STRUCTURE AND FUNCTION OF ONCOGENES AND ANTI-ONCOGENES

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Our research objective is to understand the molecular and genetic mechanisms responsible for cell growth, differentiation and neoplastic transformation. We study the oncogenes, tumor-suppressor genes and signal transducing proteins involved in BALB/c mouse plasmacytomas, B-cell lymphomas and other mouse and human experimental tumor systems. These are valuable experimental models, because they have many biological and molecular genetic features in common with human multiple myeloma, non-Hodgkins lymphomas, and other human malignancies that are in need of mechanistic understanding in order to devise more specific therapy and preventive measures. BALB/c plasmacytomas, like rat immunocytomas and human Burkitt lymphomas, are characterized by constitutive expression of messenger RNA and protein from the master oncogene, c-Myc. Most commonly, c-Myc expression in plasmacytomas is dysregulated secondary to a chromosomal translocation in the vicinity of the c-Myc gene.It is still not clear why the c-Myc oncogene is universally involved in plasma cell tumors nor how overexpression of this gene leads to many different forms of tumors in human and mouse cells. We think we have found a clue to this mechanism in that we have found that the gene encoding an important protein that drives the cell cycle, cyclin D2, is amplified and overexpressed in human and mouse tumor cells that overexpress c-Myc. In addition, we have found that a three or four days of overexpression of c-Myc is sufficient to destabilize the genome and to cause the generation of intranuclear fragments of chromatin, called extra-chromosomal elements. These can be detected with the fluorescent microscope. Hybridization techniques show that a number of genes can be found on these non-chromosomal nuclear DNAs, some of which result in elevated expression of important growth-stimulatory proteins, including cyclin D2. We are actively engaged in learning how many such genes can be amplified by this mechanism.In the study of signal transduction, we are investigating protein kinase C (PKC), a multigene family of at least 12 structurally related isoenzymes that are important mediators of many forms of signal transduction. Using a variety of expression vectors, we have overexpressed many of the PKCs in fibroblasts, lymphocytic and myeloid cell lines. This has made possible the identification of specific functions and intracellular targets for the individual PKC isoenzymes. We have been focusing on the delta and epsilon isoenzymes, which seem to have opposite effects on cell growth. We have shown that PKC-delta is responsible for myeloid differentiation and growth inhibition, while overexpressed PKC-epsilon stimulates cell growth and transforms fibroblasts into tumor cells. We are dissecting the structure of these isoenzymes to determine which protein domains control these functions. We have shown that most of the isoenzyme-specific determinants are located in the catalytic half (the carboxyl-terminal domain) of these PKCs by creating chimeric molecules that are half PKC-delta and half PKC-epsilon. Chimeric molecules that have carboxyl -terminal PKC-delta sequences are able to cause macrophage differentiation much like the parent all-PKC-delta protein. Similarly, a PKC chimera with a PKC-epsilon carboxyl -terminus, retains the neoplastic transformation potential of the all-PKC-epsilon protein. We are also studying the nature of PKCs involvement in plasmacytoma induction and apoptosis, in cytoskeletal changes in cell shape, and its relationship to metastasis of these and other types of tumors. Recently we have shown that phorbol ester-activation of overexpressed PKC-delta disrupts the actin cytoskeleton in human and mouse lymphocytes, leading to the loss of membrane ruffling, a surface alteration needed for cell movement, and the loss of the typical elongated shape of these cells. This is the first of our studies into the important interrelationship between PKC, the cytoskeleton and signal transduction.Collaborators on this research include Peter Blumberg, Ph.D. & Jane Trepel, Ph.D., NCI; Sabine Mai, Ph.D., Univ. of Manitoba, Winnipeg, Canada, and Harald Mischak, Ph.D., Medizinische Hochschule Hannover, Hannover, Germany.