Ezhevsky S A, Toyoshima H, Hunter T, Scott D W
Department of Immunology, Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.
Mol Biol Cell. 1996 Apr;7(4):553-64. doi: 10.1091/mbc.7.4.553.
Cross-linking surface immunoglobulin (Ig)M on the WEHI-231 B-cell lymphoma results in decreased cell size, G1/S growth arrest, and finally DNA cleavage into oligonucleosomal fragments that are the classical features of apoptotic cells. Treatment of WEHI-231 cells with anti-IgM in early G1 phase prevents phosphorylation of the retinoblastoma gene product (pRb) and inhibits entry into S phase. Using unsynchronized cells, we previously demonstrated that cyclin A-associated and Cdk2-dependent GST-pRb kinase activity were inhibited in WEHI-231 cells treated with anti-IgM. We now show that progression of elutriated early G1 phase WEHI-231 cells from early into late G1 phase is accompanied by an increase in the abundance of cyclin A protein and cyclin A-associated kinase activity. Treatment of early G1 cells with anti-IgM prevented this increase in cyclin A-associated kinase activity at late G1, despite minimal changes in the overall level of cyclin A and Cdk2 proteins. Late G1 cells, which already possess high cyclin A-associated kinase activity, were insensitive to anti-IgM treatment and were able to complete the cell cycle. We also found that anti-IgM-treated cells contained increased amounts of the Cdk inhibitor protein p27Kip1. Essentially all of the cyclin A in treated cells was associated with p27, a result which we propose explains the lack of cyclin A/Cdk2 kinase activity. Accumulation of p27 in cyclin A kinase complexes, however, did not decrease the amount of Cdk2 bound to cyclin A. Thus, cross-linking IgM on growth-inhibitable B-cell lymphomas affects cyclin A kinase activity by increasing the levels of p27 in this complex, thus preventing productive pRb phosphorylation and leading to cell cycle arrest and subsequent apoptosis. These results are discussed in terms of the cell cycle restriction points that regulate lymphocyte function, as well as the lineage-specific differences in cell cycle control.
交联WEHI-231 B细胞淋巴瘤表面的免疫球蛋白（Ig）M会导致细胞尺寸减小、G1/S期生长停滞，最终DNA裂解为寡核小体片段，这些都是凋亡细胞的典型特征。在G1早期用抗IgM处理WEHI-231细胞可阻止视网膜母细胞瘤基因产物（pRb）的磷酸化，并抑制进入S期。使用未同步化的细胞，我们之前证明在用抗IgM处理的WEHI-231细胞中，细胞周期蛋白A相关的和依赖Cdk2的GST-pRb激酶活性受到抑制。我们现在表明，经淘洗的G1早期WEHI-231细胞从G1早期进入晚期时，细胞周期蛋白A蛋白丰度和细胞周期蛋白A相关激酶活性会增加。在G1早期用抗IgM处理细胞可阻止晚期G1期细胞周期蛋白A相关激酶活性的这种增加，尽管细胞周期蛋白A和Cdk2蛋白的总体水平变化极小。已经具有高细胞周期蛋白A相关激酶活性的晚期G1期细胞对抗IgM处理不敏感，并且能够完成细胞周期。我们还发现，经抗IgM处理的细胞中Cdk抑制剂蛋白p27Kip1的含量增加。处理过的细胞中基本上所有的细胞周期蛋白A都与p27相关，我们认为这一结果解释了细胞周期蛋白A/Cdk2激酶活性缺乏的原因。然而，p27在细胞周期蛋白A激酶复合物中的积累并没有减少与细胞周期蛋白A结合的Cdk2的量。因此，在生长可抑制的B细胞淋巴瘤上交联IgM会通过增加该复合物中p27的水平来影响细胞周期蛋白A激酶活性，从而阻止有效的pRb磷酸化，导致细胞周期停滞并随后发生凋亡。我们从调节淋巴细胞功能的细胞周期限制点以及细胞周期调控中的谱系特异性差异方面讨论了这些结果。
