RRM1 maintains centrosomal integrity via CHK1 and CDK1 signaling during replication stress

DNA lesion-induced centrosomal abnormalities during the replication phase are relatively unknown. Here, we report that RNAi-mediated depletion of RRM1 induces cell-cycle arrest at the replication phase, along with severe DNA damage and centrosomal amplification. Interestingly, CHK1 depletion synergistically increased RRM1-depletion-induced centrosomal amplification. In response to hydroxyurea, CHK1 was delocalized from the centrosome by RRM1 depletion. Moreover, CDK1, which functions in centrosome separation and is inhibited by CHK1, was found to be essential for RRMI1-depletion-induced centrosomal amplification. Thus, we herein demonstrate that RRM1 preserves chromosomal stability via the CHK1- and CDK1-dependent stabilization of the centrosomal integrity at the replication stage.     

乳腺癌细胞中心体调控研究新进展

随着研究的进展,人们不断认识到中心体的复杂功能及其重要性.中心体异常可导致基因不稳定及细胞的恶性转化.中心体正常功能的维系以及中心体复制与细胞周期的一致有赖于多个调控因子的协调作用.目前乳腺癌细胞中,中心体调控因子主要有中心体相关激酶,泛素-蛋白酶体途径相关物质,相关癌基因/抑癌基因及其产物等.     

Centrosome abnormalities in non-small cell lung cancer: correlations with DNA aneuploidy and expression of cell cycle regulatory proteins

The aims of this study were to investigate centrosome abnormalities in non-small cell lung cancer (NSCLC), and to assess their relationship with DNA aneuploidy, the expression of the cell cycle-associated proteins, and clinicopathological profiles. Tissue microarrays were constructed from 175 NSCLCs. We analyzed centrosome abnormalities and the expression of p16^INK4a, p53, and pRb using immunohistochemistry. Centrosome abnormalities were noted in 29% of the tumors and were even observed in the normal cells adjacent to the tumor. The frequency of DNA aneuploidy was significantly higher in the tumors containing centrosome abnormalities than in the tumors with a normal centrosome. p16^INK4a expression and loss of pRb expression, but not p53 expression, were significantly associated with centrosome abnormalities. Clinically, centrosome abnormalities were not found to have any prognostic value for NSCLCs. These results suggest that centrosome abnormalities may be associated with inactive pRb-pathway and contribute to pulmonary carcinogenesis by the level of increasing chromosome instability.     

中心体异常与膀胱癌关系的研究进展

染色体不稳定性被认为是恶性肿瘤细胞的特征性改变,而细胞有丝分裂异常可以引起染色体不稳定。中心体作为细胞的主要微管组织中心,在细胞周期中建立有丝分裂纺锤体,调节细胞有丝分裂,从而对维持染色体的稳定起着重要作用。因此肿瘤细胞基因不稳定可能与中心体异常密切相关。中心体异常是恶性肿瘤细胞的普遍特征。现就有关中心体异常在恶性肿瘤特别是膀胱移行细胞癌发生进展中的作用作一综述。     

Identification of novel small molecule inhibitors of centrosome clustering in cancer cells

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain more than two, or “supernumerary” centrosomes. Such cancer cells achieve bipolar division by clustering their centrosomes into two functional poles, and inhibiting this process then leads to cancer-specific cell death. A major problem with clinically used anti-mitotic drugs, such as paclitaxel, is their toxicity in normal cells. To discover new compounds with greater specificity for cancer cells, we established a high-content screen for agents that block centrosome clustering in BT-549 cells, a breast cancer cell line that harbors supernumerary centrosomes. Using this screen, we identified 14 compounds that inhibit centrosome clustering and induce mitotic arrest. Some of these compounds were structurally similar, suggesting a common structural motif important for preventing centrosome clustering. We next compared the effects of these compounds on the growth of several breast and other cancer cell lines, an immortalized normal human mammary epithelial cell line, and progenitor-enriched primary normal human mammary epithelial cells. From these comparisons, we found some compounds that kill breast cancer cells, but not their normal epithelial counterparts, suggesting their potential for targeted therapy. One of these compounds, N2-(3-pyridylmethyl)-5-nitro-2-furamide (Centrosome Clustering Chemical Inhibitor-01, CCCI-01), that showed the greatest differential response in this screen was confirmed to have selective effects on cancer as compared to normal breast progenitors using more precise apoptosis induction and clonogenic growth endpoints. The concentration of CCCI-01 that killed cancer cells in the clonogenic assay spared normal human bone marrow hematopoietic progenitors in the colony-forming cell assay, indicating a potential therapeutic window for CCCI-01, whose selectivity might be further improved by optimizing the compound. Immunofluorescence analysis showed that treatment with CCCI-01 lead to multipolar spindles in BT-549, while maintaining bipolar spindles in the normal primary human mammary epithelial cells. Since centrosome clustering is a complex process involving multiple pathways, the 14 compounds identified in this study provide a potentially novel means to developing non-cross-resistant anti-cancer drugs that block centrosome clustering.     

