Gene Expression and Signal Transduction in Transformatio

转化中的基因表达和信号转导

• 批准号: 7337956
• 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/7337956
• 关键词: Gene; Expression; Signal; Transduction; Transformatio

The chief objective of our research is to understand the molecular and genetic mechanisms responsible for differentiation, cell growth, and neoplastic transformation. We study the oncogenes, tumor-suppressor genes and signal-transducing proteins in mouse and human experimental tumor systems, including BALB/c mouse plasmacytomas, B-cell lymphomas, and NIH 3T3 cells, among others. These are valuable experimental models, because they can be used to devise more specific therapy and preventive measures for human multiple myeloma, non-Hodgkin's lymphomas, and other human malignancies. BALB/c plasmacytomas, like human Burkitt lymphomas, are characterized by constitutive expression of the proto-oncogene, c-Myc. To determine which additional genetic alterations are required for complete transformation, we are using microarray hybridization studies of global gene expression to follow changes in gene expression during progression from pre-malignant to fully malignant plasma cell tumors. We are also using microarray hybridization studies to probe the molecular mechanisms at work in development of plasma cell tumors in mice and the mechanisms whereby certain transgenes and viral oncogenes accelerate this neoplastic process. Global gene expression studies are also underway to determine the physiological changes necessary for these tumors to adapt to growth in tissue culture. It is our hypothesis that such adaptive changes in gene expression that enable tumor cells to grow in the foreign environment of culture vessels might be analogous to those needed for human tumors to grow in alien environments following invasion or metastasis.In the study of signal transduction in differentiation and neoplastic transformation, we are investigating the isoform-specific features of the protein kinase C (PKC) family of serine/threonine kinases. We have been focusing on the delta and epsilon isoenzymes, which have opposing effects on cell proliferation. 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 reciprocal chimeric cDNAs that encode molecules that are half PKC-delta and half PKC-epsilon. We are further dissecting the structure of the catalytic domain to determine which sub-domains determine PKC isoform- specific functions, focusing on the carboxy-terminal 50 amino acids, the "V5 domain." We are also studying the nature of PKC's involvement in apoptosis, in cytoskeleton-related changes in cell shape and motility, and in cooperation with the c-Myc proto-oncogene. 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. We have demonstrated that this effect is due to PKC-mediated changes in phosphorylation of key tyrosine residues in the adaptor molecule, paxillin. Whereas the PKC-mediated effects on loss of tyrosine phosphorylation are indirect, we also have learned that PKC-delta can directly bind paxillin and phosphorylate a specific threonine, leading to homotypic aggregation.We have also shown that Myc and one of the PKC isoforms, PKC-gamma, can cooperate to transform NIH3T3 cells in vitro and in vivo, apparently not requiring intra-nuclear Myc. We are trying to understand the mechanism whereby this is accomplished.

我们研究的主要目标是了解导致分化、细胞生长和肿瘤转化的分子和遗传机制。我们研究了小鼠和人类实验肿瘤系统中的癌基因、肿瘤抑制基因和信号转导蛋白，包括BALB/c小鼠浆细胞瘤、B细胞淋巴瘤和NIH3T3细胞等。这些都是有价值的实验模型，因为它们可以用来为人类多发性骨髓瘤、非霍奇金淋巴瘤和其他人类恶性肿瘤设计更具体的治疗和预防措施。与人类Burkitt淋巴瘤一样，Balb/c浆细胞瘤的特征是原癌基因c-Myc的结构性表达。为了确定完全转化还需要哪些额外的基因改变，我们正在使用全球基因表达的微阵列杂交研究，以跟踪从癌前病变到完全恶性浆细胞瘤过程中基因表达的变化。我们还利用微阵列杂交研究来探索在小鼠浆细胞肿瘤发展中起作用的分子机制，以及某些转基因和病毒癌基因加速这一肿瘤过程的机制。全球基因表达研究也在进行中，以确定这些肿瘤适应组织培养生长所需的生理变化。我们的假设是，使肿瘤细胞在外来培养环境中生长的这种基因表达的适应性变化，可能类似于人类肿瘤在侵袭或转移后在外来环境中生长所需的变化。在分化和肿瘤转化的信号转导研究中，我们正在研究丝氨酸/苏氨酸激酶(PKC)家族的异构体特异性特征。我们一直专注于Delta和epsilon同工酶，这两种酶对细胞增殖具有相反的影响。我们已经证明，大多数同工酶特异的决定因素都位于这些PKC的催化部分(羧基末端结构域)，方法是创建编码一半PKC-Delta和一半PKC-epsilon分子的相互嵌合cDNA。我们正在进一步剖析催化结构域的结构，以确定哪些亚结构域决定了PKC异构体的特定功能，重点是羧基末端的50个氨基酸，即“V5结构域”。我们还在研究PKC参与细胞凋亡的性质，参与细胞形状和运动的细胞骨架相关变化，以及与c-Myc原癌基因的合作。我们已经证明，过表达的PKC-Delta的佛波酯激活破坏了人和小鼠淋巴细胞中的肌动蛋白细胞骨架，导致膜褶皱的丧失，细胞运动所需的表面改变，以及这些细胞的典型拉长形状的丧失。我们已经证明，这种效应是由于PKC介导的适配器分子中关键酪氨酸残基的磷酸化变化。虽然PKC对酪氨酸磷酸化缺失的影响是间接的，但我们也了解到PKC-Delta可以直接结合帕西林并磷酸化特定的苏氨酸，导致同型聚集。我们还表明，Myc和PKC亚型之一PKC-Gamma在体外和体内都可以协同转化NIH3T3细胞，显然不需要核内Myc。我们正在努力理解实现这一目标的机制。