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1.
Methods Mol Biol ; 2323: 267-280, 2021.
Article in English | MEDLINE | ID: mdl-34086287

ABSTRACT

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.


Subject(s)
Biological Therapy/methods , Neoplasms/therapy , RNA Interference , Synthetic Biology/methods , Animals , Cell Line, Tumor , Clinical Trials, Phase I as Topic , Colonic Neoplasms/microbiology , Colonic Neoplasms/therapy , Escherichia coli/genetics , Escherichia coli/physiology , Female , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Humans , Immunotherapy/methods , Immunotherapy/trends , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Neoplasms/microbiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , Real-Time Polymerase Chain Reaction/methods , Remission Induction , Salmonella typhimurium/genetics , Salmonella typhimurium/physiology , Species Specificity , Specific Pathogen-Free Organisms , Synthetic Biology/trends , Xenograft Model Antitumor Assays
2.
PLoS One ; 7(8): e43628, 2012.
Article in English | MEDLINE | ID: mdl-22928007

ABSTRACT

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.


Subject(s)
Gamma Rays , Neoplastic Stem Cells/radiation effects , Gene Expression Regulation, Neoplastic/radiation effects , Gene Knockdown Techniques , Hep G2 Cells , Humans , Neoplastic Stem Cells/metabolism , Octamer Transcription Factor-3/deficiency , Octamer Transcription Factor-3/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , SOXB1 Transcription Factors/deficiency , SOXB1 Transcription Factors/genetics
3.
PLoS Pathog ; 8(7): e1002833, 2012.
Article in English | MEDLINE | ID: mdl-22911498

ABSTRACT

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein ß (C/EBPß) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPß converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPß as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPß in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPß-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.


Subject(s)
Betapapillomavirus/pathogenicity , CCAAT-Enhancer-Binding Protein-beta/metabolism , Chemokine CCL20/genetics , Chemokine CCL20/metabolism , Keratinocytes/metabolism , Langerhans Cells/physiology , Papillomavirus Infections/immunology , Skin Neoplasms/virology , Betapapillomavirus/immunology , Betapapillomavirus/metabolism , Binding Sites , CCAAT-Enhancer-Binding Protein-beta/genetics , Cell Line, Tumor , Cell Movement , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Epidermis/metabolism , Epidermis/virology , Epidermodysplasia Verruciformis/virology , Humans , Keratinocytes/immunology , Oncogene Proteins, Viral/metabolism , Promoter Regions, Genetic
4.
Cell Cycle ; 11(14): 2691-8, 2012 Jul 15.
Article in English | MEDLINE | ID: mdl-22732500

ABSTRACT

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.


Subject(s)
Antineoplastic Agents/toxicity , Carboplatin/toxicity , Neoplastic Stem Cells/metabolism , Antineoplastic Agents/therapeutic use , Carboplatin/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Neoplastic Stem Cells/drug effects , Octamer Transcription Factor-3/antagonists & inhibitors , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , RNA Interference , RNA, Small Interfering/metabolism , SOXB1 Transcription Factors/antagonists & inhibitors , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
5.
Oncotarget ; 3(4): 365-70, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22562943

ABSTRACT

Recent advances in cancer genomics have opened up unlimited potential for treating cancer by directly targeting culprit genes. However, novel delivery methods are needed in order for this potential to be translated into clinically viable treatments for patients. Magnetic nanoparticle technology offers the potential to achieve selective and efficient delivery of therapeutic genes by using external magnetic fields, and also allows simultaneous imaging to monitor the delivery in vivo. Compared to conventional gene delivery strategies, this technique has been shown to significantly increase gene delivery to human xenograft tumors models, as well as various internal organs (e.g. liver, kidney) and the central nervous system. Magnetic nanoparticle technology, therefore, has the potential to turn the challenge of gene therapy in vivo into a new frontier for cancer treatment.


Subject(s)
Genetic Therapy , Magnetite Nanoparticles , Neoplasms/therapy , Animals , Gene Transfer Techniques/trends , Humans , Mice , Molecular Targeted Therapy , Neoplasms/genetics , Xenograft Model Antitumor Assays
6.
Methods Mol Biol ; 487: 147-60, 2009.
Article in English | MEDLINE | ID: mdl-19301646

ABSTRACT

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.


Subject(s)
Colonic Neoplasms/therapy , Gene Knockdown Techniques/methods , Gene Silencing , Genetic Therapy/methods , RNA, Small Interfering/administration & dosage , beta Catenin/antagonists & inhibitors , Animals , Cell Line, Tumor , Colonic Neoplasms/genetics , Escherichia coli/genetics , Female , Humans , In Vitro Techniques , Intestinal Mucosa/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , RNA, Small Interfering/genetics , Transplantation, Heterologous , beta Catenin/genetics , beta Catenin/metabolism
7.
Biotechnol Genet Eng Rev ; 25: 113-27, 2008.
Article in English | MEDLINE | ID: mdl-21412352

ABSTRACT

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.


