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| Robert P. Wersto, Ph.D., Staff Scientist Head, Flow Cytometry Unit |
 Dr. Robert Wersto received his Ph.D. from the Department of Biochemistry and Biophysics, Loyola University of Chicago in 1982. Dr. Wersto did his postdoctoral work in the Departments of Pathology and Hematology at the University of Rochester using the first commercially available flow cytometers and sorters. From 1985 until 1989, he was Assistant Professor of Pathology, Albert Einstein College of Medicine in the Bronx and Head of Flow Cytometry and Analytical Cytology. After a brief stay in industrial biotechnology, Dr. Wersto joined the Pulmonary Branch, National Heart, Lung, and Blood Institute (NHLBI) and played a seminal role in the first human gene therapy trial for cystic fibrosis. Most recently, he headed the flow cytometry laboratory in the non-human primate gene transfer program within the Hematology Branch, NHLBI. In mid 1999, he moved to the Flow Cytometry Unit, Research Resources Branch at the National Institute on Aging. Research Interests: Cell Cycle Progression and Aging: The effects of aging on T-cell cycle progression and arrest is the subject of an on-going investigation utilizing multiparameter flow cytometry. Age-related cell cycle properties of human T cells are assessed using simultaneous measurements of DNA content and KI-67 protein expression following co-stimulation with immobilized CD3 antibody and soluble CD28. In T-cells from elderly individuals, there is increased G0 cell cycle arrest that cannot be overcome following subsequent exposure to IL-2. Based on mitotic blocking, the delayed cell cycle entry in T-cells from older donors appears to be independent of early activation events. |
| Flow Cytometry Applications: Debris and aggregates can be prominent components of DNA histograms affecting the accuracy and reproducibility of cell cycle estimates. Debris originates from the damage and disintegration of cells following apoptosis or the fragmentation associated with the slicing of cells or nuclei during mechanical disaggregation. Aggregates can be composed of large clusters of cells or nuclei or two or more G0/1 (2N) events adhered together (G0/1 doublets) that are indistinguishable from particles with 4N, 6N, or 8N DNA content. Strategies to separate overlapping G0/1 doublets from the G2+M population have utilized the gating of correlated measurements of integral DNA fluorescence pulse (» area) with either peak pulse height or pulse duration (width), gating on the G2+M cells that lack cyclin B1 protein expression, and computer algorithms to model aggregate probability distributions in DNA histograms. While G0/1 doublets are easily discernable from G2+M singlets in cells or nuclei that are generally spherical in shape, doublet discrimination based on pulse processing or cyclin B1 measurements is nonconcordant in epithelial cells following cell cycle arrest. Significant differences in G0/1 doublet estimates is observed in breast tumor specimens, with estimates based on pulse width twice those of pulse height and nearly five times computer estimates. Differences between techniques is attributed to increasing uncertainty between the boundaries of suspected G0/1 doublets and G2+M singlet populations in biologically heterogeneous specimens. The laboratory has a strong interest in molecular cytometry such as single cell PCR sorting, and the development of new techniques that permit multiparameter analysis of both cell function and proliferation-restricted proteins. |
| Adenovirus-Based Gene Therapy: Based on the tropism of wild-type adenovirus (Ad) for the respiratory epithelia and its ability to infect nonreplicating cells, replication-defective Ad vectors were thought to be the ideal approach by gene therapy to correct the physiological defects in the airways of individuals having the inherited human disease cystic fibrosis (CF). Culminating in human clinical trials, Ad vectors have become the prototype for other gene therapy protocols targeting cancers, inherited metabolic deficiencies, and cardiovascular disease. First-generation Ad vectors that had been rendered replication defective by removal of the E1 region of the viral genome DE1)or lacking the Ad E3 region in addition to E1 sequences DE1E3) induce G2 cell cycle arrest and inhibit traverse across the G1/S boundary in primary and immortalized human bronchial epithelial cells, independent of the cDNA contained in the expression cassette. Arrest is associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34cdc2 protein levels. In some instances, infection with DE1 or DE1E3 Ad vectors produces aneuploid DNA histogram patterns and induces polyploidization resulting from successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DE1 Ad vector in which the entire early region E4 was deleted except for the sixth open reading frame. Current research focuses on the individual proteins encoded by the open reading frames in the E4 viral gene region and their interactions with cellular regulators of proliferation (signal transduction, transcription factors, oncogenes). |
| Bone Marrow Progenitor Identification: Gene transfer to hematopoietic stem cells (HSCs) has been hampered by their low frequency, the lack of positive selection markers, and the reduced potential for self-renewal and multi-lineage differentiation following ex vivo retroviral gene therapy. In mammalian bone marrow stained with the dye Hoechst 33342, bivariate flow cytometric analysis of blue and red fluorescence identifies a small cell population, termed SP cells, that constitute primitive HSCs via a mechanism thought to involve mdr P-glycoprotein. Using unfractionated non-human primate and murine bone marrow, SP cell staining was found to be an energy-dependent process involving dye efflux, consistent with the hypothesis that this phenomena is mediated by a member of the ATP Binding Cassette family of transporters. However, dye efflux was specifically inhibited by probencid or sulfinpyrazone, implicating involvement of other multi-drug resistance associated proteins or membrane transporters. Cells having the identical staining characteristics and responses as those of bone marrow SP cells are present in cultures of the HL-60 promyleocytic cell line and exhibited a dependence on G0/1 entry. SP cells are therefore not unique to bone marrow, but reflect multidrug resistance protein (MRP) functional expression that is present in a small fraction of quiescent cells. Understanding the basis for Hoechst 33342 staining and subsequent discrimination of SP cells from other blood elements provides insights into the functional characteristics of primitive multipotent hematopoietic that may be advantageous for future primate gene transfer protocols. |
| Contact Information: Research Resources Branch Gerontology Research Center 5600 Nathan Shock Drive Baltimore, MD 21224-6825 Phone 410-558-8377 Fax 410-558-8236 E mail werstor@grc.nia.nih.gov |
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