Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers.

The mitotic checkpoint is thought to be essential for ensuring accurate chromosome segregation by implementing mitotic delay in response to a spindle defect. To date, however, very little data has become available on the defects of the mitotic checkpoint in human cancer cells. In the present study, impaired mitotic checkpoint was found in four (44%) of nine human lung cancer cell lines. To our knowledge, this is the first demonstration of frequent impairment of the mitotic checkpoint in this leading cause of cancer deaths. As an initial step towards elucidation of the underlying mechanism, we further undertook a search for mutations in a key component of the mitotic checkpoint, known as hsMAD2, and its immediate downstream molecule, p55CDC. No such mutations were found, however, in either 21 lung cancer cell lines or 25 primary lung cancer cases, although we could identify silent polymorphisms and the transcribed and processed hsMAD2 pseudogene that was subsequently mapped at 14q21-q23. The present observations appear to warrant further investigations, such as search for alterations in other components, to better understand the molecular pathogenesis of this fatal disease, and warn against potential misinterpretation when performing mutational analyses for other cancer types based on cDNA templates.     

大肠癌有丝分裂关卡基因ＢＵＢ１和ＢＵＢＲ１ ｍＲＮＡ的表达分析

目的　研究大肠癌组织中有丝分裂关卡基因ＢＵＢ１和ＢＵＢＲ１ｍＲＮＡ的表达。方法　采用半定量ＲＴ-ＰＣＲ方法检测３６例大肠癌和癌旁正常组织中ＢＵＢ１和ＢＵＢＲ１ｍＲＮＡ表达水平。结果　大肠癌和癌旁正常组织中的ＢＵＢ１ｍＲＮＡ拷贝数／β-ａｃｔｉｎｍＲＮＡ拷贝数比值分别为０.６７±０.３３和１.２４±０.３７;大肠癌和癌旁正常组织中的ＢＵＢＲ１ｍＲＮＡ拷贝数／β-ａｃｔｉｎｍＲＮＡ拷贝数比值分别为０.５３±０.２５和１.０３±０.４８。ＢＵＢ１和ＢＵＢＲ１ｍＲＮＡ的表达水平在大肠癌中显著低于癌旁正常组织（Ｐ＜０.０５）。结论　ＢＵＢ１和ＢＵＢＲ１基因是大肠癌的肿瘤相关基因,可能在大肠癌发病中具有重要作用。     

Search for in vivo somatic mutations in the mitotic checkpoint gene, hMAD1, in human lung cancers

We previously reported the presence of mitotic check-point impairment in about 40% of lung cancer cell lines. To gain an insight into the molecular basis of this impairment, we examined 49 lung cancer specimens for alterations in the hMAD1 mitotic checkpoint gene and identified a somatic, non-conservative missense mutation, which substitutes alanine (GCG) for threonine (ACG) at codon 299, together with a number of amino acid substituting, single nucleotide polymorphisms. This is the first demonstration of hMAD1 mutation in any type of human cancers. The present finding marks hMAD1 as a potential target, although with low frequency, for genetic alterations in lung cancer. Thus, further studies of hMAD1 dysfunction caused by other mechanisms appear to be warranted, as well as potential involvement of other components of the mitotic checkpoint.     

Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 and hBUB3 in human cancer

During the metaphase-anaphase transition, the spindle checkpoint prevents segregation of chromosomes if the spindle assembly is perturbed. Critical components of this checkpoint are the MAD and BUB families of proteins, which prevent the proteolysis of Pds1 and B cyclins, producing mitotic arrest. In the present study, we first intended to resolve the role of the hsMAD2 gene in human cancer by determining the potential presence of hsMAD2 mutations in 44 primary bladder tumors, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas. The entire coding region of the hsMAD2 gene was analyzed using PCR-SSCP and sequencing. One of the bladder tumor samples showed a point mutation consisting of a transition, ATC-->GTC (Ile-->Val) in codon 190 of hsMAD2. However, no differences were found in the mitotic arrest between cells transfected with mutant and wild-type MAD2 cDNA. We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder tumors, 3 soft-tissue sarcomas and 1 hepatocellular carcinoma, consistent with a polymorphism at codon 143, CCA-->CCG (Pro-->Pro). Another polymorphism was identified in a hepatocellular carcinoma case at codon 22, GAG-->GAA (Glu-->Glu). In addition, a subgroup of 67 primary tumors was analyzed by Southern blot hybridization. No deletion or visible re-arrangements were detected by comparing tumor and normal DNA band signals. Two other important components of the spindle mitotic checkpoint, hBUB1 and hBUB3, were also screened for mutations: hBUB1 in 43 bladder tumors and 9 bladder cell lines and hBUB3 only in the cell lines. Two polymorphisms were found in hBUB1 at positions 144, CAG-->CAA (Gln-->Gln) in 1 primary tumor and 1 bladder cell line, and 913 (ATC-->ATT, Ile-->Ile) in 1 primary tumor. We did not find sequence alterations in hBUB3. These results suggest that mutations of the hsMAD2, hBUB1 and hBUB3 genes are very rare in bladder tumors and that hsMAD2 alterations are also infrequent in soft-tissue sarcomas and hepatocellular carcinomas.     