Subject(s)
Bacteria/genetics , Gene Transfer Techniques , RNA Interference , Animals , Genetic Engineering , Genetic Vectors , Humans , Liposomes , Mice , Neoplasms/therapy , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Viruses/genetics
8.
J Infect Dis ; 196(1): 95-103, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17538889

ABSTRACT

BACKGROUND: Helicobacter pylori infection increases the risk of gastric carcinogenesis. The aim of the present study was to determine whether H. pylori could up-regulate the expression of the epidermal growth factor receptor (EGFR), a critical gene in the carcinogenic process. METHODS: AGS gastric epithelial cells were infected with cag(+) toxigenic or cag(-) nontoxigenic strains of H. pylori or isogenic mutants. EGFR protein expression was determined by Western blotting and immunofluorescence. EGFR mRNA levels were evaluated using real-time polymerase chain reaction. The signaling pathways leading to EGFR up-regulation were examined using the ERK1/2 inhibitor PD98059, the Src inhibitor pp2, the nuclear factor- kappa B inhibitor caffeic acid phenethyl ester, EGFR neutralizing antibodies, and the EGFR kinase inhibitor AG1478. RESULTS: Infection of AGS cells by H. pylori significantly increased EGFR mRNA and protein levels. We found that this effect was limited to cag(+) H. pylori strains and that mutants with a defective type IV secretion system were unable to cause EGFR up-regulation. Increased EGFR expression was found to be dependent on EGFR receptor transactivation, ERK1/2 phosphorylation, and Src activation. CONCLUSION: Infection of gastric epithelial cells by H. pylori triggers an autocrine loop whereby EGFR transactivation leads to the up-regulation of EGFR expression. This, in turn, may contribute to unrestrained epithelial cell proliferation and carcinogenesis.


Subject(s)
Epithelial Cells/microbiology , ErbB Receptors/metabolism , Helicobacter pylori/physiology , Stomach/microbiology , Up-Regulation , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Blotting, Western , Cell Line, Tumor , ErbB Receptors/genetics , Fluorescent Antibody Technique , Gastric Mucosa/metabolism , Gene Expression Profiling , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stomach/cytology , src-Family Kinases/metabolism
9.
J Immunol ; 178(12): 8013-21, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17548638

ABSTRACT

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.


Subject(s)
Chemokines, CC/metabolism , Colon/immunology , ErbB Receptors/genetics , Glycoproteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Macrophage Inflammatory Proteins/metabolism , Transcriptional Activation , Amphiregulin , Antibodies, Monoclonal , Caco-2 Cells , Cell Proliferation , Chemokine CCL20 , Chemokines, CC/pharmacology , Colon/drug effects , EGF Family of Proteins , Glycoproteins/antagonists & inhibitors , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Macrophage Inflammatory Proteins/pharmacology , Metalloproteases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Receptors, CCR6 , Receptors, Chemokine/metabolism , Signal Transduction
10.
Am J Physiol Gastrointest Liver Physiol ; 292(5): G1263-71, 2007 May.
Article in English | MEDLINE | ID: mdl-17272517

ABSTRACT

A characteristic feature of human inflammatory bowel disease, particularly Crohn's disease, is the presence of activated CD4(+) T cells. Recently, we have shown that colonic epithelial cell production of macrophage inflammatory protein (MIP)-3alpha, a CD4 T cell-directed chemokine, is elevated in inflammatory bowel disease. However, the functional relevance of MIP-3alpha production during intestinal inflammation is poorly understood. The aim of this study was to determine whether MIP-3alpha production is increased during murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and to examine the effect of anti-MIP-3alpha neutralizing monoclonal antibody administration in this model. We found that the administration of TNBS significantly increased colonic MIP-3alpha protein levels in Balb/c mice. Consistent with this, a marked increase in the number of CCR6-bearing lamina propria CD4(+) and CD8(+) T cells was also observed in TNBS-treated animals. Treatment of mice with an anti-MIP-3alpha neutralizing monoclonal antibody significantly reduced TNBS-mediated increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and myeloperoxidase activity. TNBS-mediated increases in the number of CCR6-bearing lamina propria T cells were also substantially reduced by anti-MIP-3alpha neutralizing monoclonal antibody treatment. Taken together, our findings indicate that blockade of MIP-3alpha bioactivity can significantly reduce TNBS-mediated colonic injury and T cell recruitment, suggesting a role for this chemokine in the pathophysiology of intestinal inflammation.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Chemokines, CC/immunology , Colitis/prevention & control , Colonic Diseases/prevention & control , Macrophage Inflammatory Proteins/immunology , Animals , CD4 Lymphocyte Count , CD8 Antigens/analysis , Chemokine CCL20 , Colitis/immunology , Colonic Diseases/chemically induced , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred BALB C , Trinitrobenzenesulfonic Acid , Up-Regulation
11.
Pharmacogenomics ; 8(7): 867-71, 2007 Jul.
Article in English | MEDLINE | ID: mdl-18240911

