A composite of exhaled LTB4 , LXA4 , FeNO, and FEV1 as an "asthma classification ratio" characterizes childhood asthma.

BACKGROUND: Aberrant generation of eicosanoids is associated with asthma, but the evidence remains incomplete and its potential utility as biomarkers is unclear. Major eicosanoids in exhaled breath condensates (EBCs) were assessed as candidate markers for childhood asthma. METHODS: Ten exhaled eicosanoid species was evaluated using ELISA in the discovery phase, followed by prediction model-building and validation phases. RESULTS: Exhaled LTB4 , LTE4 , PGE2, and LXA4 showed significant difference between asthmatics (N = 60) and controls (N = 20). For validation, an expanded study population consisting of 626 subjects with asthma and 161 healthy controls was partitioned into a training subset to establish a prediction model and a test sample subset for validation. Receiver operating characteristic (ROC) analyses of the training subset revealed the level of exhaled LTB4 to be the most discriminative among all parameters, including FeNO, and a composite of exhaled LTB4 , LXA4 , together with FeNO and FEV1 , distinguishing asthma with high sensitivity and specificity. Further, the Youden index (J) indicated the cut point value of 0.598 for this composite of markers as having the strongest discriminatory ability (sensitivity = 85.2% and specificity = 83.6%). The predictive algorithm as "asthma classification ratio" was further validated in an independent test sample with sensitivity and specificity being 84.4% and 84.8%, respectively. CONCLUSIONS: In a pediatric study population in Taiwan, the levels of exhaled LTB4 , LTE4 , LXA4, and PGE2 in asthmatic children were significantly different from those of healthy controls, and the combination of exhaled LTB4 and LXA4 , together with FeNO and FEV1 , best characterized childhood asthma.

Impaired phagocytosis and susceptibility to infection in pediatric-onset systemic lupus erythematosus.

OBJECTIVES: Impaired function of polymorphonuclear cells (PMNs) in systemic lupus erythematosus (SLE) leads to severe gram-positive and gram-negative bacterial infection, and to major morbidity and mortality. Few studies have focused on the association of impaired function of PMNs and SLE patients' susceptibility to infection. This study aimed to analyze function of PMNs in peroxidase production, chemotaxis, and phagocytosis in pediatric-onset SLE with severe infection. METHODS: This study compared function of PMNs among pediatric-onset SLE patients with and without histories of severe infection and in normal control subjects. Human peripheral blood PMNs were isolated from patients and controls. Function of PMNs was measured by analyzing peroxidase, chemotaxis, and phagocytic activities. Different disease activity and severity, and drug use in newly diagnosed SLE patients were also compared. RESULTS: In total, 34 SLE patients (12 patients with severe infection, 22 patients without infection) and 25 healthy controls were analyzed. There were no differences in function of PMNs between SLE patients with or without severe infection. Regardless of infection status, medication, and disease activity, SLE patients had impaired phagocytic ability against Salmonella-specific lipopolysaccharides (LPS) compared with normal controls (p < 0.01). The use of immunosuppressants did not influence phagocytic ability against Salmonella-derived LPS. CONCLUSIONS: Immunosuppressant agents do not influence phagocytic ability against Salmonella in SLE subjects. Impaired phagocytosis against Salmonella is prominent in pediatric-onset SLE subjects, which may result in the high prevalence of Salmonella infection. There is no deficiency of peroxidase production and chemotaxis activity among SLE subjects.

Characterization of interleukin 8 in woodchucks with chronic hepatitis B and hepatocellular carcinoma.

The eastern woodchuck, Marmota monax, represents a useful animal model to study hepatitis B virus infection in humans. However, immunological studies in this model have been impeded by a lack of basic information about the components of the immune system such as cytokines and chemokines. To clarify the role(s) of interleukin 8 (IL-8) in chronic hepatitis B and hepatocellular carcinoma (HCC) in the woodchuck model, we cloned and characterized the woodchuck IL-8 cDNA and genomic DNA. Sequence analysis revealed that the organization of the wk-IL-8 gene is similar to that of the human IL-8 gene and consists of four exons and three introns. Woodchuck IL-8 protein exhibits the conserved ELRCXC motif of IL-8 and shows 87, 82, 82 and 79% similarity with rabbit, ovine, bovine and human IL-8 proteins, respectively. The biological activity of wk-IL-8 was demonstrated using neutrophil chemotaxis assays. Wk-IL-8 could be readily detected in both tumor and non-tumor tissues with higher expression in the non-tumor tissues in most cases. The results from this study will facilitate the investigation of IL-8 in the immunopathogenesis of hepadnavirus-related diseases by the woodchuck model.

Correction to: CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation.

Following publication of their article "CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation", the authors reported an error in Fig.6b. α-Tubulin image of rCCN2 treatment  (upper panel in CL1-5) only showed eight lanes, when there should be nine.

Involvement of matrix metalloproteinase-13 in stromal-cell-derived factor 1 alpha-directed invasion of human basal cell carcinoma cells.

Basal cell carcinoma (BCC) is one of the most common skin neoplasms in humans and is usually characterized by local aggressiveness with little metastatic potential, although deep invasion, recurrence, and regional and distant metastases may occur. Here, we studied the mechanism of BCC invasion. We found that human BCC tissues and a BCC cell line had significant expression of CXCR4, which was higher in invasive than non-invasive BCC types. Further, of 19 recurrent tumors among 390 BCCs diagnosed during the past 12 years, 17/19 (89.5%) had high CXCR4 expression. We found that the CXCR4 ligand, stromal-cell-derived factor 1alpha (SDF-1alpha), directed BCC invasion and that this was mediated by time-dependent upregulation of mRNA expression and gelatinase activity of matrix metalloproteinase-13 (MMP-13). The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the AP-1 component c-Jun. Finally, CXCR4-transfected BCC cells injected into nude mice induced aggressive BCCs that co-expressed CXCR4 and MMP-13. The identification of SDF-1alpha/CXCR4 as an important factor in BCC invasiveness may contribute insight into mechanisms involved in the aggressive potential of human BCC and may improve therapy for invasive BCCs.

Effect of cyclosporin A on interleukin-15-activated umbilical cord blood natural killer cell function.

BACKGROUND: Interleukin (IL)-15-activated natural killer (NK) cells may provide a graft-versus-leukemia (GvL) effect post-umbilical cord blood (CB) transplantation. The effect of cyclosporin A (CsA), a calcineurin-inhibitor used for prophylaxis of graft-versus-host disease (GvHD), on IL-15-mediated activation, cytotoxic function and target-induced apoptosis of CB NK cells, was examined in comparison with adult peripheral blood (APB) NK cells. METHODS: CsA was added to anti-CD3+/-IL-15-stimulated CB and APB mononuclear cells (MNC) for a 5-day incubation. CD3- CD56+ NK cell recovery was determined by flow cytometric analysis. Magnetic bead-purified CB and APB NK cells were stimulated with IL-15 for 18 h under the influence of CsA. NK activation (CD69), K562 cytotoxicity and NK-K562 interactions (CD54, perforin and annexin-V expression 4 h following contact with K562 cells) were assessed by flow cytometry. RESULTS: CsA decreased CD3- CD56+ NK cell recovery in anti-CD3-stimulated CB MNC 5-day cultures, an effect that could be counteracted by IL-15; comparable effects were observed with APB. Short-term (18-h) experiments revealed that CsA down-regulated K562 cytotoxicity of IL-15-activated (P=0.018) but not resting (P=0.268) purified CB NK cells. IL-15-induced CB NK CD69 expression showed increased CsA sensitivity over APB (P=0.012). CsA down-regulated K562 cell-induced CD54 (P=0.028) but not perforin (P=0.416) expression of IL-15-activated CB NK cells. Target-induced apoptosis of IL-15-activated CB (P=0.043) but not APB (P=0.144) NK cells was decreased by CsA. DISCUSSION: We have demonstrated differential CsA sensitivity of IL-15-activated CB and APB NK cells. These results may be used to improve the design of IL-15-activated NK cell adoptive immunotherapy in cancer patients receiving CsA post-CB transplantation.

Cost Analysis and Determinants of Living Donor Liver Transplantation in Taiwan.

