Ni Huey-Juin, Chang Ying-Nang, Kao Pu-Hong, Chai Shin-Pei, Hsieh Ya-Hsin, Wang Duo-Hsiang, Fong Jim C
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Biochim Biophys Acta. 2012 Dec;1820(12):1893-900. doi: 10.1016/j.bbagen.2012.08.002. Epub 2012 Aug 10.
hMMS21 is a human SUMO ligase required for DNA damage repair and mitotic progression in HeLa cervical cancer cells. Owing to the diversity of cancer, we further investigated the effect of hMMS21-depletion on MCF-7 breast cancer cells.
hMMS21-depletion was achieved by RNA interference. Cellular hMMS21 and E2F1 mRNA levels were estimated by RT-PCR and real-time PCR. Cell cycle profile was assessed by flow cytometry. Western blot and co-immunoprecipitation were used to determine the protein levels of various factors involved in G1-S transition and CDK2- or CDK4-associated p21 and p27. Kinase activity of cyclin E/CDK2 was measured in anti-cyclin E immunoprecipitate.
hMMS21-depletion induced slower cell growth and G1-S transition. While it had no effect on cyclin D1 or phospho-Rb (S807/811) levels, hMMS21-depletion provoked lower E2F1 levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity correlated with increased cellular p21(CIP1) levels and CDK2-p21 association. Moreover, ectopic expression of Flag-hMMS21 but not its ligase-inactive mutant rescued the decreased growth rates of hMMS21-depletd cells. Thus, depletion of hMMS21 seems to impair G1-S transition due to lowered E2F1 protein levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity is probably attributable to its greater association with p21 as a result of increased p21 levels. In addition, hMMS21-mediated sumoylation appears to be involved.
This study demonstrates that hMMS21 is required for G1-S transition in breast cancer cells and implies that manipulation of hMMS21-mediated sumoylation may alter the growth rates of breast cancer cells.
hMMS21是一种人类SUMO连接酶，在HeLa宫颈癌细胞的DNA损伤修复和有丝分裂进程中发挥作用。鉴于癌症的多样性，我们进一步研究了hMMS21缺失对MCF-7乳腺癌细胞的影响。
通过RNA干扰实现hMMS21缺失。采用RT-PCR和实时PCR估算细胞中hMMS21和E2F1 mRNA水平。通过流式细胞术评估细胞周期分布。利用蛋白质印迹法和免疫共沉淀法测定参与G1-S期转换以及与CDK2或CDK4相关的p21和p27的各种因子的蛋白质水平。在抗细胞周期蛋白E免疫沉淀物中测量细胞周期蛋白E/CDK2的激酶活性。
hMMS21缺失导致细胞生长和G1-S期转换减缓。虽然它对细胞周期蛋白D1或磷酸化Rb（S807/811）水平没有影响，但hMMS21缺失导致E2F1水平降低以及细胞周期蛋白E/CDK2活性降低。细胞周期蛋白E/CDK2活性降低与细胞中p21（CIP1）水平升高以及CDK2与p21的结合增加相关。此外，Flag-hMMS21的异位表达而非其连接酶失活突变体挽救了hMMS21缺失细胞降低的生长速率。因此，hMMS21缺失似乎由于E2F1蛋白水平和细胞周期蛋白E/CDK2活性降低而损害G1-S期转换。细胞周期蛋白E/CDK2活性降低可能归因于p21水平升高导致其与p21的结合增加。此外，hMMS21介导的SUMO化似乎也参与其中。
本研究表明hMMS21是乳腺癌细胞G1-S期转换所必需的，这意味着对hMMS21介导的SUMO化的调控可能会改变乳腺癌细胞的生长速率。
