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Since abnormal expression of epidermal growth factor receptor (EGFR) is frequently associated with cancer development, the analysis of EGFR gene expression at the transcriptional level as well as the transcript level is helpful to understand the abnormal nature of cancer growth. In this study, we attempted to localize EGFR transcriptional factors, EGFR specific transcription factor (ETF) and GC factor (GCF), in the frozen sections of A431 human epidermoid tumor transplated into nude mice by southwestern histochemistry. As probes for southwestern histochemistry, (+) and (-) sequences of the DNA segment (91 base pairs (bp)) including ETF and GCF regulatory element were synthesized, allowed to be annealed and then tailed by terminal deoxynucleotidyl transferase with digoxigenin (Dig)-11-dUTP. The sites of Dig were visualized enzyme-immunohistochemically with horseradish peroxidase-labeled anti-Dig. The 91 bp probe detected effectively a single ETF band with a molecular mass of 120 kD on a southwestern blot of the crude nuclear fraction extracted from A431 tumor cells, but not a GCF band. When the frozen sections of A431 tumor were fixed with 4% paraformaldehyde and reacted with the 91 bp probe, the staining of perinuclear area as well as nuclei in a speckled pattern were observed and the staining intensity was increased depending upon the concentrations of the probe and reached a plateu level at 0.5-1 mu g/ml. Moreover, the nuclear staining with the probe was dependent upon a salt concentration and the signal/noise ratio was a maximam at 150 mM NaCl. The staining with the 91 bp probe was abolished by the presence of an excess amount of unlabeled 91 bp DNA or unlabeled ETF responsive element DNA alone, but not by that of unlabeled GCF DNA, indicating that the nuclear and perinuclear staining with the 91 bp probe reflects the localization of ETF, Thus, southwestern histochemistry can be a novel tool to analyze cellular expression of gene-specific transcription regulatory factors.

Localization of Epidermal Growth Factor Receptor Enhancer Protein in A431 Epidermoid Carcinoma Cells by Southwestern Histochemistry.