ABSTRACT

Cequent Pharmaceuticals, Inc. is a recently established biopharmaceutical company that aims to develop clinically compatible therapies based on RNAi, a potent gene-silencing mechanism discovered in 1998. The company's proprietary technology, transkingdom RNAi (tkRNAi), uses nonpathogenic bacteria to produce and deliver shRNA into target cells to induce RNAi. Our initial focus is on the development of a tkRNAi-based therapy for familial adenatomous polyposis, an inherited form of colon cancer. Cequent's first tkRNAi-based drug for familial adenatomous polyposis, CEQ501, is currently in advanced preclinical testing. As part of its ongoing activities, Cequent plans to develop additional tkRNAi-based products for indications within and outside the GI tract. Our overall goal is to establish tkRNAi as a platform for developing a wide range of RNAi-based therapies.


Subject(s)
Drug Industry , Genetic Therapy/methods , RNA Interference , Adenomatous Polyposis Coli/genetics , Adenomatous Polyposis Coli/therapy , Boston , Humans
12.
Am J Physiol Gastrointest Liver Physiol ; 289(6): G1075-83, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16099872

ABSTRACT

Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.


Subject(s)
Chemokines, CXC/antagonists & inhibitors , Colitis/drug therapy , Oligodeoxyribonucleotides, Antisense/therapeutic use , Animals , Cell Line , Chemokine CXCL5 , Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Colitis/chemically induced , Colitis/pathology , Colon/pathology , Dextran Sulfate , Disease Models, Animal , Enterocytes/metabolism , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C
13.
J Biol Chem ; 279(24): 25179-88, 2004 Jun 11.
Article in English | MEDLINE | ID: mdl-15069060

ABSTRACT

Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.


Subject(s)
Chemokines, CC/genetics , Colon/metabolism , Gene Expression Regulation , Interleukin-8/genetics , Macrophage Inflammatory Proteins/genetics , Membrane Glycoproteins/physiology , Mitogen-Activated Protein Kinase Kinases/physiology , Receptors, Cell Surface/physiology , Chemokine CCL20 , Colon/cytology , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Proteins/physiology , Signal Transduction , TNF Receptor-Associated Factor 6 , Toll-Like Receptor 5 , Toll-Like Receptors
14.
J Biol Chem ; 278(2): 875-84, 2003 Jan 10.
Article in English | MEDLINE | ID: mdl-12414801

ABSTRACT

We have previously shown that colonic epithelial cells are a major site of MIP-3alpha production in human colon and that enterocyte MIP-3alpha protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3alpha gene transcription in Caco-2 intestinal epithelial cells. We show that a kappaB element at nucleotides -82 to -93 of the MIP-3alpha promoter binds p50/p65 NF-kappaB heterodimers and is a major regulator of basal and interleukin-1beta (IL-1beta)-mediated gene activation. Scanning mutagenesis of the MIP-3alpha 5'-flanking region also identified two additional binding elements: Site X (nucleotides -63 to -69) and Site Y (nucleotides -143 to -154). Site X (CGCCTTC) bound Sp1 and regulated basal MIP-3alpha gene transcription. Overexpression of Sp1 increased basal luciferase activity, whereas, substitutions in the Sp1 element significantly reduced reporter activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal and cytokine-induced gene activation and bound the Ets nuclear factor ESE-1. Substitutions in the Site Y element markedly reduced inducible MIP-3alpha reporter activity. Conversely, overexpression of ESE-1 significantly up-regulated MIP-3alpha luciferase levels. Taken together, our findings demonstrate that co-ordinate activation and binding of ESE-1, Sp1, and NF-kappaB to the MIP-3alpha promoter is required for maximal gene expression by cytokine-stimulated Caco-2 human intestinal epithelial cells.


Subject(s)
Chemokines, CC/genetics , Macrophage Inflammatory Proteins/genetics , Proto-Oncogene Proteins , Receptors, Chemokine , Trans-Activators/physiology , Base Sequence , Binding Sites , CCAAT-Enhancer-Binding Proteins/metabolism , Caco-2 Cells , Chemokine CCL20 , Cloning, Molecular , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Interleukin-1/pharmacology , Molecular Sequence Data , NF-kappa B/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-ets , Receptors, CCR6 , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation
15.
Gastroenterology ; 122(4): 1048-57, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11910356

ABSTRACT

BACKGROUND & AIMS: Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor kappaB (NF-kappaB) activation and interleukin (IL)-8 release from toxin A-exposed enterocytes. METHODS: NF-kappaB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function. RESULTS: HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P < 0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IkappaB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-kappaB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1beta was not affected. CONCLUSIONS: Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-kappaB before release of IL-8.


Subject(s)
Bacterial Toxins/pharmacology , Enterotoxins/pharmacology , I-kappa B Proteins , Interleukin-8/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/metabolism , DNA-Binding Proteins/metabolism , Glycosylation , HT29 Cells , Humans , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , rho GTP-Binding Proteins/metabolism
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