Somatic mutation of the hBUB1 mitotic checkpoint gene in primary lung cancer

Mutations in mitotic checkpoint genes have been detected in several human cancers, and these cancers exhibit chromosomal instability. Aneuploid stem cells seem to result from chromosomal instability and have been reported in many lung cancers. To determine whether alteration of mitotic checkpoint regulators is involved in carcinogenesis and tumor progression in primary lung cancer, we screened the genomic DNA sequence of 30 human lung cancer cell lines and 30 primary lung cancer tumors for a mutation in the hBUB1 mitotic checkpoint gene. First, we designed 26 sets of intron-based primers to amplify each of the 25 exons of the hBUB1 gene to examine the entire coding region of the hBUB1 gene. Using these primers, we performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis as well as direct sequencing in the mutation analysis of the hBUB1 gene. Three different nucleotide substitutions were detected in the coding region of the hBUB1 gene in some of the cancer cell lines and primary tumors as follows. The hBUB1 gene of one adenocarcinoma tumor contained a somatic missense mutation, a cytosine-to-guanine substitution in codon 51 of exon 5 that resulted in a histidine-to-aspartic acid amino acid substitution. The hBUB1 gene of three lung cancer cell lines contained a thymine-to-cytosine substitution in codon 430 of exon 12, which did not result in an amino-acid substitution. We were unable to determine whether the nucleotide substitution in exon 12 was a polymorphism or a silent mutation because matched normal tissue was not available. A polymorphism in codon 93 of exon 4, a guanine-to-thymine substitution, in hBUB1 was found in one lung cancer cell line and one primary lung tumor. This is the first report of a somatic missense mutation of a gene involved in a mitotic checkpoint in primary lung cancer. The presence of a point mutation in the hBUB1 gene is consistent with the hypothesis that alteration of mitotic checkpoint genes is involved in the development of primary lung cancers. Because the frequency of hBUB1 gene mutations was low, future studies should focus on other mechanisms of inactivation of the hBUB1 gene as well as mutation analysis of other mitotic checkpoint genes in lung cancers.     

Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer

Cytogenetic analyses have revealed that many aneuploid breast cancers have cell-to-cell variations of chromosome copy numbers, suggesting that these neoplasms have instability of chromosome numbers. To directly test for possible chromosomal instability in this disease, we used fluorescent in situ hybridization to monitor copy numbers of multiple chromosomes in cultures of replicating breast cancer-derived cell lines and nonmalignant breast epithelial cells. While most (7 of 9) breast cancer cell lines tested are highly unstable with regard to chromosome copy numbers, others (2 of 9 cell lines) have a moderate level of instability that is higher than the "background" level of normal mammary epithelial cells and MCF-10A cells, but significantly less than that seen in the highly unstable breast cancer cell lines. To evaluate the potential role of a defective mitotic spindle checkpoint as a cause of this chromosomal instability, we used flow cytometry to monitor the response of cells to nocodazole-induced mitotic spindle damage. All cell lines with high levels of chromosomal instability have defective mitotic spindle checkpoints, whereas the cell lines with moderate levels of chromosomal instability (and the stable normal mammary cells and MCF10A cells) arrest in G(2) when challenged with nocodazole. Notably, the extent of mitotic spindle checkpoint deficiency and chromosome numerical instability in these cells is unrelated to the presence or absence of p53 mutations. Our results provide direct evidence for chromosomal instability in breast cancer and show that this instability occurs at variable levels among cells from different cancers, perhaps reflecting different functional classes of chromosomal instability. High levels of chromosomal instability are likely related to defective mitotic checkpoints but not to p53 mutations.     

SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK activation-dependent p300 decrease, eventually leading to apoptosis

We previously reported that sirtuin 2 (SIRT2), a mammalian member of the NAD+-dependent protein deacetylases, participates in mitotic regulation, specifically, in efficient mitotic cell death caused by the spindle checkpoint. Here, we describe a novel function of SIRT2 that is different from mitotic regulation. SIRT2 down-regulation using siRNA caused apoptosis in cancer cell lines such as HeLa cells, but not in normal cells. The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation-dependent degradation of p300 and the subsequent MDM2 degradation. Sirtuin inhibitors are emerging as antitumor drugs, and this function has been ascribed to the inhibition of SIRT1, the most well-characterized sirtuin that deacetylases p53 to promote cell survival and also binds to other proteins in response to genotoxic stress. This study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.     

