IDENTIFICATION OF ERBB3-STIMULATED GENES USING MODIFIED REPRESENTATIONAL DIFFERENCE ANALYSIS

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific co ntribution of each of the four family members to these processes remai ns unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to ac tivation of ErbB3 and EGFR. The approach described is a modification o f the representational difference analysis technique adapted for analy sis of cDNA, which we have modified to permit identification of differ ential gene expression using as little as 20 mu g of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplico ns (small PCR-amplified fragments) which were subtracted against drive r amplicons derived from unstimulated NIH-3T3 cells expressing the EGF R-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EG FR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons, Six of these, correspond ing to known DNA sequences, were selected for further Northern blot an alysis against total RNA prepared from the starting cell lines. Of the se, the gene encoding the protein dlk (or a closely related protein, P ref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amylo id protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.