STRUCTURE AND FUNCTION OF ONCOGENES AND ANTI-ONCOGENES

癌基因和抗癌基因的结构和功能

• 批准号: 6289210
• 负责人: J F MUSHINSKI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289210
• 关键词: B lymphocyte; Retroviridae; antibody formation; antitumor antibody; apoptosis; cell cell interaction; cell differentiation; chimeric proteins; chromosome translocation; cyclins; gene expression; helper T lymphocyte; human tissue; immunoglobulin genes; interleukin 6; laboratory mouse; molecular cloning; monoclonal antibody; neoplasm /cancer genetics; neoplastic transformation; oncogenes; oncoprotein p21; plasma cell neoplasm; protein kinase C; tissue /cell culture

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.

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 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肿瘤系统中涉及的癌基因、肿瘤抑制基因和信号转导蛋白。这些都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特征，需要对其机制进行了解，以便制定更具体的治疗和预防措施。 BALB/c 浆细胞瘤与大鼠免疫细胞瘤和人伯基特淋巴瘤一样，其特征是持续表达来自主癌基因 c-Myc 的信使 RNA 和蛋白质。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，也不清楚该基因如何过度表达导致人类和小鼠细胞中多种不同形式的肿瘤。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——细胞周期蛋白D2的基因在过度表达c-Myc的人类和小鼠肿瘤细胞中被扩增和过度表达。此外，我们发现 c-Myc 过度表达三四天就足以破坏基因组的稳定性并导致核内染色质片段（称为染色体外元件）的产生。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，其中一些基因会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。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。在信号转导的研究中，我们正在研究蛋白激酶C（PKC），它是一个由至少12种结构相关的同工酶组成的多基因家族，它们是许多信号转导的重要介质。信号转导的形式。使用多种表达载体，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 PKC。这使得鉴定各个 PKC 同工酶的特定功能和细胞内靶标成为可能。我们一直关注δ和ε同工酶，它们似乎对细胞生长具有相反的作用。我们已经证明 PKC-delta 负责骨髓分化和生长抑制，而过表达的 PKC-epsilon 则刺激细胞生长并将成纤维细胞转化为肿瘤细胞。我们正在剖析这些同工酶的结构，以确定哪些蛋白质结构域控制这些功能。我们通过创建一半 PKC-δ 和一半 PKC-ε 的嵌合分子，证明大多数同工酶特异性决定簇位于这些 PKC 的催化半部分（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，就像亲本全 PKC-delta 蛋白一样。类似地，具有 PKC-ε 羧基末端的 PKC 嵌合体保留了全 PKC-ε 蛋白的肿瘤转化潜力。我们还在研究 PKC 参与浆细胞瘤诱导和凋亡、细胞形状的细胞骨架变化的性质，及其与这些和其他类型肿瘤转移的关系。最近我们发现，佛波酯激活过表达的 PKC-δ 会破坏人和小鼠淋巴细胞中的肌动蛋白细胞骨架，导致细胞膜皱褶消失、细胞运动所需的表面改变以及这些细胞典型的细长形状的丧失。细胞。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 Peter Blumberg 博士。 & Jane Trepel，博士，NCI； Sabine Mai，博士，大学加拿大温尼伯马尼托巴省医学博士和德国汉诺威汉诺威医学学院 Harald Mischak 博士。