STRUCTURE AND FUNCTION OF ONCOGENES AND ANTI-ONCOGENES

我们的研究目标是了解细胞生长、分化和肿瘤转化的分子和遗传机制。我们研究了BALB/c小鼠浆细胞瘤、b细胞淋巴瘤和其他小鼠和人类实验肿瘤系统的癌基因、肿瘤抑制基因和信号转导蛋白。这些是有价值的实验模型，因为它们与人类多发性骨髓瘤、非霍奇金淋巴瘤和其他人类恶性肿瘤有许多共同的生物学和分子遗传学特征，需要了解这些肿瘤的机制，以便设计出更具体的治疗和预防措施。与大鼠免疫细胞瘤和人伯基特淋巴瘤一样，BALB/c浆细胞瘤的特征是主要致癌基因c- myc的信使RNA和蛋白质的组成性表达。最常见的是，浆细胞瘤中c-Myc表达失调继发于c-Myc基因附近的染色体易位。目前尚不清楚为什么c-Myc癌基因普遍参与浆细胞肿瘤，也不清楚该基因的过度表达如何导致人类和小鼠细胞中许多不同形式的肿瘤。我们认为我们已经找到了这一机制的线索，因为我们发现编码驱动细胞周期的重要蛋白质的基因，细胞周期蛋白D2，在过度表达c-Myc的人类和小鼠肿瘤细胞中被放大和过度表达。此外，我们发现，c-Myc过表达三到四天足以破坏基因组的稳定，并导致核内染色质片段的产生，称为染色体外元件。这些可以用荧光显微镜检测到。杂交技术表明，在这些非染色体核dna上可以发现许多基因，其中一些基因导致重要的生长刺激蛋白的表达升高，包括细胞周期蛋白D2。我们正在积极地研究有多少这样的基因可以通过这种机制被放大。在信号转导的研究中，我们正在研究蛋白激酶C (PKC)，这是一个由至少12个结构相关的同工酶组成的多基因家族，是许多形式的信号转导的重要介质。利用多种表达载体，我们在成纤维细胞、淋巴细胞和骨髓细胞系中过表达了许多PKCs。这使得鉴定PKC同工酶的特定功能和细胞内靶标成为可能。我们一直在关注δ和ε同工酶，它们似乎对细胞生长有相反的影响。我们已经证明PKC-delta负责髓细胞分化和生长抑制，而PKC-epsilon过表达刺激细胞生长并将成纤维细胞转化为肿瘤细胞。我们正在解剖这些同工酶的结构，以确定哪些蛋白质结构域控制这些功能。我们通过创造一半PKC-delta和一半PKC-epsilon的嵌合分子，证明了大多数同工酶特异性决定因子位于这些PKCs的催化一半（羧基末端结构域）。具有羧基末端PKC-delta序列的嵌合分子能够引起巨噬细胞分化，就像亲本all-PKC-delta蛋白一样。同样，具有PKC-epsilon羧基末端的PKC嵌合体保留了全PKC-epsilon蛋白的肿瘤转化潜能。我们也在研究PKCs参与浆细胞瘤诱导和凋亡的本质，参与细胞形状的细胞骨架变化，以及它与这些和其他类型肿瘤转移的关系。最近，我们已经证明，磷酸化酯激活过表达的pkc - δ会破坏人和小鼠淋巴细胞中的肌动蛋白细胞骨架，导致膜褶皱（细胞运动所需的表面改变）的丧失，以及这些细胞典型的细长形状的丧失。这是我们第一次研究PKC、细胞骨架和信号转导之间的重要相互关系。这项研究的合作者包括Peter Blumberg博士和Jane Trepel博士，NCI；Sabine Mai博士，加拿大温尼伯马尼托巴大学，Harald Mischak博士，德国汉诺威汉诺威医学学院。

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.