Centrosomal aggregates and Golgi fragmentation disrupt vesicular trafficking of DAT

Lewy bodies containing the centrosomal protein γ-tubulin and fragmentation of Golgi apparatus (GA) have been described in nigral neurons of Parkinson's disease (PD) patients. However, the relevance of these features in PD pathophysiology remains unknown. We analyzed the impact of proteasome inhibition in the formation of γ-tubulin-containing aggregates as well as on GA structure. SH-SY5Y cells were treated with the proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) to induce centrosomal-protein aggregates. Then, microtubules (MTs) and Golgi dynamics, as well as the vesicular transport of dopamine transporter (DAT) were evaluated both in vitro and in living cells. MG132 treatment induced γ-tubulin aggregates which altered microtubule nucleation. MG132-treated cells containing γ-tubulin aggregates showed fragmentation of GA and perturbation of the trans-Golgi network. Under these conditions, the DAT accumulated at the centrosomal-Golgi region indicating that the vesicular transport of DAT was disrupted. Thus, centrosomal aggregates and fragmentation of GA are 2 closely related processes that could result in the disruption of the vesicular transport of DAT toward the plasma membrane in a model of dopaminergic neuronal degeneration.     

中心体α-微管蛋白、γ-微管蛋白在脑胶质瘤中的表达及其与Survivin表达的相关性研究

目的 探讨中心体α-微管蛋白和γ-微管蛋白在脑胶质瘤中的表达,及其与凋亡抑制基因Survivin表达的相关性。方法 应用免疫组织化学方法检测51例脑胶质瘤组织中α-微管蛋白、Survivin蛋白的表达,免疫荧光染色方法检测γ 微管蛋白的表达,并分析其之间的相关性。结果 脑胶质瘤高级别组（Ⅲ～Ⅳ级）α-微管蛋白、γ-微管蛋白及Survivin蛋白表达阳性率（88.0%、80.0%、92.0%）明显高于低级别组（Ⅰ、Ⅱ级）（22.2%、47.1%;11.1%、35.3%;33.3%、52.9%）,且差异具有统计学意义(P均＜0.0125)。α-微管蛋白、γ-微管蛋白与Survivin表达之间均呈正相关（r分别为0.440?4、0.547?8,P＜0.05)。结论 α-微管蛋白、γ-微管蛋白和Survivin在胶质瘤中的表达随恶性程度升高而增加,且相互之间均呈正相关。微管蛋白与Survivin的相互作用可能是胶质瘤发生发展过程中的重要事件。     

Association of loss of BRCA1 expression with centrosome aberration in human breast cancer

Purpose  Centrosome aberration in number and/or size is reportedly often observed in human breast cancer. The aim of this study was to investigate the relationship between centrosome aberration and chromosomal instability as well as the expression of centrosome regulators such as BRCA1, Aurora-A, and p53. Methods  Centrosome aberration in number and size was determined immunohistochemically using the anti-γ-tubulin antibody, and chromosomal instability was evaluated by fluorescence in situ hybridization analysis of chromosomes 1, 11, and 17 in paraffin sections from 50 human breast cancers. Immunohistochemical examination of BRCA1, Aurora-A, and p53 was also performed to examine the relationship of their expression with centrosome aberration. Results  Percentage of tumor cells with centrosome aberration in size varied from 0.9 to 30.4% (median 9.5%) and in number it varied from 0.5 to 86.5% (median 34.5%) in each tumor. No significant association in number or size, however, was observed between chromosomal instability and centrosome aberration. Numerical centrosome aberration was significantly associated with negative BRCA1 expression (P = 0.001). Breast tumors (n = 3) from patients with a proven BRCA1 germline mutation also showed a significant relationship with numerical centrosome aberration (P = 0.011). On the other hand, expression of Aurora-A or p53 was not significantly associated with centrosome aberration in either number or size. Conclusions  Centrosome aberration is not associated with chromosomal instability, indicating the importance of other mechanisms in the induction of chromosomal instability in human breast cancer. BRCA1, but not Aurora-A and p53, is significantly involved in the pathogenesis of centrosome aberration.     

Centrosome amplification in tumorigenesis

With regard to cancer development the centrosome has been the center of attraction of scientists for already more than a 100 years. After the initial assumption that amplified centrosomes and abnormal mitotic arrangements might be a cause of cancer at the beginning of the last century, enormous efforts have been undertaken to clarify the relevance of centrosome amplification in tumorigenesis. In the meantime, centrosome amplification has been observed in most, both solid and hematological, cancer entities and by now is viewed as a "hallmark" of cancer cells. In this review we summarize basics in centrosome biology and what is known about the emergence of amplified centrosomes. In addition, we discuss how centrosome amplification might cause aneuploidy thereby leading to malignant transformation of cells. Furthermore, we present recent insights into the role of centrosome amplification in tumor formation based on work in model systems.     

Aurora A与妇科恶性肿瘤发病关系的研究现状

Aurora A是参与有丝分裂的丝氨酸／苏氨酸激酶新成员,与中心体功能相关,其高表达与中心体异常、非整倍体以及染色体不稳定性之间存在很大程度的相关性,是一种癌基因,在许多肿瘤中均发现有高表达。随着Aurora A过表达可以产生非整倍体以及与肿瘤形成有关,Aurora A已经成为药物设计的一个潜在靶点,Aurora A激酶抑制剂,如VX-680代表了一种新的治疗癌症的方法。该文着重综述Aurora A与妇科恶性肿瘤发病关系的研究现状。