LOCALIZATION OF EPIDERMAL GROWTH-FACTOR RECEPTOR ENHANCER PROTEIN IN A431 EPIDERMOID CARCINOMA-CELLS BY SOUTHWESTERN HISTOCHEMISTRY

Since abnormal expression of epidermal growth factor receptor (EGFR) i s 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 f rozen sections of A431 human epidermoid tumor transplated into nude mi ce by southwestern histochemistry. As probes for southwestern histoche mistry, (+) 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 w ith digoxigenin (Dig)-11-dUTP. The sites of Dig were visualized enzyme -immunohistochemically with horseradish peroxidase-labeled anti-Dig. T he 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 e xtracted from A431 tumor cells, but not a GCF band. When the frozen se ctions of A431 tumor were fixed with 4% paraformaldehyde and reacted w ith the 91 bp probe, the staining of perinuclear area as well as nucle i in a speckled pattern were observed and the staining intensity was i ncreased 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 ra tio was a maximam at 150 mM NaCl. The staining with the 91 bp probe wa s 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 unl abeled GCF DNA, indicating that the nuclear and perinuclear staining w ith the 91 bp probe reflects the localization of ETF, Thus, southweste rn histochemistry can be a novel tool to analyze cellular expression o f gene-specific transcription regulatory factors.