Effects of an irinotecan derivative, ZBH­1208, on the immune system in a mouse model of brain tumor and its antitumor mechanism.

The present study aimed to evaluate the inhibitory effects of an irinotecan derivative, ZBH­1208, on brain tumors, and to explore the underlying molecular mechanisms. To determine the effects of ZBH­1208, a brain tumor mouse model was established by transplanting B22 cells. Subsequently, the visceral indices of immune organs and white blood cell counts were determined, and the effects of ZBH­1208 on the expression levels of cell cycle­related proteins were assessed by western blotting. The tumor inhibition rates of 20 and 40 mg/kg ZBH­1208 were 11.7 and 54.1%, respectively. Compared with the negative control group, ZBH­1208 barely affected visceral indices or white blood cell count. Furthermore, the expression levels of p53, p21, cyclin­dependent kinase 7 (CDK7), Wee1, phosphorylated (p)­cell division cycle 2 (CDC2) (Tyr15), p­CDC2 (Thr161) and cyclin B1 proteins were upregulated, whereas the expression levels of cyclin E were downregulated, and those of CDC2, CDK2 and CDC25C were barely altered. In conclusion, the present study demonstrated that ZBH­1208 suppressed the growth of B22 mouse brain tumor xenografts, but did not affect their visceral indices or white blood cell counts. It was suggested that ZBH­1208 exerted its effects by regulating the expression of p53, p21, Wee1, p­CDC2 (Tyr15) and cyclin E proteins.

Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice.

Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5+ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G1/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis-dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection.

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.

The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB­231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression.

Unraveling natalizumab effects on deregulated miR-17 expression in CD4+ T cells of patients with relapsing-remitting multiple sclerosis.

MicroRNAs (miRNAs) are a family of noncoding RNAs that play critical roles in the posttranscriptional regulation of gene expression. Accumulating evidence supports their involvement in the pathogenesis of multiple sclerosis (MS). Here, we compare miR-17 expressions in CD4+ T cells from relapsing-remitting (RR) MS patients treated with natalizumab versus untreated patients. miR-17 was downregulated under natalizumab treatment and upregulated during relapse, therefore supporting a possible role of miR-17 in MS immunopathogenesis. Downregulation of miR-17 was associated with upregulation of PTEN, BIM, E2F1, and p21 target genes. In vitro miR-17 inhibition was associated with upregulation of the same targets and resulted in impaired CD4+ T cell activation and proliferation. We further describe deregulated TGFBR2 expression in untreated patients versus healthy volunteers (HVs) and confirm in vitro the link between miR-17 and TGFBR2 expressions. These findings support an effect of natalizumab on expression of specific miRNA and subsequent expression of genes involved in proliferation and control of the cell cycle.

Dendritic cell phenotype can be improved by certain chemotherapies and is associated with alterations to p21(waf1/cip1.).

INTRODUCTION: Dendritic cells (DCs) possess the capacity to elicit immune responses against harmful antigens and have been used in DC-vaccines to stimulate the immune system to engage cancer cells. However, a lack of an appreciation of the quality of the DC that is used and/or the monocyte from which it is derived has limited their successful incorporation into treatment strategies. METHODS: In the current study, we explored the relationship between cytokine receptor expression on the monocytes and its subsequent development into DCs. The significance of p21 expression in DCs during differentiation was also studied, as was the effect that manipulating this with chemotherapy may have on DC quality. RESULTS: DCs separated into two groups based on their ability to respond to a maturation stimulus. This quality correlated with a particular receptor profile of granulocyte-macrophage colony-stimulating factor and interleukin 4 expressed on the monocytes from which they were derived. DC quality was also associated with p21 expression, and artificially increasing their levels in DCs by using some chemotherapy improved function. CONCLUSIONS: Overall, these studies have highlighted a role for common chemotherapy in activating p21 in DCs, which is a prerequisite for good DC function.

Reevaluating the concept of treating experimental tumors with a mixed bacterial vaccine: Coley's Toxin.

Several decades after Coley's initial work, we here systematically analyzed tumoricidal as well as immunostimulatory effects of the historical preparation Coley's Toxin (CT), a safe vaccine made of heat-inactivated S. pyogenes and S. marcescens. First, by performing in vitro analysis, established human pancreatic carcinoma cell lines responded with dose- and time-dependent growth inhibition. Effects were attributed to necrotic as well as apoptotic cell death as determined by increased Caspase 3/7 levels, raised numbers of cells with sub-G1-DNA, and induced p21(waf) expression, indicative for cell cycle arrest. Besides, CT effectively stimulated human peripheral blood leukocytes (huPBL) from healthy volunteers. Quantitative gene expression analysis revealed upregulated mRNA levels of selected Toll-like receptors. Flow cytometric phenotyping of CT-stimulated huPBLs identified raised numbers of CD25(+)-activated leukocytes. In vivo, repetitive, local CT application was well tolerated by animals and induced considerable delay of Panc02 tumors. However, systemic treatment failed to affect tumor growth. Antitumoral effects following local therapy were primarily accompanied by stimulation of innate immune mechanisms. Data presented herein prove that the historical approach of using killed bacteria as active immunotherapeutic agents still holds promise, and further careful preclinical analyses may pave the way back into clinical applications.

