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.     

Overexpression of the mitotic checkpoint genes BUB1 and BUBR1 is associated with genomic complexity in clear cell kidney carcinomas

Background: A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC).Methods: We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the MAD gene family (MAD1, MAD2L1, MAD2L2) by real-time quantitative PCR in 39 ccRCC and in 36 normal kidney tissue samples. We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC.Results: BUB1, BUBR1, MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls (BUB1, BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes (p<0.001 for both genes) and with Furhman grade of the tumors (p=0.006 and p=0.005, respectively).Conclusions: We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC.     

Functional defects in the fanconi anemia pathway in pancreatic cancer cells

Biallelic BRCA2-mutations can cause Fanconi anemia and are found in approximately 7% of pancreatic cancers. Recently, several sequence changes in FANCC and FANCG were reported in pancreatic cancer. Functional defects in the Fanconi pathway can result in a marked hypersensitivity to interstrand crosslinking agents, such as mitomycin C. The functional implications of mutations in the Fanconi pathway in cancer have not been fully studied yet; these studies are needed to pave the way for clinical trials of treatment with crosslinking agents of Fanconi-defective cancers. The competence of the proximal Fanconi pathway was screened in 21 pancreatic cancer cell lines by an assay of Fancd2 monoubiquitination using a Fancd2 immunoblot. The pancreatic cancer cell lines Hs766T and PL11 were defective in Fancd2 monoubiquitination. In PL11, this defect led to the identification of a large homozygous deletion in FANCC, the first cancer cell line found to be FANCC-null. The Fanconi-defective cell lines Hs766T, PL11, and CAPAN1 were hypersensitive to the crosslinking agent mitomycin C and some to cis-platin, as measured by cell survival assays and G(2)/M cell-cycle arrest. These results support the practical exploration of crosslinking agents for non-Fanconi anemia patients that have tumors defective in the Fanconi pathway.     

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.     

BUB1 infrequently mutated in human breast carcinomas

The BUB1 gene is a key player in the mitotic spindle checkpoint machinery that monitors proper segregation of sister chromatides during mitosis. It has been suggested that mutations in BUB1 may disrupt the spindle checkpoint and thereby cause chromosomal instability, which is a hallmark of solid tumors including those from the breast. From a series of breast carcinomas we selected 20 cases with genomic instability, as scored by Comparative Genome Hybridization (CGH), and without somatic TP53 (p53) mutations, and sequenced the entire coding region of the BUB1 gene. Two different constitutional sequence variants were found; a base substitution in exon 5, c.481G>A (CAG>CAA, a synonymous change encoding Gln144) in two samples, and a base substitution 8 bp upstream of exon 10, c.1007-8T>C in two other samples. No somatic mutations were detected. These results indicate that genomic instability scored as copy number alterations by CGH in TP53 wild type breast carcinomas is not caused by somatic mutations in the BUB1 gene.     

Allelic loss at 10q26 in osteosarcoma in the region of the BUB3 and FGFR2 genes

Loss of heterozygosity at 10q26 was mapped using microsatellite markers in 20 osteosarcomas. A four-megabase region centered on marker D10S587 was affected by allelic loss in 60 percent of osteosarcomas. The most frequently lost marker was D10S1723. Around 15 known genes are found in this region. The gene immediately adjacent to D10S1723 encodes BUB3, an element of the spindle assembly mitotic checkpoint. Loss of BUB3 function could contribute to chromosomal instability. The fibroblast growth factor receptor 2 (FGFR2) gene is located 2 Mb from the BUB3 gene and has the potential for a role in cancer. Inherited mutations of the FGFR2 gene result in skeletal dysplasias. FGFR2 alterations have also been implicated in gastric cancer. Human genome project data were used to design primers for amplifying FGFR2 in 18 genomic segments and BUB3 in 7 genomic segments. In each case, the segments encompassed coding exons and flanking intron sequences. The primers were used to search for mutations by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Several shifted bands were detected in the BUB3 exon 3 fragment. Sequencing resolved the BUB3 exon 3 fragment shifts into polymorphisms in intron 2. No mutations of BUB3 or FGFR2 were detected. It remains possible that BUB3 or FGFR2 hemizygosity alone contributes to osteosarcoma, or that one of the genes is cryptically inactivated by a higher-order modification or mutation outside the coding region. There may also be a yet undiscovered tumor suppressor gene in this region.     