Mutation analysis of hBUB1, human mitotic checkpoint gene in multiple carcinomas

hBUB1 is a human homolog of yeast mitotic check point gene that plays an important role in chromosome segregation. Recently mutations of hBUB1 were reported in colorectal cancer cell lines, indicating that inactivation of this gene could be directly involved in aneuploidy in human carcinoma cells. To obtain information of the magnitude of hBUB1 inactivation in multiple carcinomas, we examined mutations in 59 multiple carcinoma cell lines showing single base alteration, however, there was no mutation of hBUB1 with amino acid change in these carcinomas. There were four silent mutations at codon 93, codon 735, codon 430 and codon 98 in KYSE190, TE8 esophageal carcinoma cells, KATOIII gastric carcinoma cells and 697 B cell leukemia cells, respectively. Two candidates of mutation were identified in TE3 esophageal carcinoma cells and 697 B cell leukemia cell line at codon 9 and codon 285, respectively. This result suggests that the inactivation of hBUB1 may be very rare in human carcinomas, or restricted to certain cell lines of colorectal carcinomas.     

Promoter hypermethylation and silencing of CHFR mitotic stress checkpoint gene in human gastric cancers

CHFR is a recently identified mitotic stress check-point gene. CHFR is ubiquitously expressed in normal human tissues, whereas loss of CHFR expression has been observed in human tumors. Silencing of CHFR has been associated with aberrant promoter methylation and histone deacetylation in several cancer types. In this study, we investigated epigenetic CHFR inactivation in human gastric cancers by examining CHFR expression and methylation status in gastric cancer cell lines with RT-PCR analysis, bisulfite PCR and sequencing. A series of primary gastric tumors were also analyzed for CHFR methylation. Eight of 12 (66.7%) gastric cancer cell lines and 19/43 (44.2%) primary gastric tumors showed CHFR methylation. In addition, CpG methylation status correlated well with CHFR expression in the human gastric cancer cell lines, in which treatment with 5-aza-dC resulted in de novo or enhanced expression of CHFR. Combination treatment of 5-aza-dC with trichostatin A showed a synergistic effect on CHFR expression in some cases. Our results indicate that aberrant promoter methylation of the CHFR gene was observed in a significant proportion of human gastric cancers and was responsible for the inactivation of the CHFR gene in gastric cancers.     

Inactivating mutations targeting the chfr mitotic checkpoint gene in human lung cancer

A hallmark of cancer is inactivation of cell cycle checkpoints. However, very few mutations targeting mitotic checkpoint genes have been described, and in those instances, a wild-type copy of the gene was retained. chfr is a mitotic checkpoint gene that functions in early prophase delaying chromosome condensation in response to microtubule poisons. In a panel of 53 lung carcinomas for which matched normal tissue was available, we identified three missense mutations in the chfr gene, at least one of which was associated with loss of heterozygosity. In tissue culture checkpoint assays, the tumor-associated missense mutants had reduced activity or were inactive. Together with recent data suggesting that the chfr gene is frequently silenced in various tumors because of methylation of its promoter, these findings suggest that chfr is inactivated by multiple mechanisms in human cancer.     

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.     

Molecular signature and pathway analysis of human primary squamous and adenocarcinoma lung cancers

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with a poor response to chemotherapy and low survival rate. This unfavorable treatment response is likely to derive from both late diagnosis and from complex, incompletely understood biology, and heterogeneity among NSCLC subtypes. To define the relative contributions of major cellular pathways to the biogenesis of NSCLC and highlight major differences between NSCLC subtypes, we studied the molecular signatures of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), based on analysis of gene expression and comparison of tumor samples with normal lung tissue. Our results suggest the existence of specific molecular networks and subtype-specific differences between lung ADC and SCC subtypes, mostly found in cell cycle, DNA repair, and metabolic pathways. However, we also observed similarities across major gene interaction networks and pathways in ADC and SCC. These data provide a new insight into the biology of ADC and SCC and can be used to explore novel therapeutic interventions in lung cancer chemoprevention and treatment.     