STAT3 protein interacts with Class O Forkhead transcription factors in the cytoplasm and regulates nuclear/cytoplasmic localization of FoxO1 and FoxO3a proteins in CD4(+) T cells.

An important feature of the adaptive immune response is its remarkable capacity to regulate the duration of inflammatory responses, and effector T cells have been shown to limit excessive immune responses by producing anti-inflammatory cytokines such as IL-10 and IL-27. However, how anti-inflammatory cytokines mediate their suppressive activities is not well understood. In this study, we show that STAT3 contributes to mechanisms that control the duration of T cell proliferation by regulating the subcellular location of FoxO1 and FoxO3a, two Class O Forkhead transcription factors that mediate lymphocyte quiescence and inhibit T cell activation. We show that active FoxO1 and FoxO3a reside exclusively in the nucleus of naïve T cells whereas inactive pFoxO1 and pFoxO3a were most abundant in activated T cells and sequestered in their cytoplasm in association with unphosphorylated STAT3 (U-STAT3) and 14-3-3. We further show that FoxO1/FoxO3a rapidly relocalized into the nucleus in response to pSTAT3 activation by IL-6 or IL-10, and the accumulation of FoxO1/FoxO3a in their nuclei coincided with increased expression of p27(Kip1) and p21(WAF1). STAT3 inhibitors completely abrogated cytokine-induced translocation of FoxO1/FoxO3a into the nucleus. In naïve or resting STAT3-deficient T cells, expression of pFoxO1/pFoxO3a was predominantly in the cytoplasm and correlated with defects in p27(Kip1) and p21(WAF1) expression, suggesting requirement of STAT3 for importation or retention of FoxO in the nucleus and attenuation of lymphocyte proliferation. Taken together, these results suggest that U-STAT3 collaborates with 14-3-3 to sequester pFoxO1/pFoxO3a in cytoplasm and thus prolong T cell activation, whereas pSTAT3 activation by anti-inflammatory cytokines would curtail the duration of TCR activation and re-establish lymphocyte quiescence by inducing nuclear localization of FoxO1/FoxO3a and FoxO-mediated expression of growth-inhibitory proteins.

Human regulatory T cells induce T-lymphocyte senescence.

Regulatory T (Treg) cells have broad suppressive activity on host immunity, but the fate and function of suppressed responder T cells remains largely unknown. In the present study, we report that human Treg cells can induce senescence in responder naive and effector T cells in vitro and in vivo. Senescent responder T cells induced by human Treg cells changed their phenotypes and cytokine profiles and had potent suppressive function. Furthermore, Treg-mediated molecular control of senescence in responder T cells was associated with selective modulation of p38 and ERK1/2 signaling and cell-cycle-regulatory molecules p16, p21, and p53. We further revealed that human Treg-induced senescence and suppressor function could be blocked by TLR8 signaling and/or by specific ERK1/2 and p38 inhibition in vitro and in vivo in animal models. The results of the present study identify a novel mechanism of human Treg cell suppression that induces targeted responder T-cell senescence and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression.

Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling.

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.

Histone deacetylase inhibitor uses p21(Cip1) to maintain anergy in CD4+ T cells.

T cell anergy defined as antigen-specific proliferative unresponsiveness was induced in CD4+ T cells exposed to antigen (Ag) in the presence of the histone deacetylase (HDAC) inhibitors n-butyrate, trichostatin A or scriptaid. However, the ability of HDAC inhibitors to induce anergy in Th1 cells was not due to general histone hyperacetylation. Instead, the anergy induced by HDAC inhibitors was associated with upregulation of p21(Cip1), a secondary effect of histone acetylation. Induction of p21(Cip1) in the absence of histone hyperacetylation by exposure to okadaic acid also resulted in T cell anergy. In addition, Ag-specific p21(Cip1)-deficient CD4+ T cells were much less susceptible to anergy induction by n-butyrate. Thus, p21(Cip1) appears to mediate the proliferative unresponsiveness found in CD4+ T cell anergized by exposure to Ag in the presence of HDAC inhibitors.

Leukocyte activation and circulating leukocyte-derived microparticles in preeclampsia.

