MECHANISMS OF TUMOR PROMOTION AND CARCINOGENESIS

确定允许肿瘤细胞经历 不受控制的增殖对于理解致癌作用至关重要。 最近的研究表明，蛋白磷酸酶，像蛋白激酶， 在细胞周期调控中发挥重要和特定的作用;因此， 这些蛋白质的表达或调节的改变可能会影响 细胞周期的进展，甚至可以让细胞进行不受调节的 增殖 蛋白磷酸酶在肿瘤发生中的作用 过程也出现在对冈田酸，微囊藻毒素-LR， 和calyculin A，三种高度特异性和有效的抑制剂， 丝氨酸/苏氨酸蛋白磷酸酶。 我们已经鉴定、纯化和表征了一种新的丝氨酸/苏氨酸 蛋白磷酸酶属于一类新的蛋白磷酸酶， 命名为类型3（PP 3）。 PP 3，如1型（PP 1）和2A型（PP 2A）， 被冈田酸和calyculin A有效抑制， 小鼠皮肤中的促进剂。 PP 3也被微囊藻毒素有效抑制。 LR是一种肝脏肿瘤促进剂。 这表明这三类人 蛋白磷酸酶可能在肿瘤的促进过程中起作用。 这项提议旨在检验蛋白磷酸酶 在细胞周期进程中发挥重要作用;因此，干扰 它们的活性可能会导致异常的增殖行为 肿瘤细胞。 这些研究将集中在以下方面 具体目标：目标1。 克隆PP 3。 一旦克隆被鉴定， 将进行测序并推导出PP 3的氨基酸序列。 然后将cDNA用于研究PP 3在细胞中的表达， 周期进展和致癌作用。 目标二。 表征 调节PP 3活性的机制。 影响机制 PP 3的活性将使用特定的 抑制剂（即冈田酸、微囊藻毒素-LR、节球藻毒素、甲基 okadaate、calyculin A和互变霉素）、类型特异性抗体和cDNA probes. 目的3、研究PP#在EGF刺激细胞增殖中的作用 丝氨酸/苏氨酸蛋白与细胞增殖的关系 磷酸酶和TGF β 1介导的CCL 64细胞生长停滞。 具体 抑制剂、类型特异性cDNA探针和反义硫代磷酸酯 寡脱氧核苷酸将被用来确定PP 1的作用， PP 3在TGF β 1介导的CCL 64细胞生长停滞中的作用

Determining the mechanisms that allow neoplastic cells to undergo uncontrolled proliferation is crucial to understanding carcinogenesis. Recent studies indicate that protein phosphatase, like protein kinases, play important and specific roles in cell-cycle regulation; thus alterations in the expression or regulation of these proteins may affect cell-cycle progression and could even allow cells to undergo unregulated proliferation. A role for protein phosphatase in the tumor promotion process is also emerging from studies with okadaic acid, microcystin-LR, and calyculin A, three highly specific and potent inhibitor of certain serine/threonine protein phosphatase. We have identified, purified and characterized a novel serine/threonine protein phosphatase belonging to a new class of protein phosphatase, designated as type 3 (PP3). PP3, like type 1 (PP1) and type 2A (PP2A), is potently inhibited by okadaic acid and calyculin A, two potent tumor promoters in mouse skin. PP3 is also potently inhibited by microcystin- LR, a tumor promoter in liver. This suggests that these three classes of protein phosphatase may have a role in the tumor promotion process. This proposal is designed to test the hypothesis that protein phosphatase play an important role in cell cycle progression; thus, the interference of their activity may contribute to the aberrant proliferative behavior of neoplastic cells. These studies will focus around the following specific aims: Aim 1. Clone PP3. Once clones are identified, the cDNA will be sequenced and the amino acid sequence of PP3 will be deduced. The cDNA will then be used to study the expression of PP3 during cell cycle progression and carcinogenesis. Aim 2. Characterize the mechanisms regulating the activity of PP3. The mechanisms influencing the activity of PP3 will be investigated with the use of specific inhibitors (i.e. okadaic acid, microcystin-LR, nodularin, methyl okadaate, calyculin A and tautomcyin), type specific antibodies, and cDNA probes. Aim 3, Characterize the role of PP# in EGF stimulated cell proliferation and the relationship between serine/threonine protein phosphatase and TGFBeta1 mediated growth arrest in CCL64 cells. Specific inhibitors, type-specific cDNA probes and antisense phosphorothioate oligodeoxynucleotides will be employed to determine the role of PP1 and PP3 in TGFBeta1-mediated growth arrest of CCL64 cells.