Gene Expression and Signal Transduction in Transformatio

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中涉及的癌基因、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具有许多共同的生物学和分子遗传学特征，需要从机制上了解这些特征，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类Burkitt淋巴瘤一样，Balb/c浆细胞瘤的特征是主要癌基因c-Myc的信使RNA和蛋白的结构性表达。目前还不清楚完成转化还需要哪些额外的突变。Myc表达改变的一个重要后果可能是基因组的不稳定，表现为染色体内外扩增，以及随后对DNA合成和细胞增殖至关重要的几个基因的过度表达，包括细胞周期蛋白D2。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失。 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11个结构上相关的同工酶组成的多基因家族，它们是许多形式的信号转导的重要中介。利用不同的表达载体，我们已经在成纤维细胞、淋巴细胞、髓系细胞和平滑肌细胞系中过表达了许多PKCs。这使得鉴定单个PKC同工酶的特定功能和细胞内靶点成为可能。我们一直专注于Delta和epsilon同工酶，这两种酶对细胞增殖有相反的影响。我们已经证明，PKC-Delta负责髓系分化和生长抑制，而过表达的PKC-epsilon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。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，很像亲本ALL-PKC-Delta蛋白。同样，具有PKC-epsilon羧基末端的PKC嵌合体保留了All-PKC-epsilon蛋白的肿瘤转化潜力。我们正在进一步剖析催化域的结构，以确定哪些亚域决定了PKC异构体的特定功能。我们还在研究PKC参与细胞凋亡、细胞形态中与细胞骨架相关的变化以及这些和其他类型的肿瘤(包括人类前列腺癌)转移的本质。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。我们认为这种作用是由于PKC介导的适配器分子paxlin的磷酸化的变化。

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. It is still not clear which additional mutations are required for complete transformation. One mechanism that may be important consequence of altered Myc expression is genomic instability, in the form of extra- and intra-chromosomal amplification, and the subsequent overexpression of several genes that are crucial for DNA synthesis and cell proliferation, including cyclin D2. We are actively engaged in learning how many such genes can be amplified by this mechanism and what determines their expression or lack thereof. In the study of signal transduction, we are investigating protein kinase C (PKC), a multigene family of 11 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, myeloid and smooth muscle 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 have opposite effects on cell proliferation. 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 reciprocal chimeric cDNAs that encode 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 further dissecting the structure of the catalytic domain to determine which sub-domains determine PKC isoform-specific functions. We are also studying the nature of PKC's involvement in apoptosis, in cytoskeleton-related changes in cell shape, and in metastasis of these and other types of tumors, including human prostate cancer. 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 think that this effect is due to PKC-mediated changes in the phosphorylation of the adaptor molecule, paxillin.