Mutations in the mitotic check point gene, MAD1L1, in human cancers.

Aneuploidy is a characteristic of the majority of human cancers, and recent studies suggest that defects of mitotic checkpoints play a role in carcinogenesis. MAD1L1 is a checkpoint gene, and its dysfunction is associated with chromosomal instability. Rare mutations of this gene have been reported in colon and lung cancers. We examined a total of 44 cell lines (hematopoietic, prostate, osteosarcoma, breast, glioblastoma and lung) and 133 fresh cancer cells (hematopoietic, prostate, breast and glioblastoma) for alterations of MAD1L1 by RT-PCR-SSCP and nucleotide sequencing. Eight mutations consisting of missense, nonsense and frameshift mutations were found, together with a number of nucleotide polymorphisms. All the alterations in cell lines were heterozygous. Frequency of mutations was relatively high in prostate cancer (2/7 cell lines and 2/33 tumor specimens). We placed a mutant truncated MAD1L1, found in a lymphoma sample, into HOS, Ht161 and SJSA cell lines and found that it was less inhibitory than wild type MAD1L1 at decreasing cell proliferation. Co-expression experiments showed that the mutant form had a dominant-negative effect. Furthermore, this mutant impaired the mitotic checkpoint as shown by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after culture with the microtubule-disrupting agent, nocodazole. Our results suggest a pathogenic role of MAD1L1 mutations in various types of human cancer.     

Molecular classification and molecular genetics of human lung cancers

Recent advances in the molecular classification of lung carcinomas and the identification of causative genetic alterations will likely lead to improvements in the diagnosis and treatment of patients with lung cancer. It is now possible to identify gene expression profiles that associate with patient outcome in lung carcinomas, in particular adenocarcinoma. Furthermore, patient survival has been shown to correlate with lung cancer oligonucleotide microarray expression profiles. Large-scale microarray technology may allow for the identification of useful biomarkers for early cancer detection. Oligonucleotide microarray data can be optimized by relating them to protein expression levels in tissue microarrays, by annotation with mutational data, and with results of testing for post-translational modification of cellular proteins. These data may be useful in tailoring chemotherapeutic protocols to individual tumors and identifying new targets for therapeutic intervention.     

Mutation analysis of mitotic checkpoint genes (hBUB1 and hBUBR1) and microsatellite instability in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a neoplasm of T-lymphocytes, and human T-cell lymphotropic virus type-I (HTLV-I) is etiologically considered as the causative virus of ATLL. The karyotypes of ATLL are very complex in both number and structure, although no specific karyotype abnormalities have been identified. HTLV-I is thought to integrate its provirus into random sites in host chromosomal DNA and induces chromosomal instability. The BUB gene is a component of the mitotic checkpoint in budding yeast. Recently, human homologues of the BUB were identified and mutant alleles of hBUB1 and hBUBR1 were detected in two colorectal tumor cell lines, which showed microsatellite instability (MIN). In vitro, BUB proteins form a complex of monomers. These proteins interact with the human MAD1 gene product, a target of the HTLV-1 tax oncogene. We examined the role of checkpoint gene in the chromosomal abnormalities of ATLL by investigating mutations of hBUB1 and hBUBR1, and MIN of replication errors of BAX, insulin-like growth factor, and transforming growth factor beta type II. We analyzed ten cases with ATLL and eight B-cell lymphomas (five diffuse large cell lymphomas, three follicular lymphomas). Complex chromosomal abnormalities were detected in ATLL, while B-cell lymphomas showed only simple or minimal chromosomal abnormalities. Significant mutations/deletion of hBUB1 or hBUBR1 were detected in four of ten cases with ATLL, including two heterozygous point mutations, one homozygous point mutation, and one with a 47 bp deletion. In contrast, only one of eight B-cell lymphomas showed nonsense mutation of hBUBR1. None of the ATLL and B-cell lymphomas showed MIN. In the multistage process of leukemogenesis of ATLL, our findings indicate that mutations of mitotic checkpoint genes may play an important role in the induction of complex chromosomal abnormalities.     

