Transcriptional repression of the human fibronectin gene in laryngeal squamous cell carcinoma cells

Purpose: The aim of the experiments was to analyze the mRNA expression patt ern and verify the repression of FN gene expression in laryngeal squamous c ell carcinoma (SCC) cells in comparison with benign mucosal keratinocytes. Methods: Messenger RNA from SCC cells and benign keratinocytes was reverse transcribed and subjected to PCR following differential display (DD) analys is of the amplicons. Northern hybridization was carried out to confirm the reduction of the FN-mRNA expression in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. Quantitation of protein synthesis was performed with homogenates of fresh tumor biopsies an d their normal phenotypes, as well as of benign keratinocytes and laryngeal SCC cell lines, respectively, using ELISA. In the liposome-mediated transi ent transfection assay, FN promoter activity was analyzed by linking the FN promoter sequence to the chloramphenicol acetyltransferase (CAT) reporter gene. Transfection efficacy was monitored by co-transfection with pGL3 cont rol vector. Results: A 191 bp mRNA fragment revealing a 99% homology with t he human FN-mRNA was detected, the expression of which was repressed 20 tim es as much in SCC cells as compared to benign phenotypes. Northern hybridiz ation confirmed the distinctly reduced expression of FN-mRNA in both laryng eal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. The quantitation experiments showed a correlation between the rang e of FN synthesis and the expression of FN-mRNA in cell lines and the biops ies which were used. The 1.28 kb FN gene promoter drove expression of the C AT reporter gene, which was similar to the FN-mRNA expression showed by DD and Northern hybridization. Conclusions: The mechanisms leading to the low level of FN in many tumors have not yet been sufficiently investigated. Our findings suggest that the decrease of FN in laryngeal SCC cells is transcr iptionally regulated.