Professor and Chair
M.D. 1961 Columbia University
and control of DNA replications
Cytokines and Cell Proliferation
An understanding of the control of cell proliferation at the molecular level is of obvious importance for the ultimate elucidation of mechanisms involved in normal growth, aging, and neoplasia, as well as in processes of host defense and repair. The ligand-receptor interactions at the cell membrane, early transduction events, and cell cycle-dependent gene activation that play a role in cell proliferation have all been the focus of intense study. However, the final stages of the pathway(s) leading to DNA replication remain poorly understood, largely because limited information is available regarding the institution of DNA replication in mammalian systems. We found that interleukin 2 stimulated lymphoblasts, mitogen-activated lymphocytes, and neoplastic lymphoid lines contained a cytosolic protein (ADR) that can induce DNA replication in isolated nuclei, as well as sufficient quantities of the required replication proteins. We were able to utilize these preparations to initiate the replication of origin-containing DNA sequences CHE obtained from human ribosomal RNA genes. Initial results have demonstrated both substantial DNA as well as the presence of replication intermediates in addition to template DNA by 2D gel analysis. By hybridizing ddGTP chain-terminated replication products to restriction fragments of C HE , we were able to localize an origin within C HE . Hybridization of labelled nascent replication to C HE restriction fragments localized the major site of in vitro replication initiation to within a 1375 bp fragment (C BX ). Preliminary electrophoretic analysis of C HE subclones indicated the presence of two regions of intrinsicly bent DNA; one such region with C BX was mapped by circular permutation analysis to the end of a 317 bp subfragment which appears to be at the site of replication initiation. This 317 bp fragment, as well as another 230 bp fragment which is only 236 bp away, specifically binds protein from MOLT-4 nuclear extracts as determined by gel shift assay. Preliminary purification of MOLT-4 nuclear proteins using a C BX DNA affinity column followed by gel shift analysis has identified two protein doublet species of MW 36 kD - 38 kD which specifically bind these sequences. In contrast, cytosolic preparations contain a larger molecular weight binding molecule which may be a multimer of the nuclear protein. Aprotinin shift assays as well as aprotinin affinity chromatography are underway to explore the relationship of the molecular species to ADR. In addition, we are beginning the characterization of replication origins from other human genes in order to elucidate a specific minimal set of structures and sequences required for the origin of replication.
More recent studies relate to the role of mammalian chitinases in migration and proliferation of bone tumor cells, and the effect of chitinase inhibitors of these processes. This is prompted by our finding through gene array analysis of chitinase and chitinase gene expression in various osteosarcomas.
Agarwal, O., Oldenburg, M.C., Czarneski, J.E., Morse, R.M., Hameed, M.R., Cohen, S., and Fernandes, H.D. Comparison study for identifying promoter allelic polymorphisms in IL-10 and TNF ? genes. Diagnostic Molecular Pathol., 9:158, 2000.
Fernandes H., Cohen, S., and Bishayee, S. Glycosylation-induced conformational modification positively regulates receptor-receptor association: A study with an aberrant EGF receptor expressed in cancer cells. J. Biol. Chem., 276:5375, 2001.
Barbashina, V., Aviv, H., Benevenia, J., Patterson, F., Tsai, J., Cohen, S., Aisner, S., Fernandes, H., Skurnick, J., and Hameed, M. Oncoproteins and proliferation markers in synovial sarcomas: a clinicopathologic study of 17 cases. J. Canc. Res. And Clin. Onc., 128:610, 2002.
Uppal, S., Aviv, H., Patterson, F., Cohen S., Benevenia, J., Aisner, S., and Hameed, M. Alveolar soft part sarcoma-reciprocal translocation between chromosome 17q25 and Xp11: report of a case and review of the literature. Acta Ortho. Belgica., 69:182, 2002.
Coffman, F.D., He, M., Diaz, M.L., Georgoff, L., Fresa, K.L and Cohen, S. Compact core origin motifs within a human DNA replication origin. Nuc. Acids Res., (Submitted).