Structure and function of oncogenes and anti-oncogenes

我们的研究目标是了解负责细胞生长，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细胞淋巴瘤以及其他小鼠和人类实验性肿瘤系统中涉及的癌基因、抑癌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具有许多共同的生物学和分子遗传学特征，这些特征需要机械理解，以便设计出更具体的治疗和预防措施。BALB/c浆细胞瘤，如大鼠免疫细胞瘤和人伯基特淋巴瘤，其特征在于组成性表达的信使RNA和蛋白质的主癌基因，c-Myc。最常见的是，浆细胞瘤中的c-Myc表达失调继发于c-Myc基因附近的染色体易位。目前还不清楚为什么c-Myc癌基因的过度表达会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种机制的线索，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，在过表达c-Myc的人类和小鼠肿瘤细胞中被扩增和过表达。具体地说，我们已经发现c-Myc过表达三到四天就足以使基因组不稳定，并导致产生染色质的核内片段，称为染色体外元件（Ees）。在这些非染色体核DNA上可以发现许多基因，其中一些导致重要的生长刺激蛋白（包括细胞周期蛋白D2）的表达升高。其他的，如核糖核苷酸还原酶亚基2（RNR 2）被扩增但不过度表达。我们正在积极研究有多少这样的基因可以通过这种机制被扩增，以及是什么决定了它们的表达或缺乏。这项研究的另一个重要方面是发现细胞周期蛋白D2是原癌基因c-Myc表达激活的直接靶点。在信号转导的研究中，我们正在研究蛋白激酶C（PKC），这是一个多基因家族，至少有12种结构相关的同工酶，是许多形式的信号转导的重要介质。使用多种表达载体，我们已经在成纤维细胞、淋巴细胞和骨髓细胞系中过表达了许多PKC。我们一直专注于δ和δ同工酶，这似乎对细胞生长有相反的影响。我们已经表明，PKC-d负责骨髓分化和生长抑制，而过表达的PKC-e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。我们已经表明，大多数同工酶特异性决定簇位于催化的一半（羧基末端结构域），这些蛋白激酶通过创建嵌合分子，是半PKC-d和半PKC-e。具有羧基末端PKC-d序列的嵌合分子能够引起巨噬细胞分化，非常像亲本全PKC-d蛋白。类似地，具有PKC-e羧基末端的PKC嵌合体保留了全PKC-e蛋白的肿瘤转化潜力。我们正在进一步解剖催化结构域的结构，以确定哪些子域决定PKC亚型特异性功能。我们也在研究被个别PKC亚型磷酸化的靶分子。我们也在研究PKC参与浆细胞瘤诱导和凋亡的性质，细胞形状的细胞骨架变化，及其与这些和其他类型肿瘤（包括人前列腺癌）转移的关系。最近，我们已经表明，佛波酯激活过表达的PKC-d破坏了人类和小鼠淋巴细胞中的肌动蛋白细胞骨架，导致膜皱褶的损失，细胞运动所需的表面改变，以及这些细胞的典型细长形状的损失。这是我们对PKC、细胞骨架和信号转导之间重要相互关系的首次研究。这项研究的合作者包括Charles Vinson博士。& Jane Trepel，Ph.D.，NCI; Sabine Mai博士，加拿大温尼伯马尼托巴大学，拉里萨罗曼诺娃医学博士，哲学博士、哈佛医学院和Harald Mischak博士，德国，汉诺威，汉诺威医学院。

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-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.