BACKGROUND: Liver transplantation (LT) has become established therapy for end-stage liver disease and small-cell hepatocellular carcinoma (HCC), relying mainly on living donor LT (LDLT) in Taiwan. The cost of LDLT varies in different countries depending on the insurance system, the costs of the facility, and staff. In this study we aimed to investigate cost outcomes and determinants of LDLT in Taiwan. METHODS: From January 2014 to December 2015, 184 LDLT patients were enrolled in a study performed at the Kaohsiung Chang Gung Memorial Hospital. Patients' transplantation costs were defined as expense from immediately after surgery to discharge during hospitalization for LDLT. Antiviral therapy and hepatitis B immunoglobulin (HBIG) for prevention of hepatitis B virus (HBV) were included, but direct-acting antiviral (DAA) therapy for hepatitis C (HCV) was excluded. RESULTS: The median total, intensive care unit (ICU), and ward costs of LT were US$64,250, $43,357, and $16,138 (currency ratio 1:30), respectively. HBV significantly increased the total cost of LT, followed by postoperative reintubation and bile duct complications. CONCLUSION: The charges associated with anti-HBV viral therapy and HBIG increase the cost of LDLT. Disease severity of liver cirrhosis showed less importance in predicting cost. Postoperative complications such as reintubation or bile duct complications should be avoided to reduce the cost of LT.

Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway.

This study is to investigate the molecular mechanism of radiation-enhanced cell invasiveness of hepatocellular carcinoma (HCC) correlating with clinical patients undergoing radiotherapy and subsequently developing metastasis. Three HCC cell lines (HepG2, Hep3B and Huh7) and normal hepatocyte cell line (CL-48) were irradiated with different doses. The effect of radiation on cell invasiveness was determined using the Boyden chamber assay. Radiation-enhanced invasion capability was evident in HCC cells but not in normal hepatocytes. Invasion was observed in gelatin-coated but not fibronectin-coated or type I collagen-coated membranes. Radiation upregulated matrix metalloproteinase-9 (MMP-9) mRNA level, MMP-9 protein level and MMP-9 activity. MMP-9 antisense oligonucleotides inhibited radiation-induced MMP-9 expression and thereby significantly inhibited radiation-induced HCC invasion. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt chemical inhibitors LY294002 and wortmannin suppressed radiation-induced MMP-9 mRNA expression. Transient transfection with dominant-negative Akt plasmid also showed that the PI3K/Akt-signaling pathway was involved in this radiation-induced MMP-9 expression. Moreover, nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide suppressed radiation enhanced MMP-9 promoter activity completely. PI3K/Akt chemical inhibitors inhibited radiation-induced NF-kappaB-driven luciferase promoter activity. Taken together, our results indicated that sublethal dose of radiation could enhance HCC cell invasiveness by MMP-9 expression through the PI3K/Akt/NF-kappaB signal transduction pathway.

The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position -87 and CRE-2 at -70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway and is one component of the GM-CSF viability response.

mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor beta chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between -197 and -69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.

Cytosolic PKM2 stabilizes mutant EGFR protein expression through regulating HSP90-EGFR association.

This corrects the article DOI: 10.1038/onc.2015.397.

Expression of ribonucleotide reductase after ionizing radiation in human cervical carcinoma cells.

Ribonucleotide reductase (RR), the rate-limiting enzyme in the de novo synthesis of deoxynucleotide triphosphates (dNTPs), is a potential target for cancer therapy. We characterized the response of RR in a human cervical carcinoma cell line, Caski, after damage by ionizing radiation (IR). We also investigated the cell cycle regulation of both the regulatory (R1) and catalytic (R2) RR subunits in an attempt to distinguish between a direct DNA damage induction of RR by IR and a cell cycle-dependent expression of RR after IR. Confluent, growth-arrested Caski cells showed a > or = 5-fold increase in R2 mRNA and an 18-fold increase in R2 protein as cells entered S phase after serum stimulation. The R2 protein levels peaked in late S phase and returned to lower basal levels in G2-M. No changes in R1 mRNA and protein levels occurred with progression through the cell cycle after serum stimulation. In growth-arrested Caski cells treated with IR (6 Gy) without serum stimulation, a similar rise (17-fold) in R2 protein was evident at 24 h after IR and was associated with a 4-fold increase in in situ RR enzyme activity, but no increases in R1 and R2 mRNA nor R1 protein were found. E2 promoter binding factor 1 mRNA and protein levels also showed no change after IR. Growth-arrested controls (no IR and no serum stimulation) showed <4-fold elevation in R2 protein. These data suggest that RR plays a role in IR-mediated damage responses in Caski cells, which appears different than RR regulation after a proliferation (serum) stimulus. Such a response to IR in human tumor cells has not been reported previously. The use of specific R2 protein or RR enzyme inhibitors after IR may enhance IR cytotoxicity by altering this potential RR-mediated repair pathway.

The interaction of hydroxyurea and iododeoxyuridine on the radiosensitivity of human bladder cancer cells.

Biochemical modulation of iododeoxyuridine (IdUrd) incorporation into the DNA of tumor cells is a potential clinical strategy to enhance radiosensitivity and to simultaneously differentiate the sensitivity of rapidly proliferating tumor cells and more slowly proliferating adjacent normal tissues to radiation. The interactions of hydroxyurea (HU) and IdUrd were studied in a human bladder cancer cell line, 647V. Exposure of exponentially growing 647V cells to HU concentrations of 10-100 microM for one cell population doubling (24 h) resulted in no cytotoxicity as assessed by clonogenic survival. Flow cytometric analysis showed a significant increase in an early S-phase population after a 12-h exposure but a return to a normal cell cycle distribution after a 24-h exposure to 100 microM HU. Incorporation of IdUrd into DNA was increased 2-fold by coincubation with HU (100 microM) and a clinically achievable concentration of IdUrd (2 microM) for 24 h. To elucidate the mechanism of modulation, IdUTP pools were compared in 647V cells treated with 2 microM IdUrd with or without 100 microM HU. A 2-fold increase in IdUTP pools was evident within 2 h when this drug combination was used. With the use of multivariate statistical analysis, the radiosensitivity of 647V cells was compared after a 24-h exposure to various concentrations of IdUrd (0 and 2 microM) and HU (0, 10, and 100 microM). A 24-h exposure to 100 microM HU alone or to 2 microM IdUrd alone before irradiation resulted in significant (P < 0.02) radiosensitization with sensitizer enhancement ratios of 1.15 and 1.27, respectively. A 24-h exposure to 100 microM HU + 2 microM IdUrd resulted in even more significant (P = 0.001) radiosensitization, which was found to be a greater than additive response (sensitizer enhancement ratio, 1.76 observed compared with 1.37 expected). No radiosensitization was found with a 12-h exposure to 100 microM HU alone. The mechanism of biochemical modulation of IdUrd by a noncytotoxic dose of HU is proposed as increasing the IdUTP pools by stimulating enzymes in the thymidine salvage pathway and subsequently enhancing IdUrd incorporation and radiosensitization.

Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor beta-lapachone-induced apoptosis through an oxidation-dependent pathway.

Beta-lapachone (beta-Lap) has been found to inhibit DNA topoisomerases (Topos) by a mechanism distinct from that of other commonly known Topo inhibitors. Here, we demonstrated a pronounced elevation of H2O2 and O2- in human leukemia HL-60 cells treated with beta-Lap. Treatment with other Topo poisons, such as camptothecin (CPT), Vbeta-16, and GL331, did not have the same effect. On the other hand, antioxidant vitamin C (Vit C) treatment effectively antagonized beta-Lap-induced apoptosis. This suggested that a reactive oxygen species (ROS)-related pathway was involved in beta-Lap-induced apoptosis program. We also found that c-Jun NH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 was persistently activated in apoptosis induced by beta-Lap. Overexpression of a dominant-negative mutant mitogen-activated protein kinase kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotide or Vit C all prevented beta-Lap-induced JNK activation and the subsequent apoptosis. Only the expression of MEKK1-DN, not Vit C treatment, blocked the JNK activity induced by CPT, Vbeta-16, or GL331. These results confirm again that ROS acts as a mediator for JNK activation during beta-Lap-induced apoptosis. Furthermore, we found that beta-Lap can stimulate CPP32/Yama activity, which was, however, markedly inhibited by the MEKK1-DN expression or Vit C treatment. Again, CPT-induced CPP32/Yama activation can be abolished by MEKK1-DN but not by Vit C treatment. Taken together, these results indicate that beta-Lap but not other Topo inhibitors triggers apoptosis signaling, i.e., JNK and subsequent CPP32/Yama activation are mediated by the generation of ROS.

Cytosolic PKM2 stabilizes mutant EGFR protein expression through regulating HSP90-EGFR association.

Secondary mutation of epidermal growth factor receptor (EGFR) resulting in drug resistance is one of the most critical issues in lung cancer therapy. Several drugs are being developed to overcome EGFR tyrosine kinase inhibitor (TKI) resistance. Here, we report that pyruvate kinase M2 (PKM2) stabilized mutant EGFR protein by direct interaction and sustained cell survival signaling in lung cancer cells. PKM2 silencing resulted in markedly reduced mutant EGFR expression in TKI-sensitive or -resistant human lung cancer cells, and in inhibition of tumor growth in their xenografts, concomitant with downregulation of EGFR-related signaling. Mechanistically, PKM2 directly interacted with mutant EGFR and heat-shock protein 90 (HSP90), and thus stabilized EGFR by maintaining its binding with HSP90 and co-chaperones. Stabilization of EGFR relied on dimeric PKM2, and the protein half-life of mutant EGFR decreased when PKM2 was forced into its tetramer form. Clinical levels of PKM2 positively correlated with mutant EGFR expression and with patient outcome. These results reveal a previously undescribed non-glycolysis function of PKM2 in the cytoplasm, which contribute to EGFR-dependent tumorigenesis and provide a novel strategy to overcome drug resistance to EGFR TKIs.

The involvement of PI 3-K/Akt-dependent up-regulation of Mcl-1 in the prevention of apoptosis of Hep3B cells by interleukin-6.

Interleukin-6 (IL-6) is a pleitrophic cytokine that not only regulates growth and differentiation of many cell types, but also induces production of acute phase proteins (AAP) in hepatocytes. Our previous works have demonstrated that both PI 3-K/Akt and STAT3 pathways were concomitantly activated and cooperatively mediated the anti-apoptotic effect of IL-6. This investigation reports that IL-6 protected cells against apoptosis induced by a variety of agents including, TGF-beta, UV and retinoic acid (RA) in Hep3B cells, suggesting that IL-6 is a fundamental determinant of hepatic cell survival. Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated by IL-6, with a peak (approximately 3-4-fold) appearing at 4 h. Transient transfection of cells with a mcl-1 antisense vector, resulting in a 50-60% reduction of the anti-apoptotic effect of IL-6, indicating that Mcl-1 is a downstream effector of IL-6. Which signaling pathway transduced by IL-6 responsible for the Mcl-1 up-regulation was further investigated. In Hep3B cells, the JAK/STAT3, ERK, and PI 3-K/Akt pathways were activated by IL-6 stimulation. Blocking JAK/STAT3 activation with a dominant-negative mutant STAT3F or a JAK inhibitor AG490 could not influence IL-6-mediated Mcl-1 up-regulation. Similarly, PD98059 treatment, a MEK specific inhibitor, also failed to inhibit Mcl-1 expression. However, the IL-6-induced Mcl-1 up-regulation was effectively attenuated in the presence of PI 3-K inhibitors, LY294002 and wortmannin. Expression of dominant-negative Akt, but not Etk, could abrogate the IL-6-induced increase of Mcl-1. In conclusion, our results suggest that the anti-apoptotic effect of IL-6 is mediated, at least in part, by Mcl-1 expression and that is mainly through the PI 3-K/ Akt-dependent pathway.

Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency.

Interleukin-6 (IL-6) is a pleiotropic cytokine that is capable of modulating the diverse functions of cells such as acute phase responses and inflammation. Excessive or insufficient production of IL-6 may contribute to certain diseases of the skin. The aim of this study was to investigate the possible role of IL-6 in the tumorigenesis of basal cell carcinoma (BCC). Initially, we transfected IL-6 expression vector, under the control of a CMV promoter, into human BCC cells and successfully obtained IL-6-overexpressing clones (BCC/IL-6-c1 and BCC/IL-6-c2) and a mixture (BCC/IL-6). DNA synthesis assay determined using (3)H-thymidine pulse incorporation revealed that IL-6-expressing BCC cells exhibited a much higher DNA synthesis rate than the neo control or parental BCC cells. We also detected a greater abundance of IL-6-expressing cell colonies formed in soft agar than in the vector control cells. Furthermore, BCC/IL-6 cells, but not vector control cells, were resistant to UV and photodynamic therapy (PDT)-induced apoptosis, as confirmed using DNA fragmentation and morphologic change analyses. Immunoblot analysis showed that Mcl-1, an anti-apoptotic protein, was specifically up-regulated IL-6 transfectants but not in the control cells. Transient transfection of IL-6 transfectants with antisense mcl-1 greatly enhanced their apoptosis frequency by UV treatment. In tumorigenesis assay, IL-6 transfected clones formed tumors in nude mice more rapidly than the control cells. These tumors appeared to be highly vascularized using pathological examination. Supportive of this finding, we found that IL-6 transfected cells expressed elevated levels of two angiogenic factors, cyclooxygenase (Cox)-2 and vascular endothelial growth factor (VEGF). These results suggest that overexpression of IL-6 enhances the tumorigenic activity of BCC cells by both suppressing apoptosis and actively promoting angiogenesis.

Distinct p53-mediated G1/S checkpoint responses in two NIH3T3 subclone cells following treatment with DNA-damaging agents.

N3T3 and P-3T3 cells, originally isolated from a NIH3T3 cell clone on the basis of their negative and positive transformation by v-Abl, v-Src and Bcr-Abl, were previously found to show distinct cyclin activity changes following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, which is anti-mitogenic for N-3T3 cells and mitogenic for P-3T3 cells. We have found in this study that, while the G1/S arrest and cell death induced by serum starvation and TPA treatment in N-3T3 cells did not involve p53-mediated checkpoint or apoptosis, N-3T3 and P-3T3 cells evidently responded differently in these aspects of cell cycle regulation to DNA-damaging agents, methylmethane sulfonate (MMS) and gamma-radiation. In N-3T3 cells, DNA damages elicit cell growth arrest at G1/S transition with concomitant accumulation of p53 and p53-inducible Waf1/Cip1 proteins and also signs of apoptosis such as DNA ladder patterns and apoptotic (subgenomic) peak in flow cytograph. Conversely, P-3T3 cells treated with the DNA-damaging agents showed no cell cycle interruption nor accumulation of p53 or Waf1/Cip1. However, both P-3T3 and N-3T3 cells showed the same p53 protein half-life of 40 min or less, the same wild-type p53 DNA sequence and the same co-immunoprecipitable cellular proteins in complexes with p53, suggesting that an alteration in a signal transduction pathway upstream of p53 might account for the evasion of p53-mediated G1 checkpoint in P-3T3 cells.

The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K/Akt pathway.

Interleukin-6 (IL-6), a multifunctional cytokine, has recently been implicated in human cervical cancer, though the mechanism remains elusive. This study demonstrates that the anti-apoptotic protein Mcl-1 and IL-6 was concomitantly expressed in human cervical cancer tissues and cell lines, but not in normal cervix tissues. Upon IL-6 treatment, Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated peaking at 4-8 h in human cervical cancer C33A cells. Supporting this observation, using anti-IL-6 or anti-IL-6 receptor antibody to interrupt the IL-6 autocrine loop in SiHa cells significantly reduced cellular level of Mcl-1. This study hypothesizes that the expression of Mcl-1 in cervical cancer cells is regulated by IL-6. The matter of which signaling pathways transduced by IL-6 is responsible for the Mcl-1 up-regulation is further investigated herein. Blocking the STAT3 or MAPK pathway with dominant-negative mutant STAT3F or the MEK inhibitor PD98059 failed to inhibit IL-6-mediated Mcl-1 expression. Meanwhile, the IL-6-induced Mcl-1 up-regulation was effectively abolished by treatment with PI 3-K inhibitors, LY294002. Additionally, overexpression of dominant-negative (dn) Akt in C33A cells could inhibit the IL-6-induced increase of Mcl-1. Finally, overexpression of IL-6 in C33A cells caused a markable resistance to apoptosis induced by doxorubicin or cisplatin. Transient transfection of IL-6-overexpressed cells with a mcl-1 antisense vector, leading to the attenuation of their apoptosis-resistant activity. In conclusion, the data herein suggest that IL-6 regulated the mcl-1 expression via a PI 3-K/Akt-dependent pathway that may facilitate the oncogenesis of human cervical cancer by modulating the apoptosis threshold.