PROBLEM: Preeclampsia shows characteristics of an inflammatory disease including leukocyte activation. Analyses of leukocyte-derived microparticles (MP) and mRNA expression of inflammation-related genes in leukocytes may establish which subgroups of leukocytes contribute to the development of preeclampsia. METHOD OF STUDY: Blood samples were obtained from preeclamptic patients, normotensive pregnant and non-pregnant controls. sL-selectin and elastase were measured by ELISA. mRNA was isolated from leukocytes and gene expression was determined by multiplex ligation-dependent probe amplification (MLPA). MP were characterized by flow cytometry. RESULTS: Altered concentrations of sL-selectin and elastase confirmed leukocyte activation in preeclampsia. These leukocytes showed up-regulation of Nuclear Factor of Kappa light chain gene enhancer in B cells inhibitor (NFkappaB-1A) and cyclin-dependent kinase inhibitor (CDKN)-1A compared with normotensive pregnant women. Interleukin-1 Receptor Antagonist (IL-1RA) and tumor necrosis factor (TNF)-R1 were increased compared with those in non-pregnant controls. Monocyte-derived MP were elevated in preeclamptic patients compared with pregnant women. The numbers of cytotoxic T-cell-derived and granulocyte-derived MP were elevated compared with those of non-pregnant women. CONCLUSION: Leukocytes are activated in preeclampsia. A pro-inflammatory gene expression profile is not prominent, although differences in mRNA expression can be detected. Increased levels of particular subsets of leukocyte-derived MP reflect activation of their parental cells in preeclampsia.

p21(waf1/CIP1), a CDK inhibitor and a negative feedback system that controls macrophage activation.

p21(WAF1/CIP1) (p21) is a crucial CDK inhibitor that controls the cell cycle. This molecule is also involved in the regulation of apoptosis and gene expression. However, like many other cell regulators, the functional activity of p21 depends on its cellular context and is controlled through phosphorylation and protein-protein interactions. p21 is also important in cells of the immune system regulating the cell cycle and preventing apoptosis of macrophages. In this issue of the European Journal of Immunology, two reports investigate the role of p21 further determining its critical role as a negative regulator of macrophage activation, in particular inhibiting the LPS-dependent induction of TNF-alpha and IL-1beta. The inhibition mediated by p21 is shown to be related to NF-kappaB activity. Furthermore, the observation that p21(-/-) mice are more susceptible to septic shock supports the notion that p21 is a negative regulator of macrophage activation and therefore a potential new target to control inflammatory diseases.

The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

Regulation of macrophage activation and septic shock susceptibility via p21(WAF1/CIP1).

p21 is a cell-cycle inhibitor that is also known to suppress autoimmunity. Here, we provide evidence of a novel role for p21 as an inhibitor of macrophage activation. LPS stimulation of p21-deficient peritoneal macrophages induced increased activation compared with controls, with elevated production of proinflammatory mediators such as TNF-alpha and IL-1beta. The enhanced activity of LPS-stimulated p21-deficient macrophages correlated with increased activity of the transcription factor NF-kappaB. LPS stimulation of p21-deficient macrophages led to increased IkappaBalpha kinase activity, and increased IkappaBalpha phosphorylation and degradation, resulting in elevated NF-kappaB activity. The effect of p21 in macrophage activation was independent of its cell-cycle inhibitory role. p21(-/-) mice showed greater sensitivity to LPS-induced septic shock than did WT mice, indicating that p21 contributes to maintenance of a balanced response to inflammatory stimuli and suggesting biological significance for the role of p21 in macrophage activation. Our findings project a role for p21 in the control of NF-kappaB-associated inflammation, and suggest that therapeutic modulation of p21 expression could be beneficial in inflammation-associated diseases.

IL-4 blocks M-CSF-dependent macrophage proliferation by inducing p21Waf1 in a STAT6-dependent way.

Macrophages are recruited from the blood stream to the inflammatory loci to carry out their functional activities. In an early phase of the cell cycle, macrophages become activated by Th1-type cytokines (i.e. IFN-gamma), thereby producing several factors (cytokines, NO, etc.) and developing pro-inflammatory activities. When bacteria and apoptotic bodies are removed, through the interaction with Th2-type cytokines (i.e. IL-4), macrophages become anti-inflammatory and repair damaged tissues. Incubation of bone-marrow-derived macrophages with IFN-gamma or IL-4 blocked their proliferation. While M-CSF withdrawal caused cell cycle arrest at the early G(1) phase, treatment of macrophages with IFN-gamma or IL-4 caused this arrest later, at the G(1)/S boundary. Proliferation arrest was not due to an induction of apoptosis. IFN-gamma and IL-4 induced the expression of the cyclin-dependent kinase (Cdk) inhibitor p21(Waf1). Using KO mice and iRNA experiments, we found that p21(Waf1)is required for IL-4- but not for IFN-gamma-dependent inhibition of macrophage proliferation. IL-4 inhibited M-CSF-dependent Cdk-2 and Cdk-4 activities, which are necessary for entry and passage through the S phase of the cell cycle. The signal transduction used to induce the expression of p21(Waf1)after interaction of IL-4 with the corresponding receptor was mediated by STAT6. Thus, IL-4 and IFN-gamma blocked M-CSF-induced macrophage proliferation through distinct mechanisms.

