Young M R, Lozano Y, Djorjevic A, Maier G D
Department of Research Services, Hines V.A. Hospital, IL 60141.
Int J Cancer. 1993 Jul 30;54(6):1036-41. doi: 10.1002/ijc.2910540629.
Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.
环磷酸腺苷（cAMP）的升高剂，如前列腺素E2（PGE2），可激活蛋白激酶A（PKA），并诱导转移性Lewis肺癌细胞（LLC-LN7）产生PKA刺激的运动性和转移。非转移性LLC（LLC-C8）对cAMP升高无反应，尽管它们的PKA酶并不缺乏。为了确定这种PKA无反应性是否可能是由于非转移性LLC-C8内丝氨酸/苏氨酸蛋白磷酸酶（PP-1/2A）的去磷酸化增加所致，测量了PP-1/2A抑制剂冈田酸对非转移性LLC-C8细胞迁移和侵袭的影响。冈田酸刺激非转移性LLC-C8细胞的运动性，使其达到与转移性LLC-LN7细胞相当的水平。当存在冈田酸时，PGE2进一步增加了非转移性LLC-C8细胞的运动性，而在不存在冈田酸时则没有这种作用。当PKA活性被抑制时，冈田酸对运动性的刺激作用减弱。用选择性抑制PP-2A或同时抑制PP-2A和PP-1的冈田酸浓度进行剂量反应研究，结果显示非转移性LLC-C8细胞的迁移逐渐增加，这表明PP-1和PP-2A都限制了它们的运动性。相比之下，转移性LLC-LN7细胞比非转移性LLC-C8细胞更具运动性，但这种运动性仅受到冈田酸的轻微影响。对LLC变体中PP-1/2A酶活性水平的比较显示，非转移性LLC-C8中的活性高于转移性LLC-LN7细胞。通过使用冈田酸（在低浓度下选择性抑制PP-2A活性，在高浓度下抑制PP-1和PP-2A）和花萼海绵诱癌素A（在与冈田酸影响的浓度相似时抑制PP-2A，但在抑制PP-1方面更有效）来评估非转移性LLC-C8中活性增加的PP的身份。冈田酸和花萼海绵诱癌素A对PP活性的抑制显示出一种模式，表明非转移性LLC-C8细胞中同时存在PP-1和PP-2A，但转移性LLC-LN7细胞中存在PP-1且PP-2A减少。这些数据的总和表明，在非转移性LLC中，PKA刺激的运动性受到PP-1和PP-2A的限制，而这种限制的缺乏导致迁移和侵袭增加。
