Mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in human bladder cancer, screening for mutations and loss of heterozygosity

Sanne H. Olesen, Thomas Thykjaer and Torben F. Ørntoft1,

Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, DK-8200 Aarhus N, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Reference
 
Chromosomal instability is common in bladder cancer and could be caused by mutations of mitotic checkpoint genes. Therefore we screened for mutations of the mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in six aneuploid bladder cancer cell lines and 15 human bladder tumours. The screening was performed by sequence analysis of the entire coding regions of the four genes. No mutations were detected in any of the four genes. We detected several sequence variations in hBUB1, hBUB1B and TTK both new and previously published. The genetic stability of the four gene loci were tested by loss of heterozygosity (LOH) analysis in the 15 patient samples, showing one LOH for each of the hBUB1B, hBUB3 and TTK loci (6.7%) of the cases, all in different tumour samples. No LOH was detected at hBUB1. We conclude that both mutational inactivation, and loss of one allele, of the examined mitotic checkpoint genes are relatively uncommon.

Abbreviations: APC, anaphase promoting complex; BUB, budding uninhibited by benomyl; LOH, loss of heterozygosity; MAD, mitotic arrest deficiency.


    Introduction
 Top
 Abstract
 Introduction
 Reference
 
Chromosomal instability is a common feature of bladder cancer and several other cancer types, but the cause of this defect remains obscure. In early stages of bladder cancer 50% or more of the tumours have lost one, or parts of one, chromosome 9 (1,2), and in more advanced stages of disease, pronounced instability is observed (3,4). One mechanism that leads to genomic instability is the disruption of the mitotic checkpoint. This checkpoint machinery has been intensively studied in Saccharomyces cerevisiae using spindle disrupting drugs, leading to identification of two groups of genes, MAD (mitotic arrest deficiency) (5), and BUB (budding uninhibited by benomyl) (6).

The BUB group consists of three genes (four in humans), BUB1–3. BUB1p is a protein kinase which binds and phosphorylates BUB3p, both associating with unattached kinetochores in metaphase. BUB1p is necessary for control of both normal mitosis and activation of the mitotic checkpoint (7). The third BUB gene, BUB2, acts in a separate pathway. The BUB2p protein is necessary for the mitotic checkpoint arrest, but detailed knowledge of its function is not available (8). MPS1p (9) (human homologue TTK) is a protein kinase that phosphorylates MAD1p, a phosphorylation essential to the mitotic checkpoint activation (8,10). Human homologues of most of the known yeast mitotic checkpoint genes have now been identified and characterized (1115). Previously it was shown that a dominant mutation in the mitotic checkpoint gene hBUB1 in a colon cancer cell line caused chromosomal instability (16). This finding indicated that the cause of aneuploidy could be due to mutations in one or more mitotic checkpoint genes. Following this observation, further investigations of mutations in mitotic checkpoint genes have been published (12,1719). However, these reports revealed a very low mutation frequency of these genes in colon and breast cancer cell lines, sporadic digestive tract cancers and in lung cancer specimens.

Here we investigated the possible inactivation of four mitotic checkpoint genes in human bladder cancer. We screened for mutations in hBUB1, hBUB1B, hBUB3 and TTK (human homologue of MPS1) in aneuploid bladder cancer cell lines and invasive bladder tumour samples from patients with concomitant carcinoma in situ. This group was selected as they show even more chromosomal instability than patients with invasive tumours without concomitant carcinoma in situ (20). Furthermore, in the clinical samples we investigated the frequency of allelic losses by microsatellite analysis.

The following bladder cancer cell lines were used in this study: HCV29, HU609, SW780, HT1376, J82 and T24. Cell lines SW780, HT1376, J82 and T24 were obtained from the American Type Culture Collection, USA. HCV29 and HU609 were obtained from the Fibiger Institute (Denmark). T24 was cultured in McCoy media supplemented with 10% fetal bovine serum. HCV29, HU609 and SW780 were grown in D-MEM supplemented with 10% fetal bovine serum (FBS). HT1376 and J82 were cultured in MEM with 10% FBS. All media were obtained from Life Technologies (Denmark). The six bladder cancer cell lines selected for this study showed varying degrees of genomic instability with modal chromosome number ranging from 49 to 121 (2125).