Clinical and genetic heterogeneity in patients with mosaic variegated aneuploidy: delineation of clinical subtypes

Mosaic variegated aneuploidy (MVA) is a rare autosomal recessive syndrome related to BUB1B gene mutations and characterized by multiple mosaic aneuploidies, cancer predisposition, and a distinct phenotype. We report on two mildly affected sibs with MVA syndrome but without BUB1B mutation. Both patients exhibited growth retardation, frontal bossing, triangular face and micrognathia but not microcephaly or cancer. Aneuploidies were assessed both in G-banded metaphases from lymphocyte cultures and in interphase nuclei from buccal cells by FISH. Screening of 23 exons and intron-exon boundaries of BUB1B was also carried out. These patients were then compared with other 19 MVA patients screened for BUB1B mutations. Around one half of the cultured lymphocytes from our patients had aneuploidies ranging from nullisomies to heptasomies; the most frequent abnormalities were trisomies (42%) and monosomies (28%). FISH results demonstrated more chromosomal losses than gains. Screening of BUB1B in our two patients failed to identify any mutation. A review of the 21/35 patients screened for BUB1B demonstrated three clinical pictures. Patients with monoallelic BUB1B mutations were severely affected with Dandy-Walker complex (7/8), cataracts (6/6), and Wilms' tumor (7/8); premature chromatid separation (PCS) was observed in 8/8 propositi and 7/7 carrier parents. Patients without BUB1B mutations were mildly affected with no evidence of cancer, Dandy-Walker malformation or cataract, and rarely (1/7) showed PCS. Finally, patients with biallelic BUB1B mutations showed a moderate phenotype. The distinct MVA clinical groups delineated here point to involvement of at least another mitotic spindle checkpoint gene in addition to the BUB1B gene.     

Molecular cloning and characterization of OSR1 on human chromosome 2p24

During Drosophila hindgut development, bowl, caudal/CDX, brachyenteron/Brachyury/TBX, fork head/FOX, drumstick, lines, and wingless/WNT play important roles. Drosophila bowl gene is homologous to Drosophila odd-skipped (odd) gene and odd-skipped related gene (sob). Here, human OSR1, related to Drosophila odd, was isolated using bioinformatics and cDNA-PCR. OSR1 was found to encode 266 amino-acid protein with three C2H2-type zinc fingers, a tyrosine phosphorylation site (Tyr 203), and several putative PXXP SH3 binding motifs. Three zinc fingers and a tyrosine phosphorylation site were conserved among human OSR1, OSR2, Drosophila odd, sob, and bowl. OSR1 showed 63.6% total amino-acid identity with OSR2. OSR1 gene consisting of three exons was located on human chromosome 2p24. OSR1 mRNA of 2.3-kb in size was detected in adult colon, small intestine, prostate, testis, and fetal lung. OSR1 mRNA was significantly up-regulated in a pancreatic cancer cell line MIA PaCa-2, and was weakly expressed in gastric cancer cell lines OKAJIMA, MKN45, pancreatic cancer cell lines PANC-1, BxPC-3, AsPC-1, PSN-1, Hs766T, and esophageal cancer cell line TE10. Among 10 cases of primary gastric cancer, OSR1 mRNA was up-regulated in 5 cases, and was down-regulated in 2 cases. This is the first report on molecular cloning and characterization of human OSR1.     

Centrosome amplification and aneuploidy in bone marrow failure patients

Patients with bone marrow failure are at risk for development of hematopoietic progenitor clones with abnormal numbers of chromosomes (aneuploidy) and leukemia. Numerical centrosome abnormalities or mutations in genes associated with the mitotic spindle checkpoint (BUB1 and MAD2) are two important mechanisms that can induce abnormal mitosis resulting in aneuploid daughter cells. To assess the role of these mechanisms, we used fluorescence in situ hybridization techniques to determine aneuploidy and centrosome copy number and PCR-SSCP to identify gene mutations of BUB1 and MAD2 in marrow cells of 25 patients. No mutations were found in BUB1 or MAD2 genes. However, we found that cells with more than two centrosomes exhibited aneuploidy for three or more chromosomes. We conclude that centrosome amplification may be associated with the development of a clonal population of potentially preleukemic aneuploid cells.     

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.     

Expression changes of the MAD mitotic checkpoint gene family in renal cell carcinomas characterized by numerical chromosome changes

Papillary and chromophobe renal cell carcinomas are characterized by multiple trisomies and monosomies, respectively, but the molecular mechanisms behind the acquisition of these numerical chromosome changes are unknown. To evaluate the role of mitotic checkpoint defects for the karyotypic patterns characteristic of these two renal cell cancer subtypes, we analyzed the messenger RNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family (BUB1, BUBR1, BUB3) and of the mitotic arrest deficiency family (MAD1, MAD2L1, MAD2L2) by real-time quantitative polymerase chain reaction in 30 renal cell cancer samples (11 chromophobe and 19 papillary) and 36 normal kidney tissue samples. MAD1, MAD2L1, and MAD2L2 showed significant expression differences in tumor tissue compared to controls. Chromophobe tumors presented underexpression of MAD1, and MAD2L2, whereas papillary tumors showed overexpression of MAD2L1. The expression level of the BUB gene family did not differ significantly from that of normal kidney. We conclude that expression changes in mitotic arrest deficiency genes (MAD1, MAD2L1, and MAD2L2) play a role in renal carcinogenesis characterized by multiple numerical chromosome abnormalities.     

