Centrosome Amplification In Cells Research Articles

Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells.

Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.

Resveratrol attenuates diabetes-associated cell centrosome amplification via inhibiting the PKCα-p38 to c-myc/c-jun pathway.

Type 2 diabetes increases the risk for cancer. Centrosome amplification can initiate tumorigenesis. We have described that type 2 diabetes increases the centrosome amplification of peripheral blood mononuclear cells, with high glucose, insulin, and palmitic acid as the triggers, which suggests that centrosome amplification is a candidate biological mechanism linking diabetes to cancer. In this study, we aimed to further investigate the signaling pathways of the diabetes-associated centrosome amplification and to examine whether and how resveratrol inhibits the centrosome amplification. The results showed that treatment with high glucose, insulin, and palmitic acid, alone or in combination, could increase the protein levels of phospho-protein kinase C alpha (p-PKCα), phospho-p38 mitogen-activated protein kinases (p-p38), c-myc, and c-jun, as well as the mRNA levels of c-myc and c-jun. PKCα inhibitor could inhibit the treatment-induced increase in the protein levels of p-p38, c-myc, and c-jun. Inhibitor or siRNA of p38 was also able to inhibit the treatment-induced increase in the levels of p-p38, c-myc, and c-jun. Meanwhile, knockdown of c-myc or c-jun did not alter the treatment-induced increase in the phosphorylation of PKCα or p38. Importantly, inhibition of the phosphorylation of PKCα or p38 and knockdown of c-myc or c-jun could attenuate the centrosome amplification. In diabetic mice, the levels of p-PKCα, p-p38, c-myc, and c-jun were all increased in the colon tissues. Interestingly, resveratrol, but not metformin, was able to attenuate the treatment-induced increase in the levels of p-PKCα, p-p38, c-myc, and c-jun, as well as the centrosome amplification. In conclusion, our results suggest that PKCα-p38 to c-myc/c-jun is the signaling pathway of the diabetes-associated centrosome amplification, and resveratrol attenuates the centrosome amplification by inhibiting this signaling pathway.

Secreted Wnt6 mediates diabetes-associated centrosome amplification via its receptor FZD4.

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of β-catenin. The treatment also activated β-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or β-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-β-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of β-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

PPARγ promotes diabetes-associated centrosome amplification via increasing the expression of SKA1 directly at the transcriptional level.

We haverecently published that type 2 diabetes can induce cell centrosome amplification due to the action of high glucose, palmitic acid, and insulin, and ROCK1 and 14-3-3σ are signal mediators. In this study, we further investigated the molecular mechanisms of the centrosome amplification in colon cancer HCT116 cells. Treatment of the cells with high glucose, palmitic acid, and insulin increased the expression of peroxisome proliferator-activated receptor γ (PPARγ) as well as the spindle and kinetochore associated protein 1 (SKA1), knockdown of each of which resulted in the inhibition of the treatment-triggered centrosome amplification. Knockdown of PPARγ inhibited the treatment-evoked increase in the SKA1 level, whereas knockdown of SKA1 did not modify the treatment-increased PPARγ level. We found a predicted binding site for PPARγ in the promoter region of the SKA1 gene from the JASPAR database. Experimental results showed that the treatment increased the messenger RNA level of SKA1, which could be inhibited by PPARγ chemical inhibitor or small interfering RNA. Moreover, we were able to show that PPARγ could bind to the binding site in the SKA1 gene promoter, which was increased by the experimental treatment. In conclusion, it is suggested that the pathophysiological factors in type 2 diabetes, high glucose, palmitic acid, and insulin, induce the cell centrosome amplification through the PPARγ-SKA1 pathway, in which PPARγ increases the expression of SKA1 via directly enhancing the SKA1 gene transcription.

Cadmium induces cell centrosome amplification via reactive oxygen species as well as endoplasmic reticulum stress pathway.

There is evidence that cadmium can initiate carcinogenesis. However, the underlying mechanisms remain unknown. There is also evidence that moderate centrosome amplification can initiate tumorigenesis. The present study investigated whether cadmium could trigger cell centrosome amplification, and examined the underlying molecular mechanisms. We found that cadmium was able to cause cell centrosome amplification at the subtoxic concentrations, in a dose-dependent manner. It could cause centrosome amplification via the signaling of reactive oxygen species (ROS). Proteomic analysis revealed that cadmium caused differential expressions of three proteins, which included HSPA1A which is associated with endoplasmic reticulum (ER) stress. Western blot analysis confirmed that cadmium upregulated HSPA1A. Further analyses showed that cadmium upregulated Bip and decreased the phosphorylation of ASK1 as well as increased the phosphorylation of MKK7 and c-Jun N-terminal kinases (JNK). Knockdown of JNK2 using small interfering RNA inhibited the cadmium-induced centrosome amplification but not the level of ROS. N-acetylcysteine did not inhibit the cadmium-activated ER stress pathway. In conclusion, our results suggest that cadmium can induce cell centrosome amplification via ROS as well as ER stress through the Bip-TRAF2-ASK1-MKK7-JNK signaling route, in parallel. More studies are required to clarify whether centrosome amplification underlies cadmium-induced carcinogenesis.

Type 2 Diabetes Promotes Cell Centrosome Amplification via AKT-ROS-Dependent Signalling of ROCK1 and 14-3-3σ

Background/Aims: Type 2 diabetes is associated with oxidative stress and DNA damage which can cause centrosome amplification. Thus, the study investigated centrosome amplification in type 2 diabetes and the underlying mechanisms. Methods: Centrosome numbers in human peripheral blood mononuclear blood cells (PBMC) from healthy subjects and patients with type 2 diabetes were compared to access the association between type 2 diabetes and centrosome amplification. Colon cancer cells were used to investigate the molecular mechanisms underlying the centrosome amplification triggered by high glucose, insulin and palmitic acid. Western blot analysis was used to quantify the level of protein and protein phosphorylation. Immunofluorescent staining was performed to detect centrosomes. ROS was quantified using flow cytometry technique. Transcriptpmic profiling was performed using Illumina HiSeq^TM500 platform. Results: We found that centrosome amplification was increased PBMC from the type 2 diabetic patients, which correlated with the levels of fasting blood glucose and HbA1c. High glucose, insulin and palmitic acid, alone or in combinations, induced ROS production and centrosome amplification. Together, they increased AKT activation as well as the expression, binding and centrosome translation of ROCK1 and 14-3-3σ. Results from further analyses showed that AKT-ROS-dependent upregulations of expression, binding and centrosome translocation of ROCK1 and 14-3-3σ was the molecular pathway underlying the centrosome amplification in vitro triggered by high glucose, insulin and palmitic acid. Moreover, the key in vitro molecular signalling events activated by high glucose, insulin and palmitic acid were verified in PBMC from the patients with type 2 diabetes. Conclusion: Our results show that type 2 diabetes promotes cell centrosome amplification, and suggest that the diabetic pathophysiological factors-activated AKT-ROS-dependent signalling of ROCK1 and 14-3-3σ is the underlying molecular mechanism.

Repeated cleavage failure does not establish centrosome amplification in untransformed human cells

We tested whether cleavage failure as a transient event establishes an incidence of centrosome amplification in cell populations. Five rounds of ∼30% cytochalasin-induced cleavage failure in untransformed human cell cultures did not establish centrosome amplification in the short or long terms. The progeny of binucleate cells progressively dropped out of the cell cycle and expressed p53/p21, and none divided a fourth time. We also tested whether cleavage failure established centrosome amplification in transformed cell populations. Tetraploid HCT116 p53(-/-) cells eventually all failed cleavage repeatedly and ceased proliferating. HeLa cells all died or arrested within four cell cycles. Chinese hamster ovary cells proliferated after cleavage failure, but five rounds of induced cleavage failure produced a modest increase in the incidence of centrosome amplification in the short term, which did not rise with more cycles of cleavage failure. This incidence dropped to close to control values in the long term despite a 2-6% rate of spontaneous cleavage failure in the progeny of tetraploid cells.