Fifteen bladder tumour tissue samples from patients with concomitant carcinoma in situ were collected fresh from surgery, immediately embedded in guadinium thiocyanate and frozen at –80°C until use (for RNA preparation). For DNA preparation the tumour samples were snap frozen and stored at –80°C. The samples were all high stage muscle invasive tumours from patients spanning an age range of 55–81 years.

Total RNA was extracted from homogenized guadinium thiocyanate embedded frozen tissue or cultured cells. For the extraction we used the RNAZol-BTM method (WAK Chemie GMBH, Germany) following the manufacturers instructions. DNA was extracted from 10–20 mg homogenized tumour samples and 300 µl blood from the same patients as the tumour samples. The extraction was performed using the PureGeneTM DNA Extraction kit (Gentra Systems, USA).

cDNA was prepared using First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech, USA) using oligo-dT or random hexamer primers. cDNA from the four genes under investigation were amplified in overlapping segments by PCR (hBUB1, 3 segments; hBUB1B, 4 segments; hBUB3, 1 segment; TTK, 2 segments). PCR was performed using Roche Taq polymerase (1 U/reaction) and buffer, containing 2 µl cDNA, 0.2 pM each primer and 75 µM dNTP in a total volume of 50 µl. Reactions consisted of an initial denaturing step at 94°C in two minutes, followed by 20 cycles of 15 s at 94°C, 30 s at 56°C and 90 s at 72°C, then 15 cycles of 15 s at 94°C, 30 s at 56°C, and 90 s plus 20 s per cycle at 72°C. The reaction was terminated by 7 min at 72°C. The PCR products were then sequenced in both directions using a Big Dye Sequencing kit (PE Applied Biosystems, USA) according to the manufacturers instructions, and analysed on an ABI 377 Sequencer (PE Applied Biosystems). The primer sequences used for PCR amplification and sequencing of hBUB1, hBUB1B, hBUB3 and TTK are available at www.mdl.dk/supplementary-data.

The sequence analysis was performed on six bladder cancer cell lines and 15 bladder tumour samples from patients with concomitant carcinoma in situ. By using RT–PCR and sequencing of cDNA, we found that all four genes were expressed and without mutations in all six cell lines and 15 tumor specimens.

The sequence analysis revealed several sequence variations, both new and previously published (Table IGo). The new variations were as follows: hBUB1B, T348->C (Tyr to Tyr); TTK, A315->G (Ile to Val) and G1117->T (Ala to Ser). All sequence variations found during cDNA sequencing were verified by sequencing DNA extracted from blood samples from the patients.


View this table:
[in this window]
[in a new window]
 
Table I. Sequence variations detected in this study
 
The two new non-silent sequence variations are both positioned in the putative regulatory or binding region of TTK. The first variant is an A->G transition at position 315 of TTK, causing a shift from isoleucine to valine. Due to the close relationship between valine and isoleucine, both having hydrophobic side chains, it is relatively unlikely that this amino acid substitution will have any impact on the function of TTK.

The other sequence variant, a G->T transition at position 1117, changes an alanine to a serine. However, the sequence variation is homozygous in all six cell lines and all 15 tumour samples. Whether this sequence variant is due to an error in the GenBank sequence or it is a genuine sequence variation is presently unknown.

