DETECTION OF NUCLEAR RETINOIC ACID RECEPTOR MESSENGER-RNA IN HISTOLOGICAL TISSUE-SECTIONS USING NONRADIOACTIVE IN-SITU HYBRIDIZATION HISTOCHEMISTRY

Nuclear retinoic acid receptors (RARs) function as ligand-activated tr ans-acting transcription factors and mediate the effects of retinoids on gene expression, cell growth, and differentiation. Determination of the receptors' expression in premalignant and malignant lesions may p rovide prognostic value and direct the selection of receptor-specific retinoids in cancer prevention or treatment. We describe a sensitive a nd practical in situ hybridization method for the analysis of RARs in tissue sections of fixed and embedded surgical specimens. Digoxigenin- labeled antisense and sense RNA probes were prepared for nuclear RAR-a lpha, RAR-beta, and RAR-gamma. The specificity of the probes for their respective receptor mRNAs was demonstrated by Northern blot hybridiza tion to total RNA extracted from murine and human cells. Optimal condi tions for in situ localization of the RAR mRNA were established using cultured tumor cells, and these conditions were then used for the dete ction of RAR mRNA in formalin-fixed, paraffin-embedded sections of sur gical specimens from human tumors. The hybridization stain was detecte d in the cytoplasm (where it was expected to be localized) and not see n in the cell nucleus. This method provides a rapid detection procedur e with good resolution that allows one to clearly distinguish strongly and weakly stained cells. A comparison of receptor expression in head and neck squamous carcinoma specimens and in adjacent normal tissues revealed a significant decrease in the level of RAR-beta mRNA in the t umor cells.