Radiotherapy (RT) represents one of the major treatment methods for cancers. However, many studies have observed that in descendant surviving tumor cells, sublethal irradiation can promote metastatic ability, which is closely related to the tumor microenvironment. We therefore investigated the functions and mechanisms of sublethal irradiated liver nonparenchymal cells (NPCs) in hepatocellular carcinoma (HCC). In this study, primary rat NPCs and McA-RH7777 hepatoma cells were irradiated with 6 Gy X-ray. Conditioned media (CM) from nonirradiated (SnonR), irradiated (SR), or irradiated plus radiosensitizer celecoxib-treated (S[R + D]) NPCs were collected and added to sublethal irradiated McA-RH7777 cells. We showed that CM from sublethal irradiated NPCs significantly promoted the migration and invasion ability of sublethal irradiated McA-RH7777 cells, which was reversed by celecoxib. The differentially expressed genes in differently treated McA-RH7777 cells were enriched mostly in the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. SR increased the migration and invasion ability of HCC cells by inhibiting AMPK/mTOR signaling, which was enhanced by the AMPK inhibitor compound C and blocked by the AMPK activator GSK-621. Analyses of HCC tissues after neoadjuvant radiotherapy confirmed the effects of radiation on the AMPK/mTOR pathway. Cytokine antibody arrays and further functional investigations showed that matrix metalloproteinase-8 (MMP-8) partly mediates the promotion effects of SR on the migration and invasion ability of HCC cells by regulating AMPK/mTOR signaling. In summary, our data indicate that MMP-8 secreted by irradiated NPCs enhanced the migration and invasion of HCC by regulating AMPK/mTOR signaling, revealing a novel mechanism mediating sublethal irradiation-induced HCC metastasis at the level of the tumor microenvironment.
What is already known about this topic? Benzene is harmful to the hematopoietic system and can cause leukemia. However, benzene is still being used in various industries including furniture, rubber, plastic products, and metal product manufacturing. What is added by this report? The white blood cell count of workers in general equipment, special equipment, chemical raw materials, and chemical products manufacturing decreased significantly. The enterprises in which benzene concentration exceeded the occupational exposure limit were small enterprises and private enterprises. What are the implications for public health practice? Regular health examinations are necessary for benzene-exposed workers. In addition, the monitoring of benzene concentration in small enterprises and private enterprises should be strengthened.
After traumatic brain injury (TBI), an acute, robust inflammatory cascade occurs that is characterized by the activation of resident cells such as microglia, the migration and recruitment of peripheral immune cells and the release of inflammatory mediators that induce secondary cell death and impede neurological recovery. In addition, neuroinflammation can alter blood-brain barrier (BBB) permeability. Controlling inflammatory responses is considered a promising therapeutic approach for TBI. Hydroxychloroquine (HCQ) has already been used clinically for decades, and it is still widely used to treat various autoimmune diseases. However, the effects of HCQ on inflammation and the potential mechanism after TBI remain to be defined. The aim of the current study was to elucidate whether HCQ could improve the neurological recovery of mice post-TBI by inhibiting the inflammatory response via the TLR4/NF-κB signaling pathway.C57BL/6 mice were subjected to controlled cortical impact (CCI) and randomly divided into groups that received intraperitoneal HCQ or vehicle daily after TBI. TAK-242 (3.0 mg/kg), an exogenous TLR4 antagonist, was injected intraperitoneally 1 h before TBI. Behavioral assessments were performed on days 1 and 3 post-TBI, and the gene expression levels of inflammatory cytokines were analyzed by qRT-PCR. The presence of infiltrated immune cells was examined by flow cytometry and immunostaining. In addition, BBB permeability, tight junction expression and brain edema were investigated.HCQ administration significantly ameliorated TBI-induced neurological deficits. HCQ alleviated neuroinflammation, the activation and accumulation of microglia and immune cell infiltration in the brain, attenuated BBB disruption and brain edema, and upregulated tight junction expression. Combined administration of HCQ and TAK-242 did not enhance the neuroprotective effects of HCQ.HCQ reduced proinflammatory cytokine expression, and the underlying mechanism may involve suppressing the TLR4/NF-κB signaling pathway, suggesting that HCQ is a potential therapeutic agent for TBI treatment.
Vascular senescence is associated with aging and disease in various organ and systems of the human body, and is one of the main pathogenesis of chronic diseases such as heart, brain, kidney and others.Vascular cell senescence expresses a senescence-associated secretory phenotype (SASP), including proinflammatory cytokines, growth factors, chemokines and proteases.These factors accelerate the changes in vascular structure and function, and lead to vascular aging.This study focuses on the latestresearchprogressions of the mechanism, biological effects of SASP, relationship between SASP and vascular aging, and intervention on SASP.
Key words:
Aging; Blood vessels
Atherosclerosis is caused by various factors, and Glabridin may have protective effects on the cardiovascular system. The purpose of the present study was to evaluate the effects of Glabridin on atherosclerosis and evaluate whether Glabridin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light chain (MLC) kinase (MLCK)/phosphorylated (p)‑MLC system via the mitogen activated protein kinase (MAPK) signaling pathway. Male New Zealand rabbits were randomly divided into 3 groups: The control group was administered an ordinary diet, whereas the high fat group and the Glabridin (2 mg/kg/d) intervention group were administered a high fat diet. Following 12 weeks, the blood lipid levels of rabbits, the morphological structure of the arterial wall, the arterial intimal permeability, the endothelial function and the mRNA levels of MLCK were measured. Western blot analysis was used to detect the levels of MLCK, p‑c‑Jun N‑terminal kinase (JNK), p‑extracellular signal regulated kinase (ERK), and p‑p38. The high‑fat diet group exhibited significantly increased total cholesterol and triglycerides, and endothelial dysfunction, which were attenuated by Glabridin treatment. Notably, the aortic endothelial permeability was increased in the high‑fat diet group but was ameliorated in the Glabridin treatment group. Hyperlipidemia enhanced the expression of p‑MLC and MLCK, which were associated with the increased phosphorylation of ERK, p38 and JNK. These changes were also ameliorated by Glabridin. In conclusion, the results of the present study suggested that atherosclerosis may be associated with upregulated MLCK expression and activity, which was downregulated by Glabridin. The mechanism of action of Glabridin was thought to proceed through modulating MAPK pathway signal transduction. However, further studies are required to adequately illuminate the exact regulatory mechanisms involved.
In the 1980s. benzene-induced leukemia (BIL) mainly occurred in shoemaking and painting industries. Now the industry distribution of benzene-induced leukemia may have changed over time.BlL cases mainly occurred in the manufacturing industry from 2005-2019, especially in private enterprises and small/medium-sized enterprises. The industry with the largest number of new cases of BIL was the general and special equipment manufacturing. The number of leukemia cases in emerging industries such as computer/electronic product manufacturing was found to be increasing.Strengthening supervision and regulation of manufacturing, especially of small/medium-sized enterprises and emerging manufacturing industry, may be effective in reducing BIL.
Introduction: Hemangioblastomas are highly vascular benign tumors that may increase in size during pregnancy. The concurrence of cerebellar hemangioblastoma in high-risk pregnancy is extremely rare and the treatment in this situation can be challenged. Case: Here, we report a case of a 30-year-old woman in the 33rd PW who had experienced a severe headache, dizziness, vomiting, and limb weakness. Cesarean section was performed in the 34th PW, followed by neurosurgery under multidisciplinary discussion. Discussion: The pathological exam suggested hemangioblastomas. Finally, both the pregnancy and the fetus had a good outcome. Conclusion: This case emphasizes that the timing of surgery should be determined according to the neurological symptoms of the pregnancy and the gestational age (GA) and condition of the fetus.
Tumor necrosis factor-α (TNF-α) stimulates proliferation of Mo7e, CMK, HU-3, and M-MOK human leukemic cell lines. We report here the signal transduction pathway involved in TNF-α-induced Mo7e cell proliferation. Mo7e cells spontaneously die in the absence of growth factors, but treating the cells with interleukin (IL)-3, IL-6, thrombopoietin, granulocyte/macrophage colony-stimulating factor, or TNF-α promotes their survival and proliferation. Although most of these factors activate MAP kinase and Jun NH2-terminal kinase/signal transducer and activators of transcription signaling pathways, TNF-α fails to activate either pathway. When Mo7e cells were treated with TNF-α, nuclear factor κB (NF-κB) was activated transiently. The activated NF-κB consisted of heterodimers of p65 and p50 subunits. The degradation of IκBα coincided with activation of NF-κB in TNF-α-treated cells. To investigate the role of activated NF-κB in TNF-α-induced Mo7e proliferation, a cell-permeable peptide (SN50) carrying the nuclear localization sequence of p50 NF-κB was used to block nuclear translocation of activated NF-κB. Pretreating Mo7e cells with SN50 blocked TNF-α-induced nuclear translocation of NF-κB and inhibited TNF-α-induced Mo7e cell survival and proliferation. A mutant SN50 peptide did not affect TNF-α-induced Mo7e cell growth. SN50 had no effects on IL-3- or granulocyte/macrophage colony-stimulating factor-induced Mo7e cell proliferation. The results indicate that activation of NF-κB is involved in TNF-α-induced Mo7e cell survival and proliferation. Tumor necrosis factor-α (TNF-α) stimulates proliferation of Mo7e, CMK, HU-3, and M-MOK human leukemic cell lines. We report here the signal transduction pathway involved in TNF-α-induced Mo7e cell proliferation. Mo7e cells spontaneously die in the absence of growth factors, but treating the cells with interleukin (IL)-3, IL-6, thrombopoietin, granulocyte/macrophage colony-stimulating factor, or TNF-α promotes their survival and proliferation. Although most of these factors activate MAP kinase and Jun NH2-terminal kinase/signal transducer and activators of transcription signaling pathways, TNF-α fails to activate either pathway. When Mo7e cells were treated with TNF-α, nuclear factor κB (NF-κB) was activated transiently. The activated NF-κB consisted of heterodimers of p65 and p50 subunits. The degradation of IκBα coincided with activation of NF-κB in TNF-α-treated cells. To investigate the role of activated NF-κB in TNF-α-induced Mo7e proliferation, a cell-permeable peptide (SN50) carrying the nuclear localization sequence of p50 NF-κB was used to block nuclear translocation of activated NF-κB. Pretreating Mo7e cells with SN50 blocked TNF-α-induced nuclear translocation of NF-κB and inhibited TNF-α-induced Mo7e cell survival and proliferation. A mutant SN50 peptide did not affect TNF-α-induced Mo7e cell growth. SN50 had no effects on IL-3- or granulocyte/macrophage colony-stimulating factor-induced Mo7e cell proliferation. The results indicate that activation of NF-κB is involved in TNF-α-induced Mo7e cell survival and proliferation. Tumor necrosis factors (TNFs) 1The abbreviations used are: TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte/macrophage colony-stimulating factor; TPO, thrombopoietin; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; STAT, signaltransducers and activators oftranscription; JNK, Jun NH2-terminal kinase; NF-κB, nuclear factor κB; IL, interleukin; FITC, fluorescein isothiocyanate; IMDM, Iscove's modified Dulbecco's medium; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; PDTC, pyrrolidine dithiocarbamate; PI, propidium iodide; PVDF, polyvinylidene difluoride are produced by neutrophils, activated lymphocytes, macrophages, natural killer cells, endothelial cells, and smooth muscle cells, and were reported initially to induce tumor necrosis (1Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1302) Google Scholar, 2Carswell E.A. Old L.J. Kassel R.L. Green S. Fiore N. Williamson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3666-3670Crossref PubMed Scopus (3760) Google Scholar). Subsequent studies, however, indicated that TNFs also promote the proliferation and survival of some tumor cell lines (3Akiyama Y. Yamaguchi K. Sato T. Abe K. Jpn. J. Cancer Res. 1992; 83: 989-994Crossref PubMed Scopus (12) Google Scholar, 4Drexler H.G. Zaborski M. Quentmeier H. Leukemia. 1997; 11: 701-708Crossref PubMed Scopus (61) Google Scholar, 5Miura K. Teramura M. Hoshino S. Mizoguchi H. Sato T. Leuk. Res. 1992; 16: 281-285Crossref PubMed Scopus (12) Google Scholar). In patients with leukemias or lymphomas, elevated levels of serum TNF-α, which are associated with a poor prognosis, have been reported (6Cimino G. Amadori S. Cava M.C. De Sanctis V. Petti M.C. Di Gregorio A.O. Sgadari C. Vegna L. Mandelli F. Leukemia. 1991; 5: 32-35PubMed Google Scholar, 7Saarinen U.M. Koskelo E.K. Teppo A.M. Siimes M.A. Cancer Res. 1990; 50: 592-595PubMed Google Scholar, 8Kalmanti M. Karamolengou K. Dimitriou H. Tosca A. 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We and others reported that TNF-α significantly stimulates the growth of several human leukemic cell lines, including Mo7e (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar, 15Wadhwa M. Dilger P. Meager A. Walker B. Gaines-Das R. Thorpe R. Cytokine. 1996; 8: 900-909Crossref PubMed Scopus (7) Google Scholar), CMK (3Akiyama Y. Yamaguchi K. Sato T. Abe K. Jpn. J. Cancer Res. 1992; 83: 989-994Crossref PubMed Scopus (12) Google Scholar, 5Miura K. Teramura M. Hoshino S. Mizoguchi H. Sato T. Leuk. Res. 1992; 16: 281-285Crossref PubMed Scopus (12) Google Scholar), HU-3, and M-MOK (4Drexler H.G. Zaborski M. Quentmeier H. Leukemia. 1997; 11: 701-708Crossref PubMed Scopus (61) Google Scholar). TNF-α also stimulates the proliferation of primary leukemia cells isolated from patients (16Brach M.A. Herrmann F. Leukemia. 1993; 7 Suppl. 2: S22-S26PubMed Google Scholar, 17Aderka D. Maor Y. Novick D. Engelmann H. Kahn Y. Levo Y. Wallach D. Revel M. J. Cell. Biochem. 1993; 81: 2076-2084Google Scholar, 18Digel W. Stefanic M. Schoniger W. Buck C. Raghavachar A. Frickhofen N. Heimpel H. Porzsolt F. J. Cell. Biochem. 1989; 73: 1242-1246Google Scholar). In an ultrastructural study of primary leukemia cells from patients, TNF-α did not induce direct cytotoxicity or apoptosis, but instead stimulated leukemia cells (19Munker R. Greither L. Darsow M. Ellwart J.W. Mailhammer R. Wilmanns W. Acta Haematol. 1993; 90: 77-83Crossref PubMed Scopus (10) Google Scholar). Furthermore, we and others have found that low doses of TNF-α act synergistically with other cytokines, such as IL-4, to stimulate Mo7e leukemic cell growth (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar,15Wadhwa M. Dilger P. Meager A. Walker B. Gaines-Das R. Thorpe R. Cytokine. 1996; 8: 900-909Crossref PubMed Scopus (7) Google Scholar). However, the molecular mechanisms of the signaling pathway(s) in TNF-α-induced leukemic cell proliferation remain unclear. Extensive studies show that TNF-α is an extremely pleiotropic factor, which can induce activation of phagocytic and endothelial cells, induction of prostaglandins, alterations in lipid metabolism, and regulated expression of major histocompatibility complex antigens, oncogenes, and transcription factors (1Old L.J. Science. 1985; 230: 630-632Crossref PubMed Scopus (1302) Google Scholar, 2Carswell E.A. Old L.J. Kassel R.L. Green S. Fiore N. Williamson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3666-3670Crossref PubMed Scopus (3760) Google Scholar). The capacity for TNF-α to induce such a wide variety of effects is attributable, in part, to its ability to activate multiple signal transduction pathways including mitogen-activated protein kinases (MAPKs), Jun NH2-terminal kinase (JNK), nuclear factor κB (NF-κB), and/or STATs in numerous cell lines (20Winston B.W. Lange-Carter C.A. Gardner A.M. Johnson G.L. Riches D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1614-1618Crossref PubMed Scopus (134) Google Scholar, 21Flury N. Eppenberger U. Mueller H. Eur. J. Biochem. 1997; 249: 421-426Crossref PubMed Scopus (17) Google Scholar, 22Lu G. Beuerman R.W. Zhao S. Sun G. Nguyen D.H. Ma S. Kline D.G. Neurochem. Int. 1997; 30: 401-410Crossref PubMed Scopus (46) Google Scholar, 23Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5579) Google Scholar, 24Guo Y.-L. Baysal K. Kang B. Yang L.-J. Williamson J.R. J. Biol. Chem. 1998; 273: 4027-4034Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although primarily involved in cell proliferation (25Vanden Berghe W. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegeman G. J. Biol. Chem. 1998; 273: 3285-3290Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 26Holmstrom T.H. Chow S.C. Elo I. Coffey E.T. Orrenius S. Sistonen L. Eriksson J.E. J. Immunol. 1998; 160: 2626-2636PubMed Google Scholar), the activation of MAPK has also been implicated in apoptosis in some circumstances (27Watabe M. Masuda Y. Nakajo S. Yoshida T. Kuroiwa Y. Nakaya K. J. Biol. Chem. 1996; 271: 14067-14072Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 28Blagosklonny M.V. Int. J. Cancer. 1998; 78: 511-517Crossref PubMed Scopus (31) Google Scholar, 29Watabe M. Kawazoe N. Masuda Y. Nakajo S. Nakaya K. Cancer Res. 1997; 57: 3097-3100PubMed Google Scholar). TNF-α-induced recruitment of the signal transducer FADD to type I TNF receptors may mediate TNF-α-induced apoptosis, but TNF-α-induced activation of NF-κB protects against TNF-α-induced apoptosis in some cell lines (30Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). Conventional NF-κB is a heterodimer that consists of p65 and p50 subunits. Both subunits of NF-κB are members of the NF-κB/Rel family of transcription factors, which also includes c-Rel, RelB, and p52 (31Ohmori Y. Schreiber R.D. Hamilton T.A. J. Biol. Chem. 1997; 272: 14899-14907Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The activity of NF-κB is strictly regulated by an inhibitor, IκBα, that forms a complex with NF-κB and keeps NF-κB in the cytoplasm (23Baldwin Jr., A. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5579) Google Scholar). When cells receive signals that activate NF-κB, IκBα is phosphorylated and degraded through a ubiquitin/proteasome pathway. The degradation of IκBα triggers the translocation of NF-κB from the cytoplasm to the nucleus. Although increasing evidence shows that the activation of NF-κB is involved in cell activation and proliferation, several lines of evidence also indicate that activation of NF-κB may result in apoptosis (32Qin Z.H. Wang Y. Nakai M. Chase T.N. Mol. Pharmacol. 1998; 53: 33-42Crossref PubMed Scopus (163) Google Scholar, 33Bessho R. Matsubara K. Kubota M. Kuwakado K. Hirota H. Wakazono Y. Lin Y.W. Okuda A. Kawai M. Nishikomori R. Biochem. Pharmacol. 1994; 48: 1883-1889Crossref PubMed Scopus (202) Google Scholar, 34Qin Z.H. Wang Y. Nakai M. Chase T.N. Mol. Pharmacol. 1998; 53: 33-42Crossref PubMed Scopus (165) Google Scholar). The fact that TNF-α stimulates the proliferation of human leukemic cell lines as well as primary leukemia cells led us to investigate the TNF-α-induced activation of signaling pathways in Mo7e and other cell lines. Specifically, we addressed the questions of what signal transduction pathways are activated in leukemic cells treated with TNF-α and whether the activated signal transduction pathway(s) is/are involved in TNF-α-induced Mo7e cell proliferation. We found that TNF-α transiently induced activation of NF-κB, but not the MAPK or STAT signaling pathways, which are activated by IL-3, GM-CSF, and thrombopoietin (TPO) in Mo7e and other cell lines. Pretreatment of Mo7e cells with a synthesized membrane-permeable peptide, SN50, blocked the TNF-α-induced translocation of NF-κB to the nuclei in a dose-dependent manner. Pretreating Mo7e cells with SN50 specifically inhibited TNF-α-induced but not IL-3- or GM-CSF-induced Mo7e cell proliferation. Thus, the data presented here provide direct evidence that the activation and function of NF-κB is essential to TNF-α-induced proliferation of Mo7e cells. Recombinant human TNF-α, recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF), and anti-TNF-α antibody were obtained from R&D Systems (Minneapolis, MN). IL-3, IL-6, and TPO were purchased from PeproTech (Rocky Hill, NJ). [methyl-3H]Thymidine (specific activity 70–86 Ci/mmol), [32P]dATP (specific activity >3000 μCi/mmol), and [32P]cATP (specific activity >3000 μCi/mmol) were purchased from Amersham Pharmacia Biotech. Anti-goat IgG antibody labeled with fluorescein isothiocyanate (FITC) was purchased from Zymed Laboratories Inc. Antibodies against human p50, p52, p65, and B-Rel NF-κB subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-human IκBα and anti-phosphorylated IκBα antibodies were purchased from New England Biolabs (Beverly, MA). The human Mo7e megakaryoblastic leukemic cell line, originally described by Avanzi et al. (35Avanzi G.C. Lista P. Giovinazzo B. Miniero R. Saglio G. Benetton G. Coda R. Cattoretti G. Pegoraro L. Br. J. Haematol. 1988; 69: 359-366Crossref PubMed Scopus (255) Google Scholar), was maintained in Iscove's modified Dulbecco's medium (IMDM; Life Technologies, Inc.) containing 10% fetal bovine serum, 1% glutamine, and 5 ng/ml rhGM-CSF. The human Meg-01 megakaryoblastic leukemic cell line, originally described by Ogura et al. (36Ogura M. Morishima Y. Ohno R. Kato Y. Hirabayashi N. Nagura H. Saito H. J. Cell. Biochem. 1985; 66: 1384-1392Google Scholar), was maintained in RPMI 1640 medium (Life Technologies, Inc.) with 10% fetal bovine serum. The human leukemic HEL cell line, which has both erythroid and megakaryocytic characteristics (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar, 37Martin P. Papayannopoulou T. Science. 1982; 216: 1233-1235Crossref PubMed Scopus (438) Google Scholar, 38Brass L.F. Woolkalis M.J. Biochem. J. 1992; 281: 73-80Crossref PubMed Scopus (26) Google Scholar), was maintained in IMDM with 10% horse serum. Both Meg-01 and HEL cell lines were purchased from ATCC, and the Mo7e cell line was obtained from Genetics Institute (Boston, MA). In experiments to detect effects of cytokines, Mo7e cells were prepared by washing three times with serum-free medium and were starved for 18 h in medium without cytokine (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar). Meg-01 and HEL cells were cultured in serum-free medium with 1× Nutridoma HU (Roche Molecular Biochemicals) for 18 h before cytokine treatments. SN50, a peptide that has been reported to have the capacity to block the nuclear translocation of activated NF-κB (34Qin Z.H. Wang Y. Nakai M. Chase T.N. Mol. Pharmacol. 1998; 53: 33-42Crossref PubMed Scopus (165) Google Scholar,39Lin Y.-Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar), contains membrane-permeable signal sequences of Kaposi's fibroblast growth factor and the nuclear translocation motif (VQRKRQKLMP) of human NF-κB p50. SN50mt, a mutant SN50, contains membrane-permeable signal sequences of Kaposi's fibroblast growth factor and the mutant nuclear translocation motif of human NF-κB p50 (39Lin Y.-Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Both SN50 and SN50mt were synthesized commercially (Genemed Synthesis, South San Francisco, CA). The peptides were purified by reverse phase high performance liquid chromatography, and the molecular weight of the purified peptides was verified by mass spectrometry analysis. To track the intracellular localization of the membrane-permeable peptides in Mo7e cells, SN50 was labeled with FITC. The synthesized peptides were dissolved in Me2SO to a final concentration of 100 μg/μl and mixed directly with culture medium (1:500–1000) before use. To determine the effects of TNF-α on cell proliferation, DNA synthesis was measured by [3H]thymidine incorporation in freshly prepared cells. The assays were performed in triplicate, using a total of 4 × 105 Mo7e or Meg-01 cells, or 2 × 105 HEL cells. Mo7e cells in IMDM with 10% FCS, and Meg-01 and HEL cells in medium with 1× Nutridoma HU, were cultured for 72 h in the presence or absence of TNF-α or other cytokines. After that, the cells were labeled with 4 μCi/ml [3H]thymidine for an additional 4 h. The radioactivity incorporated into DNA (counts per minute) was determined by a liquid scintillation counter according to a previously described protocol (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar). All assays for [3H]thymidine incorporation were repeated at least three times. Some of our data from [3H]thymidine incorporation assays also were compared with those obtained from the Non-radioactive Cell Proliferation Wst-1 kit (Roche Molecular Biochemicals). Both methods yielded consistent results, but only the data from [3H]thymidine incorporation assays are presented. The functional activity of MAP kinase kinase (MEK) was detected by the kinase-induced phosphorylation of a kinase-defective p42mapk (K52R) labeled with [32P]ATP, following a published protocol (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar). For the detection of tyrosine phosphorylation of MAPK (mitogen-activated protein kinase), cells treated with or without cytokines were boiled in SDS buffer. Total cellular proteins (30 μg) were loaded into each lane and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to PVDF membranes and probed with an anti-PhosphoPlus MAPK antibody (New England Biolabs), which reacts only with MAPK that is phosphorylated on Thr-202 and Tyr-204, or with an anti-total (unphosphorylated and phosphorylated) MAPK antibody as a control. Phosphorylation of STAT1 and STAT3 was examined by 10% SDS-PAGE Western blotting with specific anti-phosphorylated-STAT1 and anti-phosphorylated-STAT3 antibodies (New England Biolabs), according to the methods suggested by the manufacturer. For comparison, total STAT1 and STAT3 (both phosphorylated and unphosphorylated) also were examined with anti-STAT1 and -STAT3 antibodies. Western blots were visualized with enhanced chemiluminescence. Preparation of nuclear extracts and gel mobility shift assays were performed according to methods described previously (40Liu R.Y. Li X. Li L. Li G.C. Cancer Res. 1992; 52: 3667-3673PubMed Google Scholar, 41Olashaw N.E. J. Biol. Chem. 1996; 271: 30307-30310Abstract Full Text Full Text PDF PubMed Google Scholar). The sequence of the NF-κB-binding oligonucleotide used as a radioactive DNA probe was 5′-TCGACAGAGGGGACTTTCCGAGAGGC-3′. Equal amounts of nuclear proteins (5–10 μg) for each sample were incubated with 1 ng of 32P-labeled probe. The DNA binding reaction was performed at room temperature in a volume of 25 μl, which contained binding buffer (10 mm Tris-HCl, pH 7.5, 1 μm EDTA, 100 mm NaCl, 20 μg/ml bovine serum albumin, and 0.2% Nonidet P-40, 1.8 μg/ml salmon sperm DNA), 1 ng of 3′-labeled probe, and 5–10 μg of nuclear proteins. After incubation for 15 min, the samples were electrophoresed on native 6% acrylamide, 0.25× Tris borate-EDTA gels. The gels were dried and exposed to x-ray film. Competition was performed by adding 50–100 m excess of each unlabeled DNA fragment along with the 32P-labeled probe. For gel mobility supershift assays, nuclear extracts were co-incubated with the indicated specific anti-NF-κB subunit antibodies and 32P-labeled oligonucleotide probes. The DNA-protein complexes and unbound probe were separated electrophoretically on 6% native polyacrylamide gels in 0.25× TBE buffer (44.5 mm Tris, pH 8.0, 1 mm EDTA, and 44.5 mm boric acid). The gels were fixed and dried, and the DNA-protein complexes were visualized by autoradiography at −70 °C with Kodak X-Omat film and a DuPont Cranex Lightning Plus intensifying screen. A FACScan flow cytometer was used to examine Mo7e cell cycle status, intracellular localization of SN50 peptides, and apoptosis. Mo7e cells were incubated with 50 μg/ml FITC-labeled SN50 peptide for various periods or with various amounts of FITC-SN50 for 30 min to investigate the intercellular incorporation of the peptides. The status of apoptosis was analyzed by incubating cells with FITC-labeled annexin V and propidium iodide (PI), following the manufacturer's suggested procedure. For the analysis of cell cycle status and cellular DNA ploidy, the cells were fixed with methanol and stained with PI according to the published method (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar). When the electrophoretic gel mobility shift assay (EMSA) was used to examine the activation of NF-κB in Mo7e, HEL, and K562 human leukemic cell lines, no detectable activation of NF-κB was observed in the cells without cytokine exposure (Fig.1 a, CTL). However, exposure of the cells to 5 ng/ml TNF-α for 30 min significantly induced the activation of NF-κB in Mo7e, HEL, and K562 cells (Fig.1 a, TNF). The Mo7e cells were used to further investigate the dynamics of the activation of NF-κB. When Mo7e cells were treated with 5 ng/ml TNF-α for 0–120 min, the activation of NF-κB was first detected within 5 min, attained a maximal level at 30 min, and declined thereafter (Fig. 1 b). Supershift EMSA was then used to examine the components of the DNA-protein complexes. The results showed that both anti-p50 and anti-p65 antibodies induced a supershift of the DNA-protein complex in nuclear extracts from Mo7e, HEL, and K562 cells treated with 5 ng/ml TNF-α for 30 min (Fig.2, a and b), indicating that the TNF-α-induced DNA-protein complexes in these cells contained both p50 and p65 NF-κB subunits. When the nuclear extracts from cells treated with TNF-α were incubated with anti-p52, anti-RelB, or anti-c-Rel antibodies, no supershift was observed (data not shown). Furthermore, the formation of the NF-κB-DNA complex was inhibited competitively by an unlabeled NF-κB DNA probe in a dose-dependent manner. A 100-fold molar excess of nonradioactive DNA containing the NF-κB binding site completely prevented binding of the activated NF-κB to the32P-labeled NF-κB-binding probe (Fig. 2 c), but did not affect the nonspecific DNA-protein complexes (Fig. 2,bands marked as NS). The results, therefore, demonstrate clearly that the NF-κB p65/p50 heterodimer is activated in these leukemic cell lines in response to TNF-α.Figure 2Identification of the components of activated NF-κB in the DNA-protein complex by EMSA . a, nuclear extracts from Mo7e, HEL, and K562 cells treated with 5 ng/ml TNF-α were incubated with 32P-labeled NF-κB-binding probe alone (lanes 1–3), with radioactive probe and anti-NF-κB p65 subunit antibody (lanes 4–6). b, nuclear extracts from Mo7e, HEL, and K562 cells treated with 5 ng/ml TNF-α were incubated with 32P-labeled NF-κB-binding probe alone (lanes 1–3), with radioactive probe and anti-NF-κB p50 subunit antibody (lanes 4–6).NS indicates the nonspecific DNA-binding band, and the arrow indicates the supershift complexes. c, nuclear extracts from Mo7e cells treated without TNF-α (lane 1) or with 5 ng/ml TNF-α for 30 min were incubated with32P-labeled NF-κB-binding probe alone (lane 2) or 32P-labeled probe and the indicated -fold molar excess of unlabeled probe containing the NF-κB binding site (lanes 2–6) to determine the specificity of the binding of activated transcriptional factors to the NF-κB-binding probe. The DNA-binding complexes were separated in 6% native polyacrylamide gels. Similar results were obtained in three separate experiments. The autoradiograph shows the location of NF-κB and the nonspecific band (NS).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then examined the effect of TNF-α treatment on the localization of NF-κB in Mo7e cells by Western blotting with specific antibodies against the p65 NF-κB subunit. Before stimulation, p65 was detected exclusively in the cytoplasmic lysates (Fig. 3 a,lane 1). When Mo7e cells were treated with 5 ng/ml TNF-α for 5–120 min, the level of cytoplasmic NF-κB p65 declined gradually (Fig. 3 a, lanes 2–7). When nuclear extracts were used in Western blotting to analyze the level of nuclear NF-κB p65, there was no detectable NF-κB p65 in the nucleus of Mo7e cells without TNF-α exposure (Fig.3 b, lane 1). However, NF-κB was detectable in the nucleus within 5 min, and the level of nuclear NF-κB p65 increased markedly in cells exposed to TNF-α, with a maximal level within 30–60 min of the treatment (Fig. 3 b,lanes 4–7). When nonimmune immunoglobulins were used as the first antibody, no NF-κB p65 was detected in the nuclear extracts from Mo7e cells (data not shown). The results from Western blotting analysis of cytoplasmic lysates and nuclear extracts confirm and extend the EMSA findings of activation and nuclear translocation of NF-κB in Mo7e cells treated with TNF-α. A common feature of the regulation of NF-κB is its sequestration in the cytoplasm as an inactive complex with IκBα in the basal state. To investigate the role of IκBα in the activation of NF-κB by TNF-α, phosphorylation and the total level of IκBα were analyzed by Western blotting, and the results were compared with the dynamics of NF-κB activation in Mo7e cells after TNF-α stimulation. When nuclear extracts from Mo7e cells treated with TNF-α were analyzed by Western blotting with an antibody specific for phosphorylated IκBα, the phosphorylation of IκBα became detectable in cells treated with 5 ng/ml TNF-α for 1 min, reached the maximum level at 5 min, and then declined rapidly (Fig.4 a, lanes 2–6). Western blotting analysis of cytoplasmic lysates showed that the rapid decline of nuclear IκBα in TNF-α-treated Mo7e cells was due to a decrease of total IκBα rather than dephosphorylation of IκBα, since the level of total IκBα (phosphorylated and unphosphorylated) also was decreased markedly upon TNF-α treatment (Fig. 4 b). Comparison of these results to those in Fig. 3, which showed that the activation of NF-κB induced by TNF-α was first detected within 5 min and maximal at 30 min, indicate clearly that the degradation of IκBα coincides with the activation and nuclear translocation of NF-κB in Mo7e cells. MAPK and STAT signal transduction pathways have been reported to be activated by several hemopoietic growth factors and to be involved in growth factor-induced Mo7e cell proliferation (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar, 42Liu R.Y. Fan C. Garcia R. Jove R. Zuckerman K.S. Blood. 1999; 93: 2369-2379Crossref PubMed Google Scholar). To investigate whether these signal transduction pathways are activated by TNF-α and whether they are involved in TNF-α-induced cell proliferation, we examined the effect of TNF-α on the activation of MAPK and STAT signal transduction pathways. When Mo7e cells were treated with 5 ng/ml IL-3 or GM-CSF for 30–60 min, no significant activation of NF-κB was observed (Fig. 5 a,lanes 3–6), although TNF-α treatment induced significant activation of NF-κB (Fig. 5 a, lane 2). When the activation of the STAT5 signal transduction pathway in Mo7e cells was examined by EMSA, the results, which were similar to those that we reported previously (14Liu R.Y. Fan C. Mitchell S. Chen Q. Wu J. Zuckerman K.S. Cancer Res. 1998; 58: 2217-2223PubMed Google Scholar), showed that STAT5 activation was observed in Mo7e cells treated with GM-CSF, IL-3, TPO, and IL-6, but not in cells treated with 5 ng/ml TNF-α for 10–60 min (data not shown). When the activation of STAT1 and STAT3 was examined by EMSA, supershift EMSA, or Western blotting with specific anti-phosphorylated STAT1 or STAT3 antibodies, no significant activation or phosphorylation of STAT1 or STAT3 was observed in Mo7e cells treated with 5 ng/ml TNF-α, IL-3, or GM-CSF for 10–60 min (data not shown). However, a significant activation of STAT1 and STAT3 was detected in control SK-BR-3 cells treated with 2 ng/ml epidermal growth factor for 30 min, which we reported previously as the positive control for detecting activation of STAT1 and STAT3 (42Liu R
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