Detection of nuclear retinoic acid receptor mRNA in histological tissue sections using nonradioactive in situ hybridization histochemistry.
Nuclear retinoic acid receptors (RARs) function as ligand-activated trans-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 provide prognostic value and direct the selection of receptor-specific retinoids in cancer prevention or treatment. We describe a sensitive and 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-alpha, RAR-beta, and RAR-gamma. The specificity of the probes for their respective receptor mRNAs was demonstrated by Northern blot hybridization to total RNA extracted from murine and human cells. Optimal conditions for in situ localization of the RAR mRNA were established using cultured tumor cells, and these conditions were then used for the detection of RAR mRNA in formalin-fixed, paraffin-embedded sections of surgical specimens from human tumors. The hybridization stain was detected in the cytoplasm (where it was expected to be localized) and not seen in the cell nucleus. This method provides a rapid detection procedure 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 tumor cells.
Xu
X C
XC
Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston.
Clifford
J L
JL
Hong
W K
WK
Lotan
R
R
D000818:Animals; D015152:Blotting, Northern; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D005260:Female; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D012313:RNA / Q000032:analysis; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000235:genetics*; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
D015347:RNA Probes; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D012313:RNA