Mitotic Checkpoint Regulator Research Articles

The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions

We previously demonstrated that a fraction of the human Nup107-160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107-160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107-160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107-160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore-microtubule attachment defects. Finally, we show that the presence of the Nup107-160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1-RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107-160 complex at kinetochores.

Differential Expressions of Genes in Colorectal Cancer with Liver Metastasis by using a cDNA Microarray Chip: A Pilot Study

Purpose: Liver metastasis is the most common type of failure in the treatment of colorectal cancer. The identification of differential expressions of genes in colorectal cancer and liver metastasis is important to differentiate the genetic mechanism of carcinogenesis and liver metastasis from that of a normal mucosa. The aim of this study is to find candidate genes playing roles in liver metastasis of colorectal cancer by using cDNA microarray. Methods: We screened a group of genes differentially expressed in a normal mucosa and in cancer and liver metastasis by using a 4.7 K cDNA microarray chip in 8 patients with far advanced colorectal cancer from Jan 2003 to May 2004 at Kyungpook National University Hospital. Results: A comparison of mRNA expressions of genes in normal mucosa vs. cancer, normal mucosa vs. liver metastasis, and cancer vs. liver metastasis, 76 and 27 known and unknown genes were significantly over-expressed in cancer and liver metastasis, respectively. Also 62 and 26 genes were down- regulated in cancer and liver metastasis. Among those genes, TIMP-1, SRY-box9, Rattus norvegicus fibronectin 1, mitotic check point regulator, etc. were constantly up- regulated in cancer or metastasis, and hsgk, etc. were down-regulated in cancer or liver metastasis. Conclusions: The cDNA microarray chip technique could be a useful for robust screening of candidate genes involved in carcinogenesis or metastasis of colorectal cancer.

Breast cancer risk associated with genotypic polymorphism of the mitotic checkpoint genes: a multigenic study on cancer susceptibility

Aneuploidy occurs early during tumorigenesis and may contribute to tumor formation. Tumor cells become aneuploid as a result of aberrant mitotic divisions, suggesting a tumorigenic contribution of the mechanisms in maintaining chromosomal number stability. We therefore speculated that the genes TTK, MAD2L1, BUB1, BUB1B and PTTG1 (Securin), jointly implicated in the regulation of mitotic checkpoint, might be associated with breast tumorigenesis. To test this hypothesis, this case-control study of 698 primary breast cancer patients and 1492 healthy controls examined single-nucleotide polymorphisms (SNPs) in these mitotic checkpoint genes to define their tumorigenic contribution. Because estrogen is known to promote breast cancer development via its mitogenic effect leading to malignant proliferation of breast epithelium and the mitotic checkpoint genes are involved in regulating mitosis, we were also interested in knowing whether any association between genotypes and breast cancer risk was modified by reproductive risk factors. Support for these hypotheses came from the observations that (i) two SNPs in TTK and PTTG1 were associated with breast cancer risk; (ii) haplotype and haplotype combination analyses in TTK, BUB1B and PTTG1 revealed a strong association with breast cancer risk; (iii) a trend to an increased risk of breast cancer was found in women harboring a greater number of putative high-risk genotypes/haplotypes of mitotic checkpoint genes and (iv) a significant interaction between high-risk genotypes/haplotypes and reproductive risk factors in determining breast cancer risk was defined. This study provides new support for the mutator role of mitotic checkpoint genes in breast cancer development, suggesting that breast cancer could be driven by genomic instability associated with variant mitotic checkpoint genes, the tumorigenic contribution of which could be enhanced as a result of increased mitosis due to estrogen exposure.

Epigenetic inactivation of CHFR in nasopharyngeal carcinoma through promoter methylation

Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers. Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. Epigenetic inactivation of CHFR through promoter CpG hypermethylation may lead to CIN and has been reported in several human cancers. In this study, we investigated the CHFR gene expression in a panel of nasopharyngeal carcinoma (NPC), prostate, ovarian, and breast cancer cell lines. We found that the expression of CHFR mRNA was significantly decreased or undetectable in all eight NPC cell lines as well as three human NPC xenografts, whereas non-malignant nasopharyngeal cell lines and other cancer cell lines tested expressed CHFR at relatively high levels. Hypermethylation of CHFR promoter region was also strongly correlated with decreased CHFR expression in NPC cell lines and xenografts. Treatment with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, led to restoration of CHFR expression in NPC cell lines. More importantly, hypermethylation of CHFR promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non-malignant tissues. These findings suggest that downregulation of CHFR is a common event in NPC cells which may be due to hypermethylation of the gene promoter region.

