In addition to their antiviral activity, IFNs inhibit growth of both normal and transformed cells. The mechanisms of IFN-mediated growth inhibition are not fully understood. IFNs may act by interfering with proliferative stimulus of growth factors. The primary objective of this proposal is to understood the means by which interferon (IFN) inhibits the epidermal-growth factor (EGF)-stimulated growth of a human breast tumor cell line, S4. Previous data from our laboratory indicate IFN may inhibit S4 growth in part by interrupting the mitogenic signal of EGF. To more fully characterize IFN mechanisms of action in this system, we propose the following studies:
Specific aims i nclude the following: 1. To further characterize the biological action of IFN on EGF- stimulated growth of S4 cells: We will assess the effect of IFN on logarithmically growing control and EGF-stimulated S4 cells. We will then determine the effect of IFN on EGF-induced stimulation of DNA synthesis by quiescent S4 cells. We will assess the dependence of the growth inhibitory effect of IFN on time of exposure to IFN. Kinetic studies will help determine if inhibition is cell-cycle specific. We will determine if the IFN induced inhibition of EGF response is dose-dependent and characteristic of competitive or noncompetitive interactions. We will also determine the ability of microtubule dissociating agents to overcome IFN's effect on EGF induced stimulation of DNA synthesis. 2. To determine IFN induced changes in EGF receptor (EGFR) physiology: IFN induced changes in the affinity and number of EGFR, receptor internalization, transcription of EGFR mRNA, receptor structure, and phosphorylation will be explored. IFN induced changes in early intracellular signaling pathways such as changes in cytoplasmic free Ca2+ will be examined. 3. To characterize the effect of IFN on EGF induced expression of the photooncogene c-myc: We will explore changes in steady state levels of c-myc in control and IFN treated cells after EGF stimulation. We will determine if changes in c-myc transcription is due to changes in either transcription rates (by nuclear run off transcription assays) or the stability of c-myc mRNA. IFNs are now regarded as naturally occurring negative growth regulators which may be important in restricting cell proliferation in vivo. IFNs can antagonize growth factor-mediated changes in cell proliferation. The studies proposed will clarify mechanisms of IFN-induced growth inhibition in a human tumor cell line.
