ORIGINAL ARTICLE DBC1 re-expression alters the expression of multiple components of the plasminogen pathway JP Louhelainen, CD Hurst, E Pitt, H Nishiyama, HA Pickett and MA Knowles Cancer Research UK Clinical Centre, St James’s University Hospital, Leeds, West Yorkshire, UK Deleted in bladder cancer 1 (DBC1) is a candidate gene for the bladder tumour suppressor locus at 9q33.1. The function of the gene is currently unknown but a cross- species sequence comparison suggests an important role, as it is highly evolutionarily conserved. Here, we transfected a nonexpressing human bladder cancer cell line with a set of human DBC1 cDNA constructs. The effect on global expression patterns was assessed using cDNA microarrays. The cell clone with the lowest level of DBC1 expression showed induced expression of 26 genes including plasminogen activator inhibitor 2 (SERPINB5; 4.6-fold), heparin-binding EGF-like growth factor pre- cursor (DTR; 4.2-fold), small proline-rich protein 2B (SPRR2B; 3.6-fold), metallothionein 1 isoforms (MT1B/ MT1A/MT-1F; from 2.9- to 3.2-fold), tissue-type plas- minogen activator precursor (PLAT; 2.8-fold) and uroki- nase-type plasminogen activator precursor (PLAU; 2.7- fold). In clustering analysis, both PLAT and PLAU clustered with the functionally related urokinase plasmi- nogen activator surface receptor (PLAUR; 1.9-fold). Furthermore, 14 human bladder tumours were analysed by real-time quantitative PCR using gene-specific primers for selected (n ¼ 20) genes. The expression levels of SERPINB5, PLAU, PLAUR and MT1 correlated with the DBC1 levels, suggesting previously unknown involve- ment of DBC1 in the urokinase-plasminogen pathway. Oncogene (2006) 25, 2409–2419. doi:10.1038/sj.onc.1209228; published online 12 December 2005 Keywords: DBC1; bladder cancer; tumour suppressor gene; expression microarray Introduction The common feature shared by the vast majority of bladder cancers is that they have deletions of chromo- some 9. Over the years, multiple candidate regions have been reported (reviewed in Knowles, 1999). The deleted in bladder cancer 1 (DBC1) gene, originally named DBCCR1, which represents a candidate tumour sup- pressor gene within a precisely mapped region of loss of heterozygosity on 9q33, shows loss of one allele in B60% of all bladder tumours and homozygous deletion or transcriptional silencing by promoter hypermethyla- tion in many cases (Habuchi et al., 1997, 1998; Nishiyama et al., 1999; Fujiwara et al., 2001; Stadler et al., 2001; Williams et al., 2002). It has been suggested that hypermethylation of DBC1 is one of the earliest events in the development of transitional cell carcinoma (TCC) (Habuchi et al., 2001). Recently, loss of DBC1 expression by promoter hypermethylation was reported during transformation of adult mesenchymal stem cells (hMSC) transduced with the hTERT gene (Serakinci et al., 2004), where methylation correlated with the acquisition of the tumorigenic phenotype. Thus, DBC1 alteration might be one of the essential changes during neoplastic development in hMSC. Recently, homologues of DBC1 have been found in Mus musculus (96% amino-acid identity), Rattus norvegicus (96% amino-acid identity) and several other organisms including Danio rerio, suggesting universal importance of this gene. In brain and spinal cord, DBC1 expression is high and some of the homologues have been reported as bone morphogenetic protein-2/retinoic acid-inducible neural-specific proteins (Kawano et al., 2004; Toshiyuki and Ichiro, 2004). Structurally, DBC1 harbours a membrane-attack complex (MAC)/perforin protein do- main that, in the context of the perforin protein, is involved in pore formation during complement- mediated cell lysis. However, to date, there is no evidence for an equivalent function of DBC1. Our previous studies have shown that re-expression of DBC1 in bladder tumour cells has an antiproliferative effect, but this does not appear to be due to a direct effect on apoptosis (Nishiyama et al., 2001). Recently, Wright et al. (2004) reported that DBC1 mediates cell death in cultured bladder tumour cells with a mechanism which is not of the classic apoptotic type. The same research group previously reported that acid sphingomyelinase- like protein (SMPDL3A; alternatively called ASML3a) is upregulated by DBC1 (Wright et al., 2002). At present therefore, the exact gene function of DBC1, its mechanism of action and the pathways involved have not been solved. In this study, we have re-expressed DBC1 in a human bladder cancer cell line that shows hypermethylation- induced silencing of the endogenous DBC1 gene. Cell Received 4 February 2005; revised 27 September 2005; accepted 27 September 2005; published online 12 December 2005 Correspondence: Dr M Knowles, Cancer Research UK Clinical Centre, St James’s University Hospital, Beckett Street, Leeds, West Yorkshire LS9 7TF, UK. E-mail: margaret.knowles@cancer.org.uk Oncogene (2006) 25, 2409–2419 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc