The mechanism by which epidermal growth factor (EGF) and other peptide hormones influence cellular growth control are unknown. Most efforts to isolate and identify EGF responsive genes have focused on the early phases of the EGF response. However, the earliest responses are not sufficient to elicit a growth response since EGF must be present continuously for a prolonged period (over 6-12 hours) before commitment occurs. During this time the cells undergo a large number of metabolic changes. It has proven difficult to distinguish those events which are involved in cellular activation from those required for commitment to, or stimulation of cell division. To avoid this problem, genetically related cloned cell lines which differ in their growth response to EGF (stimulated, unresponsive or inhibited) will be used to isolate cDNAs to EGF responsive genes. Although the biological response to EGF is different in each cell type, many of the early responses elicited by EGF are similar in the differentially responsive cloned cell lines; eg. the EGF-receptors are similar in their binding affinity for EGF, the extent of tyrosine phosphorylation elicited by the hormone and the rate and extent of receptor internalization. Large and representative lambda gt10 cDNA libraries were made with cDNA synthesized from each cell clone type treated with EGF for defined intervals. The libraries will be screened with cDNA probes prepared by subtractive hybridization to enrich for sequences specific to the EGF response of each cell clone type. Subtractive hybridization cDNA probes will be used to enrich for EGF responsive sequences which can be categorized as follows: 1) those uniquely expressed in each cloned cell type, 2) those which differ in abundance, and 3) those which are regulated in a similar manner in response to EGF. A comparison of the classes of EGF responsive genes provide a means to begin a systematic analysis of those which are important in the regulation of cellular proliferation. A careful analysis of the cDNA clones will be used to distinguish the EGF regulated genes which are specific to cells stimulated by, inhibited by or non-responsive to EGF. When candidate cDNA clones of the desired types are obtained, they will be used to examine normal and other transformed cells responding to EGF. With such probes in hand, studies will be initiated to identify the encoded genes and to examine the mechanism(s) by which EGF regulates their expression in both normal and transformed cells.
