Yen Chiu Lin-Lee

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-κB pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-κB site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and IκBα partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-κB. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of IκBβ (but not IκBα), and increase in NF-κB DNA binding activity. Consistent with the idea that ROS may participate inmdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-κB DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of IκBβ, and subsequent activation of NF-κB. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

BAFF-R promotes cell proliferation and survival through interaction with IKKβ and NF-κB/c-Rel in the nucleus of normal and neoplastic B-lymphoid cells

BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKbeta in the cell nucleus, enhancing histone H3 phosphorylation through IKKbeta. Nuclear BAFF-R was also associated with NF-kappaB/c-Rel and bound to NF-kappaB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-kappaB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-kappaB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.

Development of Autoimmunity in IL-14α-Transgenic Mice

Multiple genetic loci contribute to the development of systemic lupus erythematosus (SLE). In murine models for SLE, various genes on chromosome four have been implicated. IL-14 is a cytokine originally identified as a B cell growth factor. The il14 gene is located on chromosome 4. IL-14α is a cytokine encoded by the plus strand of the IL-14 gene using exons 3–10. The expression of IL-14α is increased in (NZB × NZW)F1 mice. In this study, we produced IL-14α-transgenic mice to study the role of IL-14α in the development of autoimmunity. At age 3–9 mo, IL-14α-transgenic mice demonstrate increased numbers of B1 cells in the peritoneum, increased serum IgM, IgG, and IgG 2a and show enhanced responses to T-dependent and T-independent Ags compared with littermate controls. At age 9–17 mo, IL-14α-transgenic mice develop autoantibodies, sialadenitis, as in Sjögren’s syndrome, and immune complex-mediated nephritis, as in World Health Organization class II SLE nephritis. Between the ages 14–18 mo, 95% of IL-14α-transgenic mice developed CD5+ B cell lymphomas, consistent with the lymphomas seen in elderly patients with Sjögren’s syndrome and SLE. These data support a role for IL-14α in the development of both autoimmunity and lymphomagenesis. These studies may provide a genetic link between these often related disorders.

Differential sensitivities of the MRP gene family and γ-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status

1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075-85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.

Nuclear Localization in the Biology of the CD40 Receptor in Normal and Neoplastic Human B Lymphocytes

CD40 is a tumor necrosis factor (TNF) receptor superfamily, (TNFR; TNFRSF-5) member, that initiates important signaling pathways mediating cell growth, survival, and differentiation in B-lymphocytes. Although CD40 has been extensively studied as a plasma membrane-associated growth factor receptor, we demonstrate here that CD40 is present not only in the plasma membrane and cytoplasm but also in the nucleus of normal and neoplastic B-lymphoid cells. Confocal microscopy showed that transfected CD40-green fluorescent fusion protein entered B-cell nuclei. The CD40 protein contains a nuclear localization signal sequence that, when mutated, blocks entry of CD40 into the nucleus through the classic karyopherins (importins-α/β) pathway. Nuclear fractionation studies revealed the presence of CD40 protein in the nucleoplasm fraction of activated B cells, and chromatin immunoprecipitation assays demonstrated that CD40 binds to and stimulates the BLyS/BAFF promoter, another TNF family member (TNFSF-13B) involved in cell survival in the B cell lineage. Like other nuclear growth factor receptors, CD40 appears to be a transcriptional regulator and is likely to play a larger and more complex role than previously demonstrated in regulating essential growth and survival pathways in B-lymphocytes.

MDR AND MRP GENE FAMILIES AS CELLULAR DETERMINANT FACTORS FOR RESISTANCE TO CLINICAL ANTICANCER AGENTS

The constant threat by a countless array of environmental poisons, natural products and synthetic agents, over evolutionary time has led living organisms to develop many elaborate mechanisms that combat the toxic effects of these insults. Among such mechanisms is one that decreases the intracellular accumulation of a toxic substance by directly pumping toxic molecules out of the cells, and another that modifies the metabolism of the toxic substances and effluxes the metabolized compounds. The former mechanism is typified by the mammalian multidrug resistance system mediated by P-glycoproteins (P-gp) that are encoded by the MDR gene family. The second mechanism is exemplified by the multidrug resistance protein (MRP). Both P-gp and MRP contain ATP-binding cassettes and therefore belong to the ABC superfamily of membrane transporters

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-κB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5′-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Induction of MRP1 and γ-glutamylcysteine synthetase gene expression by interleukin 1β is mediated by nitric oxide-related signalings in human colorectal cancer cells

Treatment of human colorectal cancer cells HT29 with interleukin 1β (IL‐1β) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP‐dependent glutathione S‐conjugate export (GS‐X) pump and the γ‐glutamylcysteine synthetase (γ‐GCSh) gene encoding heavy (catalytic) subunit of γ‐glutamylcysteine synthetase, the rate‐limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by NG‐methyl‐L‐arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL‐1β‐mediated MRP1 and γ‐GCSh induction involve nitric oxide (NO) ‐related signaling. Further supports to the involvement of NO in the induction of MRP1 and γ‐GCSh expression are made by the following observations. (i) Expression of MRP1 and γ‐GCSh genes were induced by treating the cells with NO donors, i.e., S‐nitro‐N‐acetyl‐D,L‐penicillamide (SNAP) and S‐nitroso‐L‐glutathione, in a concentration‐dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and γ‐GCSh. We also demonstrated that HT‐29 cells treated with either 1L‐1β or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT‐29 cells resulted in induction of γ‐GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and γ‐GCSh by IL‐1β is regulated, at least in part, by an NO‐related signaling, and induction of γ‐GCSh is by NO‐related ceramide signaling. J. Cell. Physiol. 185:293–301, 2000.