Overexpression of the mitotic checkpoint genes BUB1, BUBR1, and BUB3 in gastric cancer—association with tumour cell proliferation

The mitotic spindle assembly checkpoint modulates the timing of anaphase initiation in response to improper alignment of chromosomes at the metaphase plate. The BUB gene family encodes proteins which are part of a large multi‐protein kinetochore complex and which are believed to be key components of the checkpoint regulatory pathway. Failure of this surveillance system can lead to genomic instability and could be responsible for the increased incidence of aneuploidy in gastric cancer. Since mutations of BUB genes have not been identified in gastric cancer to date, altered BUB expression levels may significantly impair mitotic checkpoint function. To explore this possibility, the expression levels of BUB1, BUBR1, and BUB3 were determined in 43 gastric carcinomas and corresponding normal gastric mucosa by reverse transcription‐polymerase chain reaction (RT‐PCR). Gene expression levels were compared with histopathological parameters and DNA ploidy, as well as with proliferative activity, measured by Ki‐67 mRNA expression. To the authors' knowledge, this is the first study to investigate the expression levels of mitotic checkpoint genes together with DNA ploidy in gastric cancer. BUB1 was overexpressed in 84%, BUBR1 in 68%, and BUB3 in 79% of gastric cancers. This study also revealed that all three genes were simultaneously overexpressed in 61% of the tumours and that there was a statistically significant positive correlation between overexpression of BUB1, BUBR1 or BUB3 and Ki‐67 expression (p < 0.001). Eighty‐one per cent of the tumours were classified as aneuploid. However, no correlation was found between ploidy and BUB transcript expression levels. These results suggest that inactivation of the mitotic checkpoint genes BUB1, BUBR1, and BUB3 by epigenetic silencing does not seem to play a role in gastric carcinogenesis. The strong correlation of BUB expression level and tumour cell proliferation suggests that BUB overexpression is a proliferation‐dependent phenomenon in gastric cancer. However, overexpression due to lack of normal BUB protein function or due to a yet unknown additional BUB function has to be considered. Copyright © 2003 John Wiley & Sons, Ltd.

[1]  Bert Vogelstein,et al.  Mutations of mitotic checkpoint genes in human cancers , 1998, Nature.

[2]  C. Sunkel,et al.  Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila , 1999, The Journal of cell biology.

[3]  A. Yoshimura,et al.  Somatic mutation of the hBUB1 mitotic checkpoint gene in primary lung cancer , 2000, Genes, chromosomes & cancer.

[4]  M. Shichiri,et al.  Genetic and epigenetic inactivation of mitotic checkpoint genes hBUB1 and hBUBR1 and their relationship to survival. , 2002, Cancer research.

[5]  B. Roberts,et al.  The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. , 1994, Molecular and cellular biology.

[6]  G. Haroske,et al.  Fourth Updated ESACP Consensus Report on Diagnostic DNA Image Cytometry , 2001, Analytical cellular pathology : the journal of the European Society for Analytical Cellular Pathology.

[7]  P. Sorger,et al.  Chromosome Missegregation and Apoptosis in Mice Lacking the Mitotic Checkpoint Protein Mad2 , 2000, Cell.

[8]  B. Roberts,et al.  S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function , 1991, Cell.

[9]  Stephen S. Taylor,et al.  Kinetochore Localization of Murine Bub1 Is Required for Normal Mitotic Timing and Checkpoint Response to Spindle Damage , 1997, Cell.

[10]  G. Baretton,et al.  DNA-ploidy and survival in gastric carcinomas: a flow-cytometric study , 2005, Virchows Archiv A.

[11]  S. Nomoto,et al.  Molecular analysis of the mitotic checkpoint genes BUB1, BUBR1 and BUB3 in human lung cancers. , 2001, Cancer letters.

[12]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[13]  C. Cordon-Cardo,et al.  Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 and hBUB3 in human cancer , 2001, International journal of cancer.

[14]  E. Petty,et al.  Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines. , 2000, Cancer letters.

[15]  K. Choo,et al.  Bub 3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis , 2000 .

[16]  F. Spencer,et al.  Mammalian BUB1 protein kinases: map positions and in vivo expression. , 1997, Genomics.

[17]  E. Petty,et al.  Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells , 2000, Genes, chromosomes & cancer.

[18]  R. Benezra,et al.  Identification of a Human Mitotic Checkpoint Gene: hsMAD2 , 1996, Science.

[19]  Peter K. Sorger,et al.  A role for the Adenomatous Polyposis Coli protein in chromosome segregation , 2001, Nature Cell Biology.

[20]  T. Ørntoft,et al.  Mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in human bladder cancer, screening for mutations and loss of heterozygosity. , 2001, Carcinogenesis.

[21]  Y. Shiratori,et al.  Mutational Inactivation of Mitotic Checkpoint Genes, hsMAD2 and hBUB1, Is Rare in Sporadic Digestive Tract Cancers , 1999, Japanese journal of cancer research : Gann.

[22]  B. Schaar,et al.  Characterization of the Kinetochore Binding Domain of CENP-E Reveals Interactions with the Kinetochore Proteins CENP-F and hBUBR1 , 1998, The Journal of cell biology.

[23]  P. Laurén,et al.  THE TWO HISTOLOGICAL MAIN TYPES OF GASTRIC CARCINOMA: DIFFUSE AND SO-CALLED INTESTINAL-TYPE CARCINOMA. AN ATTEMPT AT A HISTO-CLINICAL CLASSIFICATION. , 1965, Acta pathologica et microbiologica Scandinavica.

[24]  F. Mauro,et al.  Cytometrically determined relative DNA content as an indicator of neoplasia in gastric lesions. , 1984, Cytometry.

[25]  L. Secker-Walker.  Catalog of Chromosome Aberrations in Cancer , 1996, British Journal of Cancer.

[26]  J. Zieske,et al.  Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ. , 1996, Investigative ophthalmology & visual science.

[27]  K. Fukasawa,et al.  Human Bub1: a putative spindle checkpoint kinase closely linked to cell proliferation. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[28]  W. Dai,et al.  BUBR1 phosphorylation is regulated during mitotic checkpoint activation. , 1999, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[29]  Y. Kakeji,et al.  DNA ploidy and its clinical implications in gastric cancer. , 2002, Surgery.

[30]  A. Pereverzev,et al.  Isoforms of α1E voltage-gated calcium channels in rat cerebellar granule cells Detection of major calcium channel α1-transcripts by reverse transcription–polymerase chain reaction , 1999, Neuroscience.

[31]  K. Choo,et al.  Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. , 2000, Genes & development.

[32]  J. Jen,et al.  Mutation analysis of hBUB1 in aneuploid HNSCC and lung cancer cell lines. , 1999, Cancer letters.

[33]  Stephen S. Taylor,et al.  The Human Homologue of Bub3 Is Required for Kinetochore Localization of Bub1 and a Mad3/Bub1-related Protein Kinase , 1998, The Journal of cell biology.

[34]  Rey-Huei Chen,et al.  Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity , 2001, The Journal of cell biology.

[35]  J. Minna,et al.  Infrequent Mutation of the hBUB1 and hBUBR1 Genes in Human Lung Cancer , 2000, Japanese journal of cancer research : Gann.

[36]  J. Hescheler,et al.  Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction. , 1999, Neuroscience.