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.     

Targeting Mitosis for Anti-Cancer Therapy

Basic research that has focused on achieving a mechanistic understanding of mitosis has provided unprecedented molecular and biochemical insights into this highly complex phase of the cell cycle. The discovery process has uncovered an ever-expanding list of novel proteins that orchestrate and coordinate spindle formation and chromosome dynamics during mitosis. That many of these proteins appear to function solely in mitosis makes them ideal targets for the development of mitosis-specific cancer drugs. The clinical successes seen with anti-microtubule drugs such as taxanes and the vinca alkaloids have also encouraged the development of drugs that specifically target mitosis. Drugs that selectively inhibit mitotic kinesins involved in spindle and kinetochore functions, as well as kinases that regulate these activities, are currently in various stages of clinical trials. Our increased understanding of mitosis has also revealed that this process is targeted by inhibitors of farnesyl transferase, histone deacetylase, and Hsp90. Although these drugs were originally designed to block cell proliferation by inhibiting signaling pathways and altering gene expression, it is clear now that these drugs can also directly interfere with the mitotic process. The increased attention to mitosis as a chemotherapeutic target has also raised an important issue regarding the cellular determinants that specify drug sensitivity. One likely contribution is the mitotic checkpoint, a failsafe mechanism that delays mitotic exit so that cells whose chromosomes are not properly attached to the spindle have extra time to correct their errors. As the biochemical activity of the mitotic checkpoint is finite, cells cannot indefinitely sustain the delay, as in cases where cells are treated with anti-mitotic drugs. When the mitotic checkpoint activity is eventually lost, cells will exit mitosis and become aneuploid. While many of the aneuploid cells may die because of massive chromosome imbalance, survivors that continue to proliferate will no doubt be selected. This is clearly an undesirable outcome, thus efforts to obtain fundamental insights into why some cells that arrest in mitosis die without exiting mitosis will be exceedingly important in enhancing our understanding of the drug sensitivity of cancer cells.     

Mouse hematopoietic stem cells, unlike human and mouse embryonic stem cells, exhibit checkpoint-apoptosis coupling

Previously, we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human embryonic stem cells (ESCs). This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex vivo hematopoietic stem cell (HSC) expansion. Here we address, for the first time, whether HSCs are more like ESCs or somatic cells with respect to SAC-apoptosis coupling. Using multiparametric permeablized cell flow-cytometric analysis to identify and analyze the mouse sca 1(+)/c-kit(+)/lin(-) (LSK) population, we found the mitotic spindle checkpoint to be functional in primary murine LSK cells, a population enriched in primitive hematopoietic stem/progenitor cells, after prolonged activation of the SAC by microtubule-depolymerizing agents such as nocodazole. HSCs can efficiently initiate apoptosis after activation of the SAC in LSK cells as indicated by increased hypodiploidy and increased levels of activated caspase 3, suggesting that HSCs behave more like somatic cells instead of ESCs with respect to this important cell cycle checkpoint. We conclude that mouse HSCs are not subject to the same kinds of chromosomal instability as are ESCs, knowledge that might aid in optimizing in vitro culture and expansion of human bone marrow or cord blood HSC for clinical applications.     

BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells

BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (?-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.     

A double missense variation of the BUB1 gene and a defective mitotic spindle checkpoint in the pancreatic cancer cell line Hs766T

To determine sequence variations of the BUB1 and BUB1B genes in pancreatic cancer, the entire coding regions of the BUB1 and BUB1B genes were sequenced in pancreatic cancer cell lines and xenografts. Although only polymorphic alterations were found in the BUB1B gene, the aneuploid pancreatic cell line Hs766T had two novel missense variants (p.[Y259C;H265N]) in the BUB1 gene. These mutations were on the same allele, accompanied by a wild-type BUB1 allele. This change was not found in other samples, the literature, or 110 additional chromosomes from a reference population. Compared to two cell lines having microsatellite instability (MIN), the TP53 wild-type pancreatic cell line Hs766T had a defective mitotic spindle checkpoint, indicative of a cell line with chromosomal instability (CIN). Evidence that this checkpoint pathway can be abrogated by mutations in the BUB1 gene (Cahill et al., 1998) supports the suggestion the missense mutations of the BUB1 gene in the Hs766T cell line may contribute to its observed mitotic checkpoint defect.     