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.     

Strabismus (STB)/Vang-like (VANGL) gene family (Review)

Strabismus 1 (STB1/VANGL2) and Strabismus 2 (STB2/VANGL1), which have been cloned and characterized using bioinformatics and cDNA-PCR, are human homologues of Drosophila tissue polarity gene strabismus (stbm)/Van Gogh (Vang). STB1 and STB2 are tetra-membrane-spanning proteins with 73.1% total-amino-acid identity. Serine-rich domain and Strabismus-homology (STH1 and STH2) domains are conserved among human STB1, STB2, Xenopus Stbm, and Drosophila Stbm. STH2 domain with the C-terminal Ser/Thr-X-Val motif is implicated in binding with Dishevelled (DVL) proteins. STB1 gene is clustered with CASQ1 gene on human chromosome 1q21-q23, while STB2 gene is clustered with CASQ2 gene on human chromosome 1p13. STB1 and STB2 genes are located around cancer susceptibility loci or recombination hot spots in the human genome. STB1 is moderately expressed in K-562 (leukemia), G-361 (melanoma), and MKN7 (gastric cancer) cells. STB2 is highly expressed in MKN28, MKN74 (gastric cancer), BxPC-3, PSN-1, and Hs766T (pancreatic cancer) cells. On the other hand, STB1 and STB2 are significantly down-regulated in several cancer cell lines and primary tumors. Xenopus homologue of human STB1 and STB2 regulates negatively the WNT - beta-catenin signaling pathway. Loss-of-function mutations of genes encoding negative regulators of WNT - beta-catenin signaling pathway lead to carcinogenesis. Based on functional aspects and human chromosomal loci, STB1 gene and STB2 gene are predicted to be potent tumor suppressor gene candidates. STB1 and STB2 might be suitable targets for tissue engineering in the field of re-generative medicine and for chemoprevention and treatment in the field of clinical oncology.     

Ataxia-telangiectasia, cancer and the pathobiology of the ATM gene

Ataxia-telangiectasia (A-T) is a pleiotropic inherited disease characterized by neurodegeneration, cancer, immunodeficiencies, radiation sensitivity, and genetic instability. Although A-T homozygotes are rare, the A-T gene may play a role in sporadic breast cancer and leukemia. ATM, the gene responsible for A-T, is homologous to several cell cycle checkpoint genes from other organisms. ATM is thought to play a crucial role in a signal transduction network that modulates cell cycle checkpoints, genetic recombination, apoptosis, and other cellular responses to DNA damage. New insights into the pathobiology of A-T have been provided by the creation of Atm-/- mice and by in vitro studies of ATM function. Analyses of ATM mutations in A-T patients and in sporadic tumors suggest the existence of two classes of ATM mutation: null mutations that lead to A-T and dominant negative missense mutations that may predispose to cancer in the heterozygous state.     

Generation of loss of heterozygosity and its dependency on p53 status in human lymphoblastoid cells

Loss of heterozygosity (LOH) is a critical event in the development of human cancers. LOH is thought to result from either a large deletion or recombination between homologous alleles during repair of DNA double-strand breaks (DSBs). These types of genetic alterations produce mutations in the TK gene mutation assay, which detects a wide mutational spectrum, ranging from point mutations to LOH-type mutations. TK6, a human lymphoblastoid cell line, is heterozygous for the thymidine kinase (TK) gene and has a wild-type p53 gene. The related cell lines, TK6-E6 and WTK-1, which are p53-deficient and p53-mutant (Ile237), respectively, are also heterozygous for the TK gene and LOH-type mutation can be detected in these cells. Therefore, comparative studies of TK mutation frequency and spectrum with these cell lines are useful for elucidating the role of p53 in generating LOH and maintaining genomic stability in human cells. We demonstrate here that LOH and its associated genomic instability strongly depend on the p53 status in these cells. TK6-E6 and WTK-1 are defective in the G1/S checkpoint and in apoptosis. Unrepaired DSBs that escape from the checkpoint can potentially initiate genomic instability after DNA replication, resulting in LOH and a variety of chromosome changes. Moreover, genomic instability is enhanced in WTK-1 cells. It is likely that the mutant p53 protein in WTK-1 cells increases LOH in a dominant-negative manner due to its abnormal recombination capacity. We discuss the mutator phenotype and genomic instability associated with p53 inactivation with the goal of elucidating the mechanisms of mutation and DNA repair in untargeted mutagenesis.     

When chromosomal dynamics control cell division]

In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.