Sinha R, Kiley S C, Lu J X, Thompson H J, Moraes R, Jaken S, Medina D
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Cancer Lett. 1999 Nov 15;146(2):135-45. doi: 10.1016/s0304-3835(99)00250-5.
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 microM) 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-alpha was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-delta was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-e (in cytosolic and membrane fractions) and PKC-zeta (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.
甲基硒代半胱氨酸（MSC）是一种有机硒化合物，是一种有效的化学预防剂，可在体内和体外抑制乳腺细胞生长，但其作用机制仍不清楚。我们之前已经证明，MSC能够在16小时时间点抑制同步化的TM6小鼠乳腺上皮肿瘤细胞系的生长，并在48小时后诱导细胞凋亡。细胞周期蛋白依赖性激酶2（cdk2）激酶活性的降低与细胞在S期的长期停滞相一致。目前的这组实验表明，在16小时时间点，MSC处理的细胞中cdk2磷酸化降低了72%。仅在16小时时间点之后，MSC处理后，生长停滞和DNA损伤诱导基因（gadd）34、45和153的表达升高了2.5至7倍。为了研究MSC可能的上游靶点，我们在这个模型中分析了蛋白激酶C（PKC）。在处理30分钟内，MSC（50微摩尔）使TM6细胞中的总PKC活性降低，对于钙依赖性和非钙依赖性PKC，分别在胞质（55.4%和77.6%）和膜（35.2%和34.1%）部分。佛波酯（PMA）显著提高了膜部分的PKC活性（P<0.01），而MSC抑制这种激活超过57%。MSC的作用具有硒特异性，因为硒代蛋氨酸和硫甲基-L-半胱氨酸（SMC）在胞质或膜部分均未改变PKC活性。免疫印迹分析表明，PKC-α被PMA转运至膜，而MSC并未改变这种转运。在对照和MSC处理细胞的膜部分中，PKC-δ仅能微弱检测到。MSC处理略微降低了PKC-ε（在胞质和膜部分）和PKC-ζ（胞质部分）的水平。本文提供的数据表明，PKC是MSC的一个潜在上游靶点，可能触发一种或所有下游效应；即cdk2激酶活性降低、DNA合成减少、gadd基因表达升高，最终导致细胞凋亡。
