Retinoid Receptors mRNA levels in normal bronchial mucosa and in lung cancer cell lines
Centre de Ressources Biologiques, Faculté de Médecine, "Oncogenese Thoracique", Vandoeuvre-Les-Nancy, France
Lung cancer remains a public health problem for which early screening remains in infancy. Aim: since we reported that Retinoid Receptors (RR) expression is down regulated in early lung cancer, it is our goal, to try to restore a normal RARbeta expression in the bronchial mucosa of patients at risk to develop lung cancer. Second generation retinoids, aerosolized on site, could be used in such chemoprevention if we could measure RR mRNA levels. Methods: we first conceived and design an appropiate RR assay in order to measure efficiently the normal bronchial mucosa level of each RRs mRNA by real-time quantitative relative RT-PCR. RR primers and actin were choosen for their capacity to generate amplicons at the same pairing temperature; actin being the standard reference. All experiments were repeated thrice. Then, sixteen cancer cell lines (5 mesothelioma, 4 small cell lung cancers and 7 lung adenocarcinoma) were also screened to identify model cell lines with which RR restoration can be studied. Real time duplex PCRs evaluated the methylation status of RARbeta and P16 promoters in the same samples. Results: we do know now what are the normal RR value for human bronchial mucosa: RXRbeta is the most expressed RR at about 400 folds less than actin. Then and in this order: RXRalpha, RARbeta, RXRgamma, RARgamma and RAR alpha expressions are even lower. Several cell lines lack completely of one or more RR mRNA. Among them, two-third had also P2 RARbeta hypermethylation and low RARbeta mRNA levels. It was twice as frequent as P16 promoter hypermethylation. Conclusion: Multiplex real time PCR can now be developed to screen sputum from at risk subjects.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 997 (Chemoprevention).