To investigate the genomic stability of the hBUB1, hBUB1B, hBUB3 and TTK loci we performed loss of heterozygosity (LOH) analysis on the clinical samples. DNA from tumour samples and blood from the same patient was used for the LOH analysis. Microsatellites used for the LOH analysis were chosen as near as possible to the gene loci, using the mapping information from Genemap99. The microsatellite markers used were as follows: hBUB1, D2S293; hBUB1B, D15S146; hBUB3, D10S587; and TTK, D6S284. Fluorescence labelled primers were obtained from DNA Technology (Denmark). The PCR reaction, containing 50 ng DNA, 0.1 pM of each primer (one primer fluorescence labeled), 10 mM Tris (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 150 µM dNTP and 0.5 U Taq polymerase (Amersham Pharmacia Biotech, USA) was carried out in a total volume of 20 µl. The PCR mix was subjected to an initial 5 min at 95°C, followed by 35 cycles of 50 s at 95°C, 90 s at 58°C and 90 s at 72°C. The reaction was terminated by 5 min at 72°C. The PCR products were subsequently analysed on an ABI 377 Sequencer (PE Applied Biosystems) using GeneScanTM software. LOH was defined as >50% decrease in chromotogram peak height of the affected allele.

At the hBUB1 locus we detected no LOH in the 15 tumour samples studied, whereas we detected one LOH for each of the hBUB1B, hBUB3 and TTK loci. LOH was found in three different tumor samples.

Taking into account the low number of LOH found in this investigation and the frequency of aneuploidy, it does not seem likely that a gene dose effect is an important factor in mitotic checkpoint failure in bladder cancer. What is obvious from the present data, and from all previous mutational investigations of mitotic checkpoint genes, is that it is unlikely that mutations in the known mitotic checkpoint genes are the primary source of genetic instability in cancer cells. An explanation for the observed chromosomal instability in bladder cancer and other types of cancer could be due to mutations in presently unknown checkpoint genes, maybe in combination with other mechanisms of inactivation, such as promoter methylation or defects in possible post-transcriptional regulatory mechanisms. It has been previously shown that a mitotic checkpoint-deficient breast cancer cell line showed severely reduced expression of the mitotic checkpoint gene hMAD2 (15). Several sequence variations resulting in amino acid differences are found in the mitotic checkpoint genes in this and previous reports. It is a possibility that specific combinations of these sequence variations may result in reduced function of the mitotic checkpoint apparatus and thereby an increased cancer risk.


    Notes
 
1 To whom correspondence should be addressed Email: orntoft{at}kba.sks.au.dk Back


    Acknowledgments
 
We would like to thank Jette Jensen for excellent technical assistance. This study was supported by The Danish Cancer Society and The University of Aarhus.


