1. ¹Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, UK
2. ²Laboratoire d'Oncogénétique – INSERM E0017, Centre René Huguenin, 35, rue Dailly, F-92210 St-Cloud, France
3. ³Department of Neurosurgery, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Fetscherstrae 74, 01307 Dresden, Germany
4. ⁴Department of Surgery, University of Birmingham, Birmingham B15 2TT, UK
5. ⁵Cancer Research UK Renal Molecular Oncology Research Group, University of Birmingham, Birmingham B15 2TG, UK

Correspondence: Dr F Latif, Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, UK. E-mail: flatif@hgmp.mrc.ac.uk

Received 25 May 2004; Revised 19 August 2004; Accepted 21 September 2004; Published online 9 November 2004.

Abstract

In Drosophila, the Slit gene product, a secreted glycoprotein, acts as a midline repellent to guide axonal development during embryogenesis. Three human Slit gene orthologues have been characterised and recently we reported frequent promoter region hypermethylation and transcriptional silencing of SLIT2 in lung, breast, colorectal and glioma cell lines and primary tumours. Furthermore, re-expression of SLIT2 inhibited the growth of cancer cell lines so that SLIT2 appears to function as a novel tumour suppressor gene (TSG). We analysed the expression of SLIT3 (5q35–34) and SLIT1 (1q23.3–q24) genes in 20 normal human tissues. Similar to SLIT2 expression profile, SLIT3 is expressed strongly in many tissues, while SLIT1 expression is neuronal specific. We analysed the 5' CpG island of SLIT3 and SLIT1 genes in tumour cell lines and primary tumours for hypermethylation. SLIT3 was found to be methylated in 12 out of 29 (41%) of breast, one out of 15 (6.7%) lung, two out of six (33%) colorectal and in two out of (29%) glioma tumour cell lines. In tumour cell lines, silenced SLIT3 associated with hypermethylation and was re-expressed after treatment with 5-aza-2'-deoxycytidine. In primary tumours, SLIT3 was methylated in 16% of primary breast tumours, 35% of gliomas and 38% of colorectal tumours. Direct sequencing of bisulphite-modified DNA from methylated tumour cell lines and primary tumours demonstrated that majority of the CpG sites analysed were heavily methylated. Thus, both SLIT2 and SLIT3 are frequently methylated in gliomas and colorectal cancers, but the frequency of SLIT3 methylation in lung and breast cancer is significantly less than that for SLIT2. We also demonstrated SLIT1 promoter region hypermethylation in glioma tumour lines (five out of six; 83%), the methylation frequency in glioma tumours was much lower (two out of 20; 10%). Hence, evidence is accumulating for the involvement of members of the guidance cues molecules and their receptors in tumour development.