The role of survivin as a new target of diagnosis and treatment in human cancer

Survivin has recently been identified as a novel inhibitor of apoptosis (IAP). Unlike other members of the IAP family, survivin is characterized by a unique structure that contains a single baculovirus IAP repeat and no really interesting new gene (RING) finger motifs, and it is expressed in many common human cancers, but not in normal tissues. Survivin regulates the G(2)/M phase of the cell cycle by associating with mitotic spindle microtubules, and it directly inhibits caspase-3 and caspase-7 activity. During tumorigenesis, survivin expression is inversely correlated with apoptosis inhibition and positively correlated with proliferation and angiogenesis. Inhibition of apoptosis by survivin predicts poor prognosis and shorter survival in human cancers. The molecular detection of occult cancer by the targeting of survivin as a novel molecular marker is useful, and micrometastasis detected by immunohistochemical staining for survivin reveals inhibition of apoptosis and the acceleration of cell proliferation. In in-vitro and in-vivo studies, survivin targeting with antisense and survivin mutants induces apoptosis, reduces tumor growth potential, and sensitizes cells to chemotherapeutic drugs and X-irradiation. These results suggest that survivin may have the potential to function as a new target for the diagnosis and treatment of cancer.     

Aneuploidy and malignancy: an unsolved equation

Aneuploidy is frequently noted in malignant tumours. There is much controversy about its cause and effect in relation to malignant tumours. Failure of the spindle checkpoint caused by mutation of the responsible genes may be one of the important factors for the development of aneuploidy. Telomere dysfunction may also be a possible source of failure of cytokinesis resulting in aneuploidy. Evidence such as tumour specific aneuploidy, presence of aneuploidy in various preneoplastic conditions, increased frequency of genetic instability in aneuploid cell lines compared with diploid cells, and mutation of mitotic checkpoint genes suggests that aneuploidy possibly plays an active role in carcinogenesis. In this brief review, the various aspects of aneuploidy with special emphasis on its mechanism of development and impact on progression of cancer are discussed.     

Polo-box domain inhibitor poloxin activates the spindle assembly checkpoint and inhibits tumor growth in vivo

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first small-molecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.     

Role of DNA mismatch repair in apoptotic responses to therapeutic agents

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.     

Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation

Karyotype and fluorescence in situ hybridization analyses have demonstrated the frequent presence of an altered static state of the number of chromosomes (ie, aneuploidy) in lung cancer, but it has not been directly established whether aneuploidy is in fact associated with a persistent increase in the rate of chromosomal losses and gains (ie, chromosome instability, or CIN). The study presented here used a panel of 10 lung cancer cell lines to provide for the first time direct evidence that CIN is a common feature in lung cancer cell lines in association with the presence of significant aneuploidy. In addition, we found that the CIN phenotype correlates well with the presence of p53 mutations. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactivation of p53 itself is unlikely to directly induce CIN in lung cancer cells. Interestingly, however, significant CIN could be induced in conjunction with the generation of aneuploid populations when the mitotic spindle formation was transiently abrogated in p53-inactivated cells. These results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cycle under certain forms of mitotic stresses, which would result in the induction of the CIN phenotype in conjunction with the generation of aneuploidy.     

Inhibitory effects of bisphosphonates on the proliferation of human ovarian cancer cell lines and the mechanism

Bisphosphonates are now well established as successful agents for the prevention and treatment of postmenopausal osteoporosis and corticosteroid-induced bone loss. Bisphosphonates have also recently become important in the management of cancer-induced bone disease, and they now have a widely recognized role for patients with multiple myeloma and bone metastases secondary to breast cancer and prostate cancer. Recent studies suggest that, besides the strong antiosteoclastic activity, the efficacy of such compounds in the oncological setting could also be due to direct anti-tumor effect. However, the effect of bisphosphonates to ovarian and endometrial cancers has not been elucidated. Thus, we examined the direct effect of bisphosphonates on the various ovarian cancer cell lines. Except for etidronate, all of bisphosphonates examined had the direct inhibitory effects on proliferation of all ovarian cancer cell lines used. Especially, pamidronate had the most marked inhibitory effect and inhibited dose-dependently the proliferation of ovarian cancer cell lines. KFr 13 cells among ovarian cancer cell lines used was the most sensitive to pamidronate and the caspase 3 activity was markedly stimulated by treatment with pamidronate, suggesting induction of apoptosis.     

