Human breast carcinoma cells MCF-7 surviving repeated 2 Gy radiation exposures in vitro exhibit an altered phenotype, isolated as the stable cell line MCF-IR-3, with 9-3-fold increase in EGF-R/TGF-alpha expression and a 3-fold reduction in ER levels. While these cells have survived a cumulative dose of 60 Gy in daily 2 Gy fractions they exhibit characteristics of proliferation stimulation and increased radiosensitivity in standard survival experiments. Our additional finding of radiation-induced EGF-R Tyr phosphorylation after 2 Gy single radiation exposures linked to a 2-fold up-regulation of EGF-R suggests that EGF-4 functions both as an immediate-early and late reacting gene in MCF-7 cells. This proposal will examine the following: (a) Role of plasma membrane associated radiation-induced events of protein Tyr phosphorylation, phospholipase activation, and cytosolic Ca2+ channel activation. (b) The involvement of secondary messenger systems through use of specific inhibitors of PK-C, PK-A, and CaM-dependent kinases. (c) The effects of radiation doses less than 2 Gy on EGF-R expression/Tyr phosphorylation, alteration of EGF-R/TGF-alpha expression, the influence of pharmacological modulation of ER and EGF-R expression, and the role Ca2+ in this process. MCF-7 and MCF-IR-3 cells will be used to isolate and quantify Tyr- phosphorylated EGF-R, PL-A2 and -Cgamma. The role of cytoplasmic protein kinases will be examined through specific inhibitors. A CAT reporter system has been established as EGF-R-cat/MCF-7 constructs which will serve as a sensitive system to study the effects of radiation-induced activation. The gene expression studies will be correlated with measures of cellular radiosensitivity as assessed by survival/repair capacity analyses.
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