Activated Rac1 regulates the degradation of IκBα and the nuclear translocation of STAT3–NFκB complexes in starved cancer cells

In several human tumors, signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB) are activated and interact; how these STAT3–NFκB complexes are transported to the nucleus is not fully understood. In this study, we found that Rac1 was activated in starved cancer cells and that activated Rac1 coexisted with STAT3 and NFκB. Rac1 knockdown and overexpression of the dominant-negative mutant Rac1N19 inhibited the degradation of IκBα, an inhibitor of NFκB. MG132, an inhibitor of the ubiquitin proteasome pathway, increased the amount of non-phosphorylated IκBα, but not serine-phosphorylated IκBα, indicating that IκBα degradation by Rac1 in starved cancer cells is independent of IκBα serine phosphorylation by IKK. Rac1 knockdown also inhibited the nuclear translocation of STAT3–NFκB complexes, indicating that this translocation requires activated Rac1. We also demonstrated that the mutant STAT3 Y705F could form complexes with NFκB, and these unphosphorylated STAT3–NFκB complexes translocated into the nucleus and upregulated the activity of NFκB in starved cancer cells, suggesting that phosphorylation of STAT3 is not essential for its translocation. To our knowledge, this is the first study demonstrating the crucial role of Rac1 in the function of STAT3–NFκB complexes in starved cancer cells and implies that targeting Rac1 may have future therapeutic significance in cancer therapy.

Phosphatidylinositol 4-phosphate 5-kinase alpha is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5Kalpha) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kalpha is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kalpha mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kalpha knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1beta and TNFalpha. PIP5Kalpha knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-kappaB and the degradation of IkappaB-alpha, indicating that PIP5Kalpha knockdown interfered with the ganglioside-activated NF-kappaB signaling. Together, these results suggest that PIP5Kalpha is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-kappaB activation in ganglioside-stimulated astrocytes.

Effects of HMGB-1 Overexpression on Cell-Cycle Progression in MCF-7 Cells

High mobility group-1 (HMGB-1) enhances the DNA interactions and possesses a transcriptional activation potential for several families of sequence-specific transcriptional activators. In order to examine the effect of HMGB-1 on the cell cycle progression in MCF-7 cells, the HMGB-1 expression vector was transfected into synchronized MCF-7 cells, and the effect of HMGB-1 overexpression on the cell cycle was examined. The HMGB-1 protein level in the transfected cells increased 4.87-fold compared to the non-transfected cells. There were few changes in the cell cycle phase distribution after HMGB-1 overexpression in the MCF-7 cells. Following the estrogen treatment, the cell cycle progressed in both the HMGB-1 overexpressed MCF-7 and the mock-treated cells. However, a larger proportion of HMGB-1 overexpressing MCF-7 cells progressed to the either S or G2 phase than the mock-treated cells. The mRNA levels of the cell cycle regulators changed after being treated with estrogen in both the HMGB-1 overexpressing MCF-7 and the mock-treated cells, but the changes in the expression level of the cell cycle regulator genes were more prominent in the HMGB-1 overexpressing MCF-7 cells than in the mock-treated cells. In conclusion, HMGB-1 overexpression itself does not alter the MCF-7 cell cycle progression, but the addition of estrogen to the HMGB-1 overexpressing MCF-7 cells appears to accelerate the cell cycle progression.

Screening and Cloning of Genes Preferentially Expressed in Aminopterin-Treated Myeloma Cell Apoptosis / 대한암학회지

PURPOSE: When murine myeloma cells P3-X63-Ag8.653 (V653) of this model treated with amin opterin, an anticancer drug, they can't synthesize nucleic acid via de novo or salvage pathway and selectively eliminated due to apoptosis. This study was aimed to clone specific known and novel genes preferentially expressed in aminopteirn-treated tumor cell apoptosis. MATERIALS AND METHODS: This study was aimed to clone specific known and novel genes pre ferentially expressed in aminopteirn-treated tumor cell apoptosis by using subtraction-PCR technique. RESULTS: By using this technique 868 clones were obtained. Of these 427 clones were positive with insert DNA. By using cross-hybridization Southern blotting, final 101 clones were selected. All of these genes were sequenced and analyzed by using genebank DNA database. Total 101 clones of genes preferentially expressed in apoptotic tumor cells were classified into 10 groups, which included ribosomal proteins, nuclear proteins, mitdegrees Chondrial proteins, signal transductional proteins, retroviral proteins, cell surface receptor proteins, cell structural proteins, unclassified miscellaneous proteins, and novel genes. Especially, Unknown novel genes preferentially ex pressed in this apoptotic tumor cells included clone numbers S1-63, 1-1, 1-3, 1-16, 1-18, 1-20, 3-33, 3-41, 3-44, 3-48, 3-55, 3-60, 6-17, 6-25, 8-12, 50-7, 50-23, and 100-35. CONCLUSION: It seemed that known and unknown novel genes cloned in this study would con tribute to the future studies regarding apoptosis of tumor cells and cancer treatment therepy.