The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5.

Kaposi's sarcoma-associated herpesvirus encodes four genes with homology to the family of interferon regulatory factors (IRFs). At least one of these viral IRFs, vIRF-3, is expressed in latently Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma (PEL) cells and is essential for the survival of PEL cells. We now report that vIRF-3 interacts with cellular IRF-5, thereby inhibiting binding of IRF-5 to interferon-responsive promoter elements. Consequently, vIRF-3 blocked IRF-5-mediated promoter activation. A central double helix motif present in vIRF-3 was sufficient to abrogate both DNA binding and transcriptional transactivation by IRF-5. Upon DNA damage or activation of the interferon or Toll-like receptor pathways, cytoplasmic IRF-5 has been reported to be translocated to the nucleus, which results in induction of both p53-independent apoptosis and p21-mediated cell cycle arrest. We report here that IRF-5 is present in the nuclei of PEL cells without interferon stimulation. Silencing of vIRF-3 expression in PEL cells was accompanied by increased sensitivity to interferon-mediated apoptosis and up-regulation of IRF-5 target genes. In addition, vIRF-3 antagonized IRF-5-mediated activation of the p21 promoter. The data presented here indicate that vIRF-3 contributes to immune evasion and sustained proliferation of PEL cells by releasing IRF-5 from transcription complexes.

Early growth response gene 2 (Egr-2) controls the self-tolerance of T cells and prevents the development of lupuslike autoimmune disease.

Maintaining tolerance of T cells to self-antigens is essential to avoid autoimmune disease. How self-reactive T cells are kept functionally inactive is, however, unknown. In this study, we show that early growth response gene 2 (Egr-2), a zinc-finger transcription factor, is expressed in CD44(high) T cells and controls their proliferation and activation. In the absence of Egr-2, CD44(high), but not CD44(low) T cells, are hyperreactive and hyperproliferative in vivo. The accumulation of activated CD4(+)CD44(high) T cells leads to the development of a late onset lupuslike autoimmune disease characterized by the accumulation of interferon (IFN)-gamma and interleukin (IL)-17-producing CD4(+) T cells, loss of tolerance to nuclear antigens, massive infiltration of T cells into multiple organs and glomerulonephritis. We found that the expression of cyclin-dependent kinase inhibitor p21cip1 was impaired in Egr-2-deficient T cells, whereas the expression of IFN-gamma and IL-17 in response to T cell receptor ligation was significantly increased, suggesting that Egr-2 activates the expression of genes involved in the negative regulation of T cell proliferation and inflammation. These results demonstrate that Egr-2 is an intrinsic regulator of effector T cells and controls the expansion of self-reactive T cells and development of autoimmune disease.

Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non-suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.

Biliary epithelial cells (BECs) of chronic non-suppurative destructive cholangitis (CNSDC) in primary biliary cirrhosis (PBC) reportedly express p21(WAF1/Cip1) and p16(INK4a), which may induce cell cycle arrest and are related to progressive loss of BECs of PBC. Given that the ATM pathway plays a role in the induction of p21(WAF1/Cip1), we examined its possible involvement in bile duct damage of PBC. The expression of phosphorylated-ATM (p-ATM) reflecting the activation of ATM, p21(WAF1/Cip1) and 8-hydroxy-deoxyguanosine (8-OHdG), an oxidative stress marker, was examined immunohistochemically in the liver tissues of 20 cases of stage 1/2 PBC, 9 extrahepatic biliary obstruction (EBO), 35 chronic viral hepatitis (CVH), 17 nonalcoholic steatohepatitis (NASH), and 18 histologically normal liver. p21(WAF1/Cip1), p-ATM and 8-OHdG were frequently and extensively co-expressed in the nuclei of CNSDC in PBC, and their expressions were correlated. In contrast, the expression of these three molecules was absent or faint in small bile ducts in normal livers, CVH, and EBO, and these molecules were clearly expressed in the nuclei of hepatocytes of NASH, in which oxidative stress is involved in hepatocellular damage. In conclusion, oxidative stress-induced p21(WAF1/Cip1) expression in BECs in PBC is closely associated with activation of the ATM pathway and the resultant reduced regeneration or cell cycle arrest of BECs may be related to the progressive loss of small bile ducts of PBC.

Deacetylase activity is required for STAT5-dependent GM-CSF functional activity in macrophages and differentiation to dendritic cells.

After interaction with its receptor, GM-CSF induces phosphorylation of the beta-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21(waf-1) expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.