Abstract 131: Biological effect of miR-210 overexpression on renal carcinoma cells

Abstract MiR-210 is significantly upregulated in clear cell renal cell carcinoma (CCC), but the mechanism and biological consequences of miR-210 upregulation are poorly understood. Here, we show that miR-210 is highly expressed in renal carcinoma cell lines and that its expression is clearly correlated with accumulation of hypoxia inducible factor 1α (HIF1α) under normoxia as well as hypoxia, suggesting that miR-210 upregulation in renal carcinoma cells is most likely due to accumulation of HIF1α. To reveal the effects of miR-210 upregulation, the miR-210 precursor was transfected into renal carcinoma cells. After transfection, the cells accumulated at G2/M phase of the cell cycle and their viability was decreased, suggesting that miR-210 overexpression may trigger an event that hinders normal cell division. Immunocytochemistry demonstrated a multipolar spindle accompanied by centrosome amplification in cells overexpressing miR-210. It has been reported that centrosome amplification induces chromosome mis-segregation, finally leading to chromosome instability and aneuploidy. Indeed, the proportion of aneuploid cells (>4n) was increased in miR-210 overexpressed cells. By using the TargetScan and PicTar algorithms, E2F3 was identified as one of the possible targets of miR-210, and was suppressed at the protein level by miR-210. Moreover, the proportion of aneuploid cells was increased in E2F3 siRNA transfected cells. On the basis of these results, we propose that miR-210 upregulation due to HIF1α accumulation may induce aneuploidy via E2F3 downregulation at least in part, and may play a role in tumorigenesis and/or progression of CCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 131. doi:10.1158/1538-7445.AM2011-131

Overexpression of miR‐210, a downstream target of HIF1α, causes centrosome amplification in renal carcinoma cells

MiR-210 is significantly up-regulated in clear cell renal cell carcinoma (CCC), but the mechanism and biological consequences of miR-210 up-regulation are poorly understood. Here, we show that miR-210 is highly expressed in renal carcinoma cell lines and that its expression is clearly correlated with accumulation of hypoxia-inducible factor 1α (HIF1α) under normoxia as well as hypoxia, suggesting that miR-210 up-regulation in renal carcinoma cells is most likely due to accumulation of HIF1α. To reveal the effects of miR-210 up-regulation, the miR-210 precursor was transfected into renal carcinoma cells. After transfection, the cells accumulated at the G2/M phase of the cell cycle and their viability was decreased, suggesting that miR-210 overexpression may trigger an event that hinders normal cell division. Immunocytochemistry demonstrated a multipolar spindle accompanied by centrosome amplification in cells overexpressing miR-210. It has been reported that centrosome amplification induces chromosome mis-segregation, finally leading to chromosome instability and aneuploidy. Indeed, the proportion of aneuploid cells (>4n) was increased in miR-210 overexpressed cells. By using the TargetScan and PicTar algorithms, E2F3 was identified as one of the possible targets of miR-210 and was suppressed at the protein level by miR-210. Moreover, the proportion of aneuploid cells was increased in E2F3 siRNA transfected cells. On the basis of these results, we propose that miR-210 up-regulation due to HIF1α accumulation may induce aneuploidy via E2F3 down-regulation at least in part, and may play a role in tumourigenesis and/or progression of CCC.

Centrosome amplification induced by the antiretroviral nucleoside reverse transcriptase inhibitors lamivudine, stavudine, and didanosine

In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division.

Roles of cyclins A and E in induction of centrosome amplification in p53-compromised cells

