c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel-and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin-and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content 44N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.

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IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.