Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.
Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.
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DOI：
10.1016/s0304-3835(99)00250-5
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发表时间：
1999-11
期刊：
影响因子：
9.7
通讯作者：
Raghu Sinha;S. Kiley;Junxuan Lu;Henry J. Thompson;Ricardo C. Moraes;S. Jaken;Daniel Medina
中科院分区：
文献类型：
--
作者：
Raghu Sinha;S. Kiley;Junxuan Lu;Henry J. Thompson;Ricardo C. Moraes;S. Jaken;Daniel Medina
甲基硒代半胱氨酸（MSC）是一种有机硒化合物，在体内和体外都是一种有效的乳腺细胞生长的化学预防剂，但其作用机制尚不清楚。我们先前已经证明MSC能够在16小时时间点抑制同步化的TM 6小鼠乳腺上皮肿瘤细胞系的生长，随后在48小时抑制凋亡。cdk 2激酶活性的降低与细胞在S期的延长停滞一致。本组实验显示，在MSC处理的细胞中，cdk 2磷酸化在16 h时间点降低了72%。MSC处理后仅在16小时时间点后，gadd 34、45和153的表达升高2.5至7倍。为了研究MSC可能的上游靶点，我们分析了该模型中的蛋白激酶C（PKC）。MSC（50 μM）在处理30 min内降低了TM 6细胞中总PKC活性，对于钙依赖性和非依赖性PKC，胞浆（55.4%和77.6%）和膜（35.2%和34.1%）组分分别降低。PMA使细胞膜PKC活性显著升高（P<0.01），MSC可抑制其活性达57%以上。MSC的作用是硒特异性的，因为硒代蛋氨酸和硫甲基-l-半胱氨酸（SMC）不改变胞浆或膜组分中的PKC活性。免疫印迹分析显示PKC-α被PMA转位到细胞膜上，MSC不改变这种转位。PKC-δ在对照和MSC处理的细胞的膜组分中微弱地检测到。MSC处理略微降低了PKC-ε（在胞质和膜部分中）和PKC-ε（胞质部分）的水平。本文提供的数据表明，PKC是MSC的潜在上游靶标，其可以触发一种或所有下游效应;即cdk 2激酶活性降低、DNA合成减少、gadd基因表达升高以及最终凋亡。
Methylselenocysteine (MSC), an organic selenium compound is an effective chemopreventive agent against mammary cell growth both in vivo and in vitro but its mechanism of action is still not understood. We have previously demonstrated that MSC is able to inhibit growth in a synchronized TM6 mouse mammary epithelial tumor cell line at 16 h time point followed by apoptosis at 48 h. The decrease in cdk2 kinase activity was coincident with prolonged arrest of cells in S-phase. The present set of experiments showed that cdk2 phosphorylation was reduced by 72% in the MSC-treated cells at 16 h time point. Expression for gadd34, 45 and 153 was elevated 2.5 to 7 fold following MSC treatment only after 16 h time point. In order to investigate a possible upstream target for MSC, we analyzed protein kinase C (PKC) in this model. Total PKC activity was reduced in TM6 cells by MSC (50 μM) within 30 min of treatment, both in cytosolic (55.4 and 77.6%) and membrane (35.2 and 34.1%) fractions for calcium-dependent and independent PKCs, respectively. PMA significantly elevated the PKC activity in membrane fraction (P<0.01) and MSC inhibited this activation by more than 57%. The effect of MSC was selenium specific as selenomethionine and sulfurmethyl-l-cysteine (SMC) did not alter PKC activity either in cytosolic or membrane fraction. Immunoblot analysis showed that PKC-α was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-δ was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-ε (in cytosolic and membrane fractions) and PKC-ζ (cytosolic fractions). The data presented herein suggest that PKC is a potential upstream target for MSC that may trigger one or all of the downstream effects; i.e. the decrease of cdk2 kinase activity, decreased DNA synthesis, elevation of gadd gene expression and finally apoptosis.