Abnormal amplification of centrosomes, which occurs frequently in cancers, leads to high frequencies of mitotic defect and chromosome segregation error, profoundly affecting the rate of tumor progression. Centrosome amplification results primarily from overduplication of centrosomes, and p53 is involved in the regulation of centrosome duplication partly through controlling the activity of cyclin-dependent kinase (CDK) 2-cyclin E, a kinase complex critical for the initiation of centrosome duplication. Thus, loss or mutational inactivation of p53 leads to an increased frequency of centrosome amplification. Moreover, the status of cyclin E greatly influences the frequency of centrosome amplification in cells lacking functional p53. Here, we dissected the roles of CDK2-associating cyclins, namely cyclins E and A, in centrosome amplification in the p53-negative cells. We found that loss of cyclin E was readily compensated by cyclin A for triggering the initiation of centrosome duplication, and thus the centrosome duplication kinetics was not significantly altered in cyclin E-deficient cells. It has been shown that cells lacking functional p53, when arrested in either early S-phase or late G(2) phase, continue to reduplicate centrosomes, resulting in centrosome amplification. In cells arrested in early S phase, cyclin E, but not cyclin A, is important in centrosome amplification, whereas in the absence of cyclin E, cyclin A is important for centrosome amplification. In late G(2)-arrested cells, cyclin A is important in centrosome amplification irrespective of the cyclin E status. These findings advance our understandings of the mechanisms underlying the numeral abnormality of centrosomes and consequential genomic instability associated with loss of p53 function and aberrant expression of cyclins E and A in cancer cells.

Gadd45a Interacts with Aurora-A and Inhibits Its Kinase Activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.

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Abnormal centrosome amplification in cells through the targeting of Ran-binding protein-1 by the human T cell leukemia virus type-1 Tax oncoprotein

Human T cell leukemia virus type-1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T cell leukemia. The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. Because adult T cell leukemia like many other human cancers is a disease of genomic instability with frequent gains and losses of chromosomes, to understand this disease it is important to comprehend how HTLV-1 engenders aneuploidy in host cells. In this regard, loss of cell cycle checkpoints permits tolerance of aneuploidy but does not explain how aneuploidy is created. We show here that HTLV-1 Tax causes abnormal centrosome fragmentation in the mitotic phase of the cell cycle. We report that Tax directly binds Ran and Ran-binding protein-1, locates to centrosomes/spindle poles, and causes supernumerary centrosomes.

Induction of centrosome amplification and chromosome instability in p53-null cells by transient exposure to subtoxic levels of S-phase-targeting anticancer drugs.

Chromosome instability (CIN) is one of the most important phenotypes in tumor progression, introducing multiple mutations required for acquisition of further malignant characteristics. Abnormal amplification of centrosomes, which is frequently observed in human cancer, has been shown to contribute to CIN by increasing the frequency of mitotic defects. Here, we show that transient exposure to subtoxic concentrations of commonly used anticancer drugs that target DNA synthesis induces centrosome amplification in cells lacking p53 tumor suppressor protein, by allowing continuous centrosome duplication in the absence of DNA synthesis. When these cells are released from cell cycle arrest by removal of drugs, cells suffer extensive destabilization of chromosomes. Considering that p53 is the most frequently mutated gene in human cancer and that CIN is known to be associated with acquisition of malignant phenotypes, our observations may explain why recurrent tumors, after chemotherapy, often exhibit more malignant characteristics than the original tumors. The tumor cells that are exposed to subtoxic levels of DNA synthesis-targeting drugs will be arrested and undergo centrosome amplification. Upon cessation of chemotherapy, these cells will re-enter cell cycling, and experience extensive CIN due to the presence of amplified centrosomes. This in turn promotes generation of tumor cells equipped with further malignant characteristics.

Activation of Cdc2/cyclin B and inhibition of centrosome amplification in cells depleted of Plk1 by siRNA.

The events of the cell cycle, the stages at which the cell proliferates and divides, are facilitated and controlled by multiple signaling pathways. Among the many regulatory enzymes that contribute to these processes is the polo-like kinase (Plk). Plks have been reported to mediate multiple mitotic processes, including bipolar spindle formation, activation of Cdc25C, actin ring formation, centrosome maturation, and activation of the anaphase-promoting complex. To investigate its functions in mammalian cells further, we used the recently developed small interfering RNA technique specifically to deplete Plk1 in cultured cells. We find that Plk1 depletion results in elevated Cdc2 protein kinase activity and thus attenuates cell-cycle progression. About 45% of cells treated with Plk1 small interfering RNA show the formation of a dumbbell-like DNA organization, suggesting that sister chromatids are not completely separated. About 15% of these cells do complete anaphase but do not complete cytokinesis. Finally, Plk1 depletion significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells. These data provide direct evidence that Plk is required for multiple mitotic processes in mammalian cells and their significance is discussed.