Dual roles of human BubR1, a mitotic checkpoint kinase, in the monitoring of chromosomal instability

In this study, we show that the formation of polyploidy following sustained mitotic checkpoint activation appears to be preceded by the ubiquitin-dependent proteolysis of hBubR1. In addition, the level of hBubR1 is significantly reduced not only in polyploid cells created by sustained mitotic spindle damage, but also in 21 (31.3%) of 67 human colon adenocarcinomas tested. Importantly, the introduction of hBubR1 triggers the apoptosis of polyploid cells formed by aberrant exit from mitosis and inhibits the growth of tumors established with these cells in athymic nude mice. These results suggest that hBubR1-mediated apoptosis prevents the propagation of cells that breach the mitotic checkpoint and that the control of hBubR1 protein level is an important factor in the acquisition of preneoplastic polyploidy.     

Altered expression of the early mitotic checkpoint protein, CHFR, in breast cancers: implications for tumor suppression

Checkpoint with FHA and Ring Finger (CHFR) is hypothesized to mediate a delay in cell cycle progression early in mitosis in response to microtubule stress, independent of the spindle assembly checkpoint. As a potential regulator of cell cycle progression, CHFR naturally becomes an interesting target for understanding cancer cells. In recent years, there has been increasing evidence supporting the role of CHFR as a tumor suppressor, most of which report loss of expression, occasionally due to promoter hypermethylation, in cancers compared with patient-matched normal tissues. We studied both a panel of breast cancer cell lines as well as primary tissue samples from breast cancer patients to investigate CHFR as a relevant tumor suppressor in breast cancer and to determine whether CHFR expression was associated with clinical and pathologic variables. We report that 41% of cell lines and 36% of patient samples showed low or negative CHFR protein expression or staining. In addition, lack of CHFR detection was associated with increased tumor size and weakly correlated with estrogen receptor-negative tumors from patients. To study the effects of low CHFR expression in vitro, we stably expressed a short hairpin RNA construct targeting CHFR in two lines of immortalized human mammary epithelial cells. Notably, decreased CHFR expression resulted in the acquisition of many phenotypes associated with malignant progression, including accelerated growth rates, higher mitotic index, enhanced invasiveness, increased motility, greater aneuploidy, and amplified colony formation in soft agar, further supporting the role of CHFR as a tumor suppressor in breast cancer.     

DNA methylation-associated inactivation of TGFbeta-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers

The transforming growth factor beta (TGFbeta)-signalling pathway is deregulated in many cancers. We examined the role of gene silencing via aberrant methylation of DRM/Gremlin and HPP1, which inhibit TGFbeta signalling, and RUNX3, which facilitates TGFbeta-signalling, of all genes that are thought to be tumour suppressors, are aberrantly expressed, and are thus thought to have important role in human cancers. We examined DRM/Gremlin mRNA expression in 44 cell lines and the promoter methylation status of DRM/Gremlin, HPP1, and RUNX3 in 44 cell lines and 511 primary tumours. The loss of DRM/Gremlin mRNA expression in human cancer cell lines is associated with DNA methylation, and treatment with the methylation inhibitor-reactivated mRNA expression (n=13). Methylation percentages of the three genes ranged from 0-83% in adult tumours and 0-50% in paediatric tumours. Methylation of DRM/Gremlin was more frequent in lung tumours in smokers, and methylation of all three genes was inversely correlated with prognosis in patients with bladder or prostate cancer. Our results provide strong evidence that these TGFbeta-related genes are frequently deregulated through aberrant methylation in many human malignancies.     

Molecular cloning of human TAK1 and its mutational analysis in human lung cancer

In previous reports, we described that DPC4/Smad4 and Smad2 are mutated in a fraction of human lung cancers and suggested possible roles of the downstream mediators of transforming growth factor-β (TGF-β)–elicited signals in the pathogenesis of this most common cancer. In the present study, we investigated whether another downstream mediator, human TGF-β-activated kinase 1 (hTAK1), also is altered in lung cancer. For this purpose, the hTAK1 gene was cloned with the aid of an expression sequence tag database search and cDNA library screening, and hTAK1 was found to be expressed ubiquitously in 2 distinct isoforms regulated in a tissue-specific manner in fetal and adult normal tissues. Interestingly, hTAK1 was assigned to the chromosome region 6q14-21, which is deleted frequently in various human malignancies, including lung cancer. Despite our extensive search for alterations in 39 lung cancer specimens as well as in 16 lung cancer cell lines, somatic mutations of hTAK1 were not identified, indicating that hTAK1 itself is not a frequent target for genetic alterations in lung cancer. Int. J. Cancer 75:559–563, 1998. © 1998 Wiley-Liss, Inc.