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-Hodgkin's 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 overexpression of the c-Myc oncogene 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 specific, 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 (Ees). 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. Others, such as ribonucleotide reductase subunit 2 (RNR2) are amplified but not overexpressed. We are actively engaged in learning how many such genes can be amplified by this mechanism and what determines their expression or lack thereof. Another important aspect of this study is the discovery that cyclin D2 is a direct target of expression activation by the proto-oncogene, c-Myc. 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. We have been focusing on the delta and epsilon isoenzymes, which seem to have opposite effects on cell growth. We have shown that PKC-d is responsible for myeloid differentiation and growth inhibition, while overexpressed PKC-e 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-d and half PKC-e. Chimeric molecules that have carboxyl-terminal PKC-d sequences are able to cause macrophage differentiation much like the parent all-PKC-dprotein. Similarly, a PKC chimera with a PKC-e carboxyl-terminus, retains the neoplastic transformation potential of the all-PKC-e protein. We are further dissecting the structure of the catalytic domain to determine which sub-domains determine PKC isoform-specific functions. We are also studying the target molecules that are phosphorylated by individual PKC isoforms. We are also studying the nature of PKC's involvement in plasmacytoma induction and apoptosis, in cytoskeletal changes in cell shape, and its relationship to metastasis of these and other types of tumors, including human prostate cancer. Recently we have shown that phorbol ester-activation of overexpressed PKC-d 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 Charles Vinson, Ph.D. & Jane Trepel, Ph.D., NCI; Sabine Mai, Ph.D., Univ. of Manitoba, Winnipeg, Canada, Larisa Romanova, M.D., Ph.D., Harvard Medical School and Harald Mischak, Ph.D., Medizinische Hochschule Hannover, Hannover, Germany.