Phenotypic and proteomic alterations of acquired trastuzumab resistance

3121 Background: Trastuzumab, a monoclonal antibody to the HER2 receptor tyrosine kinase leads to clinical responses as a single agent and improves survival when added to chemotherapy for advanced HER2-positive breast cancer. However, some patients do not respond to trastuzumab, and most eventually develop clinical resistance. Mechanisms of intrinsic and acquired trastuzumab resistance are poorly understood, and we therefore undertook a cell line-based approach to delineate genetic and protein alterations associated with resistance. Methods: Two HER2-positive breast cancer cell lines (BT474 and SKBR3) were serially passaged in the presence of trastuzumab until in vitro resistance was documented. DNA histograms and western blotting for signal transduction and cell cycle proteins were performed. Gene expression profiles before and after trastuzumab exposure were assessed using Affymetrix HG-U133A/B human gene arrays. Cell lysate analysis using differential chromatographic fractionation followed by matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) was also done using prOTOF 2000 (Perkin Elmer, Boston, MA). Results: Resistant cell lines emerged after 12 months and exhibited a 3-fold more rapid growth rate in the absence of trastuzumab. Following trastuzumab exposure, G0/G1 arrest was observed in sensitive compared to resistant cells (84 vs. 68%), with fewer cells in S-phase (3 vs. 14%). Resistant cell lines exhibited fewer changes in gene expression with trastuzumab as well as upregulation of the chemokine receptor CXCR4 and mitotic checkpoint regulators, and downregulation of PTEN compared to sensitive cells. Preliminary MALDI-MS data analyzed by an iterative search algorithm identified proteomic patterns that revealed differences in cell lines at baseline and after trastuzumab treatment; these are being further characterized and updated data will be presented. Conclusions: Acquired resistance to trastuzumab develops in HER2-positive human breast cancer cell lines. Intrinsic growth rates in the absence of trastuzumab are higher in resistant cells, with multiple alterations in signal transduction and cell cycle control observed. Causative changes could be targeted to reverse resistance. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech Genentech Genentech

Genomic instability and colon cancer.

Colorectal cancer affected approximately 135,000 people in the United States in 2001, resulting in 57,000 deaths. At the cellular level, colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations that lead to the transformation of normal colonic epithelial cells to colon adenocarcinoma cells. The loss of genomic stability appears to be a key molecular and pathogenetic step that occurs early in the tumorigenesis process and serves to create a permissive environment for the occurrence of alterations in tumor suppressor genes and oncogenes. At least three forms of genomic instability have been identified in colon cancer: (1) microsatellite instability (MSI), (2) chromosome instability (i.e. aneusomy, gains and losses of chromosomal regions) (CIN), and (3) chromosomal translocations. Microsatellite instability occurs in approximately 15% of colon cancers and results from inactivation of the mutation mismatch repair (MMR) system by either MMR gene mutations or hypermethylation of the MLH1 promoter. MSI promotes tumorigenesis through generating mutations in target genes that possess coding microsatellite repeats, such as TGFBR2 and BAX. CIN is found in the majority of colon cancers and leads to a different pattern of gene alterations that contribute to tumor formation. CIN appears to result primarily from deregulation of the DNA replication checkpoints and mitotic-spindle checkpoints. The mechanisms that induce and influence genomic instability in cancer in general and more specifically in colon cancer are only partly understood and are consequently under intense investigation. These studies have revealed mutation of the mitotic checkpoint regulators BUB1 and BUBR1 and amplification of STK15 in a subset of CIN colon cancers. The etiology of CIN in the other unexplained cases of colon cancer remains to be determined. Hopefully, discovery of the cause and specific role of genomic instability in colon cancer will yield more effective chemotherapy strategies that take advantage of this unique characteristic of cancer cells.

Alterations of anaphase-promoting complex genes in human colon cancer cells.

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation.

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

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.

MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy.

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1-1 mutation. Strains containing mob2Delta also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans.

We describe the isolation and characterization of a novel mouse gene, tsg24, which displays striking sequence similarities to the Aspergillus nidulans bimE gene. The bimE gene has been shown to be a mitotic checkpoint regulator, negatively regulating entry into mitosis in A. nidulans. The tsg24 gene was found to contain a long open reading frame of 1944 amino acids, encoding a polypeptide with a calculated molecular mass of 216,087 Da. We have developed an antibody directed against the product of the tsg24 gene and identified a protein with a molecular mass of approximately 200 kDa. This protein was found to be uniformly expressed throughout interphase of the cell cycle, whereas the level of this protein was lower in protein extracts prepared from mitotic cells.