Raf kinase inhibitor protein (RKIP) has been shown to be a metastasis suppressor in many kinds of malignant tumors. But its function in breast cancer was not yet clarified completely. We detected RKIP expression in clinical samples of primary breast cancer, breast cancer metastases, and in different breast cancer cells. Compared with the normal breast epithelia, benign breast epithelia, or in situ ductal carcinoma, the expression level of RKIP is decreased in invasive carcinoma and significantly reduced or lost in the metastasis lymph node matched to the invasive carcinoma. To explore the potential role of RKIP in breast cancer metastasis, we studied the effect of RKIP on the malignant phenotypes of the breast cancer cells with ectopically overexpression or knockdown of RKIP. Cell proliferation, soft-agar colony formation, in vitro adhesion assay, invasion, and migation assays were done to examine the malignant phenotypes of the transfected cells. Consequently, RKIP has no effect on in vitro proliferation rate or colony-forming ability of MDA-MB-435 cells. In vitro cell invasion and migration assays indicated that the RKIP expression was inversely associated with the invasiveness of MDA-MB-435 cells. Consistent with these results, in the orthotopic murine models, we observed that overexpression of RKIP in breast cancer cells impaired invasiveness and metastasis, whereas down-regulation of RKIP expression promoted invasiveness and metastasis. These results indicate that RKIP is a metastasis suppressor gene of human breast cancer. (Mol Cancer Res 2009;7(6):832–40)

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17b-estradiol (E2) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)a and ERb expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E2 not only enhanced IL-6 and IL-8 production via NF-jB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely bolished E2-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E2-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ERa increase and ERb decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ERa pathway.

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer,and melanoma cells. However,the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis,we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently,the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result,the cell proliferation, anchorage-independent growth,cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore,the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation,migration,or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA. (Mol Cancer Res 2008;6(6):917–28)

Aim: The aim of this study was to analyze the expression levels of plasma soluble Fas (sFas) and soluble Fas ligand (sFasL) in patients with orthotopic liver transplantation (OLT) procedures routinely performed without venovenous bypass. Methods: The sFas and sFasL were analyzed in the blood of 20 consecutive patients who underwent transplantation. Blood amples were drawn from the radial artery at serial time points before, during, and after surgery. Plasma levels of sFas and sFasL were detected by Enzyme Linked-Immuno-Sorbent Assay. Plasma aspartate transaminase (AST) and alanine transaminase (ALT) were assayed by routine clinical chemistry esting. Results: Marked elevation of plasma AST and ALT were detected at the reperfusion and postoperation time points (P < 0.001), with a peak on the first postoperative day. The mean plasma concentration of sFas and sFasL remained unchanged from preoperative to anhepatic phase (T1 to T3) (P P 0.268). The sFas and sFasL concentrations were significantly higher at 15 and 60 min after reperfusion compared to the preoperative value (P 6 0.048). Postoperatively, sFas and sFasL concentration were decreased to preoperative levels on the first postoperative day (PP 0.127). Conclusion: The sFas and sFasL seem to be involved in reperfusion injury during OLT. he understanding of Fas may provide new insights into the mechanisms of ischemia/reperfusion injury during OLT.

Transcription and pre-mRNA splicing are the key nuclear processes in eukaryotic gene expression, and identification of factors common to both processes has suggested that they are functionally coordinated. p100 protein has been shown to function as a transcriptional co-activator for several transcription factors. p100 consists of staphylococcal nuclease (SN)-like and Tudor-SN (TSN) domains of which the SN-like domains have been shown to function in transcription, but the function of TSN domain has remained elusive. Here we identified interaction between p100 and small nuclear ribonucleoproteins (snRNP) that function in pre-mRNA splicing. The TSN domain of p100 specifically interacts with components of the U5 snRNP, but also with the other spliceosomal snRNPs. In vitro splicing assays revealed that the purified p100, and specifically the TSN domain of p100, accelerates the kinetics of the spliceosome assembly, particularly the formation of complex A, and the transition from complex A to B. Consistently, the p100 protein, as well as the separated TSN domain, enhanced the kinetics of the first step of splicing in an in vitro splicing assay in dose-dependent manner. Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and splicing.

Signal transducer and activator of transcription 6 (STAT6) is a regulator of transcription for interleukin-4 (IL-4)-induced genes. The ability of STAT6 to activate transcription depends on functional interaction with other transcription factors and coactivators. We have characterized the mechanism of STAT6-mediated transcriptional activation by identifying STAT6 transcription activation domain (TAD) interacting nuclear proteins. The first of the identified proteins was coactivator protein p100, which regulates IL-4-induced transcription by connecting STAT6 with other transcriptional regulators. Here, we describe RNA helicase A (RHA) as a novel component of STAT6 transcriptosome. In vitro and in vivo experiments indicated that RHA did not directly interact with STAT6, but p100 protein was found to mediate the assembly of the ternary complex of STAT6-p100-RHA. In chromatin immunoprecipitation studies RHA together with p100 enhanced the binding of STAT6 on the human Ig« promoter after IL-4 stimulation. RHA enhanced the IL-4-induced transcription, and the participation of RHA in IL-4-regulated transcription was supported by RNAi experiments. Our results suggest that RHA has an important role in the assembly of STAT6 transcriptosome. As RHA is also known to interact with chromatin modifying proteins, the RHA containing protein complexes may facilitate the entry of transcriptional apparatus to the IL-4 responsive promoters.

induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. Coactivator proteins CBP/p300 have been implicated in regulation of transcription in all STATs. CBP is also required for STAT6-mediated gene activation, but the underlying molecular mechanisms are still elusive. In this study we investigated the mechanisms by which STAT6 recruits CBP and chromatin-modifying activities to the promoter. Our results indicate that while STAT1-interacted directly with CBP, the interaction between STAT6 and CBP was found to be mediated through p100 protein, a coactivator protein that has previously been shown to stimulate the transcription of IL-4-induced genes. The staphylococcal nuclease-like (SN)-domains of p100 directly interacted with amino acids 1099–1758 of CBP, while p100 did not associate with SRC-1, another coactivator of STAT6. p100 was found to recruit histone acetyltransferase (HAT) activity to STAT6 in vivo. Chromatin immunoprecipitation studies demonstrated that p100 increases the STAT6-p100-CBP ternary complex formation in the human Ig promoter. p100 also increased the amount of acetylated histone H4 at the Ig promoter, and siRNAs directed against p100 effectively inhibited Ig reporter gene expression. Our results suggest that p100 has an important role in the assembly of STAT6 transcriptosome, and that p100 stimulates IL-4-dependent transcription by mediating interaction between STAT6 and CBP and recruiting chromatin modifying activities to STAT6-responsive promoters.

The high-af®nity receptor for IgG (FcgRI) is a myeloid cell-speci®c and IFN-g-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-speci®c gene responses. The expression of FcgRI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural FcgRI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-g-induced FcgRI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and FcgRI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for FcgRI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the FcgRI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results de®ne functional cooperativity between PU.1 and Stat1 in FcgRI promoter activation where PU.1 serves as an ampli®er and bridging factor with the basal transcription machinery.