Localization of cytokeratin 4 mRNA in human oesophageal epithelium by non-radioactivein situ hybridization

Abstract

An RNA probe complementary to human cytokeratin 4 mRNA was produced by in vitro transcription, and labelled with digoxigenin-UTP. Using this probe, an in situ hybridization protocol was established, tested and optimized for the localization of the cytokeratin 4 mRNA in formalin- or paraformaldehyde-fixed, paraffin-embedded human oesophageal mucosa. The hybridization signal was exclusively detected in the cytoplasm of epithelial cells as coarse brown granules. The positive signal always showed a consistent and distinct pattern throughout the epithelium, starting from the third to fourth layer of epithelial cells, and reaching into the superficial cell layers, but was never found in the basal cell layer, the suprabasal layers, and the upper adluminal strata. Some intermediate cell layers of the epithelium lining the upper part of the secretory duct of submucosal glands were also positive. Similar results could also be obtained in sections from routinely fixed, paraffin-embedded tissue samples. The described signal pattern was found at all levels of the oesophagus. The signal distribution was in agreement with previous biochemical and immunohistochemical studies on the spreading of the cytokeratin 4 protein throughout the oesophageal epithelium. We may conclude that also the expression of the gene(s) encoding cytokeratin 4 in the human oesophageal epithelium depends on the vertical cell differentiation and the detachment from the basal lamina. The developed simplified but sensitive in situ hybridization method may be a useful tool in routine histology, physiopathology, clinical research, and clinical diagnosis, concerning normal oesophageal epithelium structure and differentiation, and development of oesophageal malignancies.