PI-3' kinase and NF-kappaB cross-signaling in human pancreatic cancer cells.

Because tumor necrosis factor-alpha (TNF-alpha) and some chemotherapeutic agents activate both apoptosis and NF-kappaB-dependent antiapoptotic genes, they may neutralize their own antitumor effects. The cell-signaling mechanisms for such chemoresistance are not clear but may involve phosphotidylinositol-3' kinase (PI3K). To clarify this we examined whether cross-signaling between PI3K and NF-kappaB enhances the antitumor effect of TNF-alpha in human pancreatic cancer cells. Quiescent pancreatic cancer cells (Panc-1, MiaPaCa-2) with TNF-alpha, Ly294002 (PI3K inhibitor), alone or combined, were restimulated with mitogen (10% fetal calf serum [FCS] to induce cell cycle entry). Proliferation (monotetrazolium), cell cycle progression (ApoBrDU and fluorescence-activated cell sorter analysis), and apoptosis (PARP cleavage; caspase-3 activation) were measured. Akt activation (Akt kinase assay) and IkappaBalpha degradation were determined by Western blot analysis. Translocation of NF-kappaB into the nucleus was examined by EMSA, whereas an NF-kappaB/luciferase reporter gene was used to quantify NF-kappaB-dependent gene expression. Statistical analysis was carried out by means of two-tailed t test (P <0.05). PI3K inhibition significantly enhanced the antiproliferative and proapoptotic effects of TNF-alpha in both cell lines, Ly294002 also blocked TNF-alpha-induced Akt activation but failed to alter cytoplasmic IkappaBalpha degradation or subsequent NF-kappaB nuclear translocation. NF-kappaB-dependent gene expression, however, was ultimately suppressed by Ly294002, suggesting that PI3k-dependent activation of NF-kappaB is IkappaBalpha independent. PI3K inhibition can block NF-kappaB-dependent gene expression regardless of cytoplasmic IkappaBalpha/NF-kappaB activation. Because it also regulates the antitumor effects of TNF-alpha, PI3K may in part determine NF-kappaB-induced chemoresistance in human pancreatic cancer.

micF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli.

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.