However, this appears contradictory with the fact that the CAL27 and CAL33 CYP1B1-VAR cells display phenotypic properties generally associated with the epithelial-to-mesenchymal transition, especially their enhanced motility and invasiveness.20 This distortion underlines the complexity of the transcriptional regulation of the epithelial-to-mesenchymal transition. The CAL27 cell line expressing the variant CYP1B1 genotype displays marked stemness properties as compared with the wild-type cell line. stemness features and resistance to DNA-damaging agents. In vivo, tumours expressing the variant CYP1B1 had higher growth rates and were markedly drug-resistant. In the clinical study, overall survival was significantly associated MEN2B with the genotypes, wild-type patients presenting a longer median survival (13.5 months) than the variant patients (6.3 months) (polymorphism and drug sensitivity1 by using the data of the CCLE cell line collection. The CCLE collection17 contains 1036 cell lines of all tumour types, among which 494 have been studied pharmacologically through evaluation GSK1059865 of the cytotoxicity of 24 anticancer drugs. Gene expression data as well as mutational profiles of a limited number of genes involved in oncogenesis are freely available on the website of the Broad Institute (http://www.broadinstitute.org/ccle/home#). In particular, data on the V432L (rs1056836) are available for 991 cell lines (494 cell lines with pharmacological data). Since 44 cell lines of the NCI collection are included in the CCLE collection, we verified that the genotypes that we GSK1059865 had determined on the NCI collection by pyrosequencing (1) were all identical to those of the same cell lines in the CCLE database. A special attention was brought to epithelial and mesenchymal marker genes, as identified by Kohn et al.18 The list of the genes considered as epithelial or mesenchymal markers is provided in Supplementary Table?2. Statistics When comparisons were made between two parameters, significance was estimated using Student test. When three parameters were to be compared (p15, WT and VAR cells or tumours), we used ANOVA with post hoc multiple comparisons. Survival data were compared using the log-rank test. Results Characterisation of the isogenic CYP1B1-expressing cells To characterise the phenotype associated with wild-type and variant CYP1B1 genotypes, we selected two HNSCC cell lines, CAL27 and CAL33, which do not express CYP1B1 at a detectable level as estimated by Western blot (Supplementary Fig.?1A, upper panel). This lack of protein expression was associated with a high level of methylation of the CpG islands studied in the promoter GSK1059865 of the gene (data not shown). Lentiviral infection of both cell lines with the two polymorphic forms of CYP1B1 (wild-type V432 and variant L432) or with an empty vector (p15) generated two series of isogenic cell lines, the CYP1B1-WT, the CYP1B1-VAR and the p15 control cell lines. After DNA extraction, the exogenic CYP1B1 sequence was confirmed by pyrosequencing (Supplementary Fig.?1B). mRNAs were also extracted and retro-converted to cDNAs, and pyrosequencing was also in agreement to what was expected. In addition, CYP1B1 expression was determined by quantitative RT-PCR, which showed that the levels of expression of the two variants were similar in the two isogenic pairs (Supplementary Fig.?1C). CYP1B1 protein expression in the two types of cell lines, CYP1B1-WT and CYP1B1-VAR, was also similar (Supplementary Fig.?1A, lower panel). We then studied a series of phenotypic characteristics of the cell lines in terms of proliferation, migration, cloning efficiency, invasion capacity and chemosensitivity. Cells expressing the CYP1B1 wild-type protein (CYP1B1-WT) had a slightly higher proliferation rate than the cell lines infected with the empty vector (p15), but this difference remained non-significant over 72?h. In contrast, cells re-expressing CYP1B1 under its variant form (L432) (CYP1B1-VAR) had a significantly higher proliferation rate than p15 cells and CYP1B1-WT cells (Fig.?1a). Open in a separate window Fig. 1 Proliferation and migration capacity of the isogenic CYP1B1 cell lines.a Growth curves of the CYP1B1-WT and CYP1B1-VAR cell lines as compared with control p15 in CAL27 and CAL33 cell lines. GSK1059865 In total, 105 cells were seeded on 60-mm Petri dishes and counted every 24?h for 3 days. Variations between CYP1B1-WT or CYP1B1-VAR.