantation of adrenocortical cells restores glucocorticoid and mineralocorticoid hormone levels in adrenalectomized immunodeficient mice. In this model, clones of cells that had been immortalized with hTERT were shown to be able to replace the function of the animals'adrenal glands by forming vascularized tissue structures when cells were transplanted beneath the capsule of the kidney. hTERT-modified cells showed no tendency for neoplastic changes. Moreover, a series of experiments showed that hTERT does not cooperate with known oncoproteins in tumorigenesis either in adrenocortical cells or in human fibroblasts. Nevertheless, hTERT was required for tumorigenesis when cells were implanted subcutaneously rather than in the subrenal capsule space. Changes in gene expression make hTERT-modified cells more robust. Understanding these changes is important so as to be able to separately control immortalization and other desirable properties of cells that could be used in cell therapy. Alternatively, desirable properties of transplants might be provided by co-transplanted mesenchymal cells: mesenchymal cell-assisted cell therapy. For both hTERT modification and mesenchymal cell-assisted cell therapy, genomics approaches will be needed to define what genetic modifications are desirable and safe in cells used in cell therapy.
PMCID: PMC1797893  PMID: 17123586
2.  The Minimal Set of Genetic Alterations Required for Conversion of Primary Human Fibroblasts to Cancer Cells in the Subrenal Capsule Assay1 
Neoplasia (New York, N.Y.)  2005;7(6):585-593.
Based on previous studies, a minimal set of genetic alterations that is required to convert normal human fibroblasts into cancer cells has been defined. Essential roles for telomere maintenance and alterations in phosphatase 2A activity were inferred from experiments in which tumorigenicity was tested by injecting cells under the skin of immunodeficient mice. However, in the present experiments, the combination of SV40 large T antigen and activated Ras, without hTERT or SV40 small t antigen, was sufficient to convert nine different primary human fibroblast cell strains to a fully malignant state. The malignant behavior of the cells was demonstrated by growth of the cells into invasive tumors when the cells were injected beneath the kidney capsule of immunodeficient mice. Lung metastases and circulating tumor cells were also detected. These tumors were not immortal; cells entered crisis, from which they could be rescued by expression of hTERT. However, the same cell populations were not tumorigenic when they were injected under the skin. In this site, tumorigenicity required the expression of hTERT and SV40 small t antigen as well as SV40 large T antigen and Ras. The cellular pathways targeted by SV40 large T antigen (p53 and pRb) and those targeted by activated Ras represent a minimal set of genetic alterations required for the conversion of normal human fibroblasts into cancer cells.
PMCID: PMC1501282  PMID: 16036109
replicative senescence; crisis; telomeres; human fibroblasts; immunodeficient mice
3.  MRG15 Regulates Embryonic Development and Cell Proliferation 
Molecular and Cellular Biology  2005;25(8):2924-2937.
MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15−/− embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15−/− mouse embryonic fibroblasts. The hearts of the Mrg15−/− embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15−/− embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.
PMCID: PMC1069611  PMID: 15798182
4.  Cooperation of hTERT, SV40 T Antigen and Oncogenic Ras in Tumorigenesis: A Cell Transplantation Model Using Bovine Adrenocortical Cells1 
Neoplasia (New York, N.Y.)  2002;4(6):493-500.
Expression of TERT, the reverse transcriptase component of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, “telomerization” by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not perturb the functional tissue-forming capacity of the cells. This cell transplantation model was used to study the cooperation of hTERT with SV40 T antigen (SV40 TAg) and oncogenic Ras in tumorigenesis. Only cells expressing all three genes were tumorigenic; this required large T, but not small t, antigen. These cells produced a continuously expanding tissue mass; they were invasive with respect to adjacent organs and eventually destroyed the kidney. Cells expressing only hTERT or only Ras produced minimally altered tissues. In contrast, SV40 TAg alone produced noninvasive nodules beneath the kidney capsule that had high proliferation rates balanced by high rates of apoptosis. The use of cell transplantation techniques in a cell type that is able to form tissue structures with or without full neoplastic conversion allows the phenotypes produced by individual cooperating oncogenes to be observed.
PMCID: PMC1503663  PMID: 12407443
telomerase; cell transplantation; adrenal cortex; SV40 T antigen; oncogenic Ras

Results 1-4 (4)