    Reference
 Top
 Abstract
 Introduction
 Reference
 

  1. Keen,A.J. and Knowles,M.A. (1994) Definition of two regions of deletion on chromosome 9 in carcinoma of the bladder. Oncogene, 9, 2083–2088.[ISI][Medline]
  2. Orlow,I., Lianes,P., Lacombe,L., Dalbagni,G., Reuter,V.E. and Cordon-Cardo,C. (1994) Chromosome 9 allelic losses and microsatellite alterations in human bladder tumors. Cancer Res., 54, 2848–2851.[Abstract]
  3. Kallioniemi,A., Kallioniemi,O.P., Citro,G., Sauter,G., DeVries,S., Kerschmann,R., Caroll,P. and Waldman,F. (1995) Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization. Cancer, 12, 213–219.
  4. Presti,J.C.J., Reuter,V.E., Galan,T., Fair,W.R. and Cordon-Cardo,C. (1991) Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res., 51, 5405–5409.[Abstract]
  5. Hardwick,K.G. and Murray,A.W. (1995) Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J. Cell Biol., 131, 709–720.[Abstract]
  6. Hoyt,M.A., Totis,L. and Roberts,B.T. (1991) Saccharomyces cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell, 66, 507–517.[ISI][Medline]
  7. Taylor,S.S., Ha,E. and McKeon,F. (1998) The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase. J. Cell Biol., 142, 1–11.[Free Full Text]
  8. Fesquet,D., Fitzpatrick,P.J., Johnson,A.L., Kramer,K.M., Toyn,J.H. and Johnston,L.H. (1999) A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast. EMBO J., 18, 2424–2434.[Abstract/Free Full Text]
  9. Hardwick,K.G., Weiss,E., Luca,F.C., Winey,M. and Murray,A.W. (1996) Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science, 273, 953–956.[Abstract]
  10. Farr,K.A. and Hoyt,M.A. (1998) Bub1p kinase activates the Saccharomyces cerevisiae spindle assembly checkpoint. Mol. Cell Biol., 18, 2738–2747.[Abstract/Free Full Text]
  11. Li,Y., Gorbea,C., Mahaffey,D., Rechsteiner,M. and Benezra,R. (1997) MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. Proc. Natl Acad. Sci. USA, 94, 12431–12436.[Abstract/Free Full Text]
  12. Cahill,D.P., da Costa,L.T., Carson-Walter,E.B., Kinzler,K.W., Vogelstein,B. and Lengauer,C. (1999) Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics, 58, 181–187.[ISI][Medline]
  13. Chan,G.K., Schaar,B.T. and Yen,T.J. (1998) Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBURB1. J. Cell Biol., 143, 49–63.[Abstract/Free Full Text]
  14. Li,Y. and Benezra,R. (1996) Identification of a human mitotic checkpoint gene: hsMAD2. Science, 274, 246–248.[Abstract/Free Full Text]
  15. Mills,G.B., Schmandt,R., McGill,M., Amendola,A., Hill,M., Jacobs,K., May,C., Rodricks,A.M., Campbell,S. and Hogg,D. (1992) Expression of TTK, a novel human protein kinase, is associated with cell proliferation. J. Biol. Chem., 267, 16000–16006.[Abstract/Free Full Text]
  16. Cahill,D.P., Lengauer,C., Yu,J., Riggins,G.J., Willson,J.K., Markowitz,S.D., Kinzler,K.W. and Vogelstein,B. (1998) Mutations of mitotic checkpoint genes in human cancers. Nature, 392, 300–303.[ISI][Medline]
  17. Imai,Y., Shiratori,Y., Kato,N., Inoue,T. and Omata,M. (1999) Mutational inactivation of mitotic checkpoint genes, hsMAD2 and hBUB1, is rare in sporadic digestive tract cancers. Jpn. J. Cancer Res., 90, 837–840.[ISI][Medline]
  18. Myrie,K.A., Percy,M.J., Azim,J.N., Neeley,C.K. and Petty,E.M. (2000) Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines. Cancer Lett., 152, 193–199.[ISI][Medline]
  19. Takahashi,T., Haruki,N., Nomoto,S., Masuda,A., Saji,S. and Osada,H. (1999) Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers. Oncogene, 18, 4295–4300.[ISI][Medline]
  20. Primdahl,H., von der Maase,H., Christensen,M., Wolf,H. and Orntoft,T.F. (2000) Allelic deletions of cell growth regulators during progression of bladder cancer. Cancer Res., 60, 6623–6629.[Abstract/Free Full Text]
  21. Orntoft,T.F., Meldgaard,P., Pedersen,B. and Wolf,H. (1996) The blood group ABO gene transcript is down-regulated in human bladder tumors and growth-stimulated urothelial cell lines. Cancer Res., 56, 1031–1036.[Abstract]
  22. Kyriazis,A.A., Kyriazis,A.P., McCombs,W.B. and Peterson,W.D.J. (1984) Morphological, biological and biochemical characteristics of human bladder transitional cell carcinomas grown in tissue culture and in nude mice. Cancer Res., 44, 3997–4005.[Abstract]
  23. Rasheed,S., Gardner,M.B., Rongey,R.W., Nelson-Rees,W.A. and Arnstein,P. (1977) Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses. J. Natl Cancer Inst., 58, 881–890.[ISI][Medline]
  24. O'Toole,C., Price,Z.H., Ohnuki,Y. and Unsgaard,B. (1978) Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma. Br. J. Cancer, 38, 64–76.[ISI][Medline]
  25. Fogh,J. (1978) Cultivation, characterization and identification of human tumor cells with emphasis on kidney, testis and bladder tumors. Natl Cancer Inst. Monogr., 49, 5–9.[Medline]
Received October 26, 2000; revised December 18, 2000; accepted December 19, 2000.