Gene Expression and Signal Transduction in Transformation and Differentiation

转化和分化中的基因表达和信号转导

• 批准号: 7592581
• 负责人: J F MUSHINSKI
• 金额: $ 115.2万
• 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/7592581
• 关键词: Actins; Adherence; Affect; Alien; Amino Acids; Animals; B-Cell Lymphomas; Binding; Biological Process; Burkitt Lymphoma; Cancer Patient; Catalytic Domain; Cell Proliferation; Cell Shape; Cells; Clinical; Cluster Analysis; Cytoskeleton; Data; Development; Elements; Environment; Event; Experimental Models; Experimental Neoplasms; Family; Gene Expression; Generations; Genes; Genomic Instability; Goals; Growth; Human; In Vitro; Inbred BALB C Mice; Isoenzymes; Learning; Lesion; Lymphocyte; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Membrane; MicroRNAs; Molecular; Molecular Genetics; Molecular Probes; Molecular Profiling; Multiple Myeloma; Mus; Mutation; NIH 3T3 Cells; Nature; Neoplasm Metastasis; Neoplastic Cell Transformation; Neoplastic Processes; Non-Hodgkin' s Lymphoma; Nuclear; Oils; Oncogenes; Patients; Pattern; Phorbol Esters; Phosphorylation; Phosphotransferases; Physiological; Plasma Cell Neoplasm; Plasma Cells; Plasmacytoma; Premalignant; Preventive; Protein Isoforms; Protein Kinase C; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Publishing; Research; Role; Signal Transduction; Structure; Surface; System; Threonine; Transgenes; Tumor Promoters; Tumor Suppressor Genes; Tyrosine; Tyrosine Phosphorylation; Up-Regulation; Viral Oncogene; Work; base; c-myc Genes; c-myc Proto-Oncogenes; cell growth; cell motility; cell transformation; comparative genomic hybridization; in vivo; malignant breast neoplasm; man; neoplastic cell; novel; outcome forecast; paxillin; protein kinase C epsilon; protein kinase C gamma; protein kinase C-delta; tissue culture; tumor; tumor progression

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 and array-based Comparative Genomic Hybridization to follow genetic changes and 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. As an extension of this project, we observed marked differences in gene expression profiles between mouse plasma cell tumors growing in tissue culture and the sme tumors growing in intact animals. We wre able to show that these gene expression differences offered a means of predicting liklihood of survival for patients with several different forms of cancer. Our analysis focused mainly on published data from patients with breast cancer. Our studies that compared global gene expression patterns of B-cell lymphomas in mouse and man with expression patterns of plasma cell neoplasms of murine (plasma cell tumors) and human multiple myeloma showed significant and characteriztic differences not only between B-cell lymphomas and plasma cell neoplasms, but also among different subtypes of human and mouse plasma cell neoplasms. Unsupervised hierarchical cluster analysis of expression patterns of the human and murine plasma cell neoplasms showed a similarity between rapid-appearing mouse plasma cell tumors and MM3 and MM4 multiple myelomas, which had particularly poor clinical prognoses. 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 PKC 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 studying how alterations in PKC's V5 region affect its kinase activity and its isoform-specific biological function. We are also studying the nature of PKC's ability to cooperate with neoplastic transformation by 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负责分化、细胞生长和肿瘤转化的分子和遗传机制。我们研究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 NIH 3T3 细胞等。这些都是有价值的实验模型，因为它们可用于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。 BALB/c 浆细胞瘤与人伯基特淋巴瘤一样，其特征是原癌基因 c-Myc 的组成型表达。为了确定完全转化需要哪些额外的遗传改变，我们正在使用全局基因表达的微阵列杂交研究和基于阵列的比较基因组杂交来跟踪从恶性前浆细胞肿瘤到完全恶性浆细胞肿瘤进展过程中的遗传变化和基因表达的变化。我们还利用微阵列杂交研究来探讨小鼠浆细胞肿瘤发展的分子机制以及某些转基因和病毒癌基因加速这种肿瘤过程的机制。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中生长所需的生理变化。我们的假设是，基因表达的这种适应性变化使肿瘤细胞能够在培养容器的外来环境中生长，这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。作为该项目的延伸，我们观察到在组织培养中生长的小鼠浆细胞肿瘤和在完整动物中生长的中小企业肿瘤之间的基因表达谱存在显着差异。我们能够证明，这些基因表达差异提供了一种预测患有几种不同形式癌症的患者的生存可能性的方法。我们的分析主要集中于乳腺癌患者的已发表数据。我们的研究将小鼠和人类 B 细胞淋巴瘤的整体基因表达模式与小鼠浆细胞肿瘤（浆细胞肿瘤）和人类多发性骨髓瘤的表达模式进行了比较，结果显示，不仅 B 细胞淋巴瘤和浆细胞之间存在显着的特征性差异肿瘤，而且还包括人类和小鼠浆细胞肿瘤的不同亚型。对人和小鼠浆细胞肿瘤表达模式的无监督层次聚类分析显示，快速出现的小鼠浆细胞肿瘤与 MM3 和 MM4 多发性骨髓瘤之间存在相似性，而后者的临床预后特别差。在分化和肿瘤转化中的信号转导研究中，我们正在研究丝氨酸/苏氨酸激酶的蛋白激酶 C (PKC) 家族的亚型特异性特征。我们一直关注 PKC δ 和 epsilon 同工酶，它们对细胞增殖具有相反的作用。我们通过创建编码一半 PKC-delta 和一半 PKC-epsilon 分子的相互嵌合 cDNA，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）。我们正在进一步剖析催化结构域的结构，以确定哪些子结构域决定 PKC 同工型特异性功能，重点关注羧基末端 50 个氨基酸，即“V5 结构域”。我们正在研究 PKC V5 区域的改变如何影响其激酶活性及其异构体特异性生物学功能。我们还在研究 PKC 与 c-Myc 原癌基因的肿瘤转化合作的能力的本质。我们已经证明，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。我们已经证明，这种效应是由于 PKC 介导的接头分子桩蛋白中关键酪氨酸残基的磷酸化变化所致。尽管 PKC 介导的酪氨酸磷酸化损失的影响是间接的，但我们还了解到 PKC-delta 可以直接结合桩蛋白并磷酸化特定的苏氨酸，从而导致同型聚集。我们还表明，Myc 和 PKC 同种型之一 PKC-gamma 可以在体外和体内协同转化 NIH3T3 细胞，显然不需要核内 Myc。我们正在尝试了解实现这一目标的机制