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癌症的发生至关重要。 最近的研究表明，蛋白磷酸酶，就像蛋白激酶一样， 在细胞周期调节中发挥重要而具体的作用；因此 这些蛋白的表达或调节的变化可能会影响 细胞周期进程，甚至可能允许细胞经历不受调控的 扩散。蛋白磷酸酶在肿瘤促进中的作用 对冈田酸、微囊藻毒素-LR、 和Calyculin A，三种高度特异和有效的某些 丝氨酸/苏氨酸蛋白磷酸酶。 我们已经鉴定、提纯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， 指定为类型3(PP3)。PP3，就像类型1(PP1)和类型2A(PP2A)一样， 被冈田酸和花盏花素A这两种有效的肿瘤有效地抑制 老鼠皮肤中的启动子。微囊藻毒素也能有效地抑制PP3- 肝脏肿瘤促进剂LR。这表明这三个班级 蛋白磷酸酶的活性可能在肿瘤的促进过程中起作用。 这一提议旨在检验蛋白质磷酸酶 在细胞周期进程中发挥重要作用；因此，干扰 它们的活性可能导致异常的增殖行为 肿瘤细胞的数量。这些研究将围绕以下几个方面展开 具体目标：目标1.克隆PP3。一旦鉴定出克隆，cDNAs 将被测序，并推导出PP3的氨基酸序列。 然后，该cDNA将用于研究PP3在细胞中的表达 周期进展与癌症的发生。目标2.描述 PP3活性的调控机制。影响因素的机制 PP3的活性将通过使用特定的 抑制剂(即冈田酸、微囊藻毒素-LR、根瘤菌素、甲基 Okadaate、calyculin A和tautomcyin)、类型特异性抗体和cDNA. 探测器。目的3，研究PP#在EGF刺激细胞中的作用 细胞增殖与丝氨酸/苏氨酸蛋白的关系 磷酸酶和TGFBeta1介导CCL64细胞生长停滞。特定的 抑制剂、类型特异性cdna探针和反义硫代磷酸 将使用寡脱氧核苷酸来确定PP1和 PP3在TGFBeta1介导的CCL64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.