Expression of BUB1 protein in gastric cancer correlates with the histological subtype, but not with DNA ploidy or microsatellite instability

During mitosis, the spindle checkpoint delays the onset of anaphase until all chromosomes have attached properly to the mitotic spindle, preventing chromosome missegregation. BUB (budding uninhibited by benzimidazole) 1 is one of the key components of this checkpoint. BUB1 mutations are rare in cancer tissues and no mutations have been identified in gastric cancer. In mice, immunodepletion of BUB1 abolished the spindle checkpoint. Thus, aberrant expression of BUB1 protein could impair mitotic checkpoint function, resulting in aneuploidy, a common phenomenon in gastric cancer. In the present study, an antibody was generated against BUB1 and its expression was studied in gastric cancer tissue sections (n = 80) by immunohistochemistry. Nuclear BUB1 expression was found in all gastric cancer cases. The proportion of tumour cells expressing BUB1 was significantly greater in diffuse-type than in intestinal-type gastric carcinoma (p < 0.001). No correlation was found between BUB1 expression and deoxyribonucleic acid (DNA) ploidy, microsatellite instability or any other histopathological parameters investigated. To the authors' knowledge, this is the first study of BUB1 protein expression in gastric cancer tissues. Different BUB1 protein expression levels in intestinal- and diffuse-type gastric cancer may provide further evidence of a potential link between different genetic pathways and morphological phenotype in gastric carcinogenesis. However, further studies are needed to establish whether there is an association between BUB1 protein expression level and mitotic spindle checkpoint function in gastric cancer.     

Increased chromosome instability but not cancer predisposition in haploinsufficient Bub3 mice

Mitotic spindle checkpoint proteins have been shown to play a crucial role in the accurate segregation of chromosomes during cell division. Bub3 is a member of a group of mitotic checkpoint proteins that are essential for this process. To investigate the role of Bub3 in chromosome segregation and cancer development, we analyzed haploinsufficient cells in mice. Heterozygous Bub3 embryonic fibroblasts displayed increased aneuploidy and premature sister-chromatid separation. In addition, when challenged with the microtubule disruptor nocodazole, the cells showed a slight increase in chromatid breakage and a decrease in the mitotic index. No substantial differences were observed between wild-type and Bub3 heterozygous mice in the frequency or the rate at which tumors appeared. Crossing Bub3(+/-) mice onto a heterozygous tumor-suppressor background of Trp53 or Rb1 similarly revealed no substantial differences in either the number or the rate at which tumors appeared. These results suggest that haploinsufficiency of Bub3 causes a slight increase in chromosome instability but is not clearly associated with a noticeable rise in the probability of tumor formation in the animal, possibly because of a partially functional mitotic checkpoint, or cells exhibiting chromosome instability could have activated the apoptosis pathway and thus escaped tumor induction and detection.     

Overexpression of BUBR1 is associated with chromosomal instability in bladder cancer

BUBR1, a mitotic checkpoint protein, is a key component of the mitotic spindle checkpoint machinery. Defective BUBR1 has been proposed to contribute to chromosomal instability (CIN). To elucidate the relationship of BUBR1 expression with CIN, expression of BUBR1, numbers of centrosomes, numerical aberrations of chromosomes, and DNA ploidy were examined, and BUBR1 expression status was compared with clinicopathological parameters in 104 human urothelial bladder carcinomas. Expression of BUBR1 and numbers of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 were evaluated by fluorescence in situ hybridization. Cancers with a large intercellular variation in centromere copy number were designated as CIN cancers. Tumors with BUBR1 overexpression were associated with CIN, DNA aneuploidy, and centrosome amplification. Array CGH revealed that BUB1B amplification and loss rarely occurred, indicating that the overexpression of BUBR1 in these bladder cancers was independent of BUB1B copy number. Overexpression of BUBR1 significantly correlated with higher histological grade, advanced pathological stage, and high cell proliferation. Overexpression of BUBR1 predicted tumor recurrence and disease progression. These data suggest that overexpression of BUBR1 is potentially a new tumor marker for estimating biological characteristics of bladder cancer.