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1.
Res Sq ; 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38746220

RESUMO

Background: Based on preclinical data showing addition of CDK4/6 inhibitors to gemcitabine is synergistic, ribociclib was evaluated in combination with gemcitabine to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT). Methods: In this single arm multicohort phase I trial, we evaluated the safety and efficacy of Ribociclib plus Gemcitabine in patients with advanced solid tumors. Patients received Gemcitabine intravenously on days 1 and 8 followed by Ribociclib days 8-14, with treatment repeated every 3 weeks. Results: The study enrolled 43 patients between October 2017 and September 2019. The escalation phase (19 patients) determined the MTD and recommended phase II dose (RP2D) to be ribociclib 800mg daily and gemcitabine 1000mg/m2 for the expansion phase (24 patients). One patient experienced Grade 4 thrombocytopenia. Eleven patients experienced Grade 3 adverse events (AE), the most common being neutropenia, thrombocytopenia, and anemia. No partial or complete responses were observed. 15/22 (68%) of efficacy evaluable patients who received the MTD achieved best response of stable disease. Conclusions: The addition of Ribociclib to Gemcitabine was tolerated well and yielded stability of tumors in both cohorts. Ribociclib and gemcitabine could have synergistic activity in certain tumor types, and our data provides support for the combination. Clinical Trial Registration: NCT03237390.

2.
Adv Sci (Weinh) ; : e2401797, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38728624

RESUMO

Gene knock-in refers to the insertion of exogenous functional genes into a target genome to achieve continuous expression. Currently, most knock-in tools are based on site-directed nucleases, which can induce double-strand breaks (DSBs) at the target, following which the designed donors carrying functional genes can be inserted via the endogenous gene repair pathway. The size of donor genes is limited by the characteristics of gene repair, and the DSBs induce risks like genotoxicity. New generation tools, such as prime editing, transposase, and integrase, can insert larger gene fragments while minimizing or eliminating the risk of DSBs, opening new avenues in the development of animal models and gene therapy. However, the elimination of off-target events and the production of delivery carriers with precise requirements remain challenging, restricting the application of the current knock-in treatments to mainly in vitro settings. Here, a comprehensive review of the knock-in tools that do not/minimally rely on DSBs and use other mechanisms is provided. Moreover, the challenges and recent advances of in vivo knock-in treatments in terms of the therapeutic process is discussed. Collectively, the new generation of DSBs-minimizing and large-fragment knock-in tools has revolutionized the field of gene editing, from basic research to clinical treatment.

3.
Pharmaceuticals (Basel) ; 17(3)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38543111

RESUMO

COVID-19, caused by SARS-CoV-2, has emerged as the most destructive emerging infectious disease of the 21st century. Vaccination is an effective method to combat viral diseases. However, due to the constant mutation of the virus, new variants may weaken the efficacy of vaccines. In the current field of new coronavirus research, viral protease inhibitors have emerged as a highly regarded therapeutic strategy. Nevertheless, existing viral protease inhibitors do not fully meet the therapeutic needs. Therefore, this paper turned to traditional Chinese medicine to explore new active compounds. This study focused on 24 isolated compounds from Acorus calamus L. and identified 8 active components that exhibited significant inhibitory effects on SARS-CoV-2 PLpro. Among these, the compound 1R,5R,7S-guaiane-4R,10R-diol-6-one demonstrated the best inhibitory activity with IC50 values of 0.386 ± 0.118 µM. Additionally, menecubebane B and neo-acorane A exhibited inhibitory activity against both Mpro and PLpro proteases, indicating their potential as dual-target inhibitors. The molecular docking results confirmed the stable conformations of these compounds with the key targets and their good activity. ADMET and Lipinski's rule analyses revealed that all the small molecule ligands possessed excellent oral absorption properties. This study provides an experimental foundation for the discovery of promising antiviral lead compounds.

4.
Cancer Med ; 12(23): 21229-21239, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37960985

RESUMO

BACKGROUND: Despite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK-228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL. METHODS: We conducted a single-arm multi-center Phase II study of sapanisertib monotherapy (3 mg orally daily of the milled formulation for 21 days every 28 days) in patients with ALL through the Experimental Therapeutics Clinical Trials Network (NCI-9775). RESULTS: Sixteen patients, 15 of whom were previously treated (median 3 prior lines of therapy), were enrolled. Major grade 3-4 non-hematologic toxicities included mucositis (3 patients) and hyperglycemia (2 patients) as well as hepatic failure, seizures, confusion, pneumonitis, and anorexia (1 patient each). Grade >2 hematological toxicity included leukopenia (3), lymphopenia (2), thrombocytopenia, and neutropenia (1). The best response was stable disease in 2 patients (12.5%), while only 3 patients (19%) were able to proceed to Cycle 2. Pharmacokinetic analysis demonstrated drug exposures similar to those observed in solid tumor patients. Immunoblotting in serially collected samples indicated limited impact of treatment on phosphorylation of mTOR pathway substrates such as 4EBP1, S6, and AKT. CONCLUSION: In summary, single-agent sapanisertib had a good safety profile but limited target inhibition or efficacy in ALL as a single agent. This trial was registered at ClinicalTrials.gov as NCT02484430.


Assuntos
Benzoxazóis , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico
7.
PLoS One ; 14(9): e0221829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31479473

RESUMO

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband's fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Síndrome Congênita de Insuficiência da Medula Óssea/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Síndrome de Kearns-Sayre/genética , Doença de Leigh/genética , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Doenças Musculares/genética , Mutação , Acil-CoA Desidrogenase de Cadeia Longa/genética , Adolescente , Sequência de Aminoácidos , Linhagem Celular , Criança , Síndrome Congênita de Insuficiência da Medula Óssea/complicações , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Heterozigoto , Humanos , Síndrome de Kearns-Sayre/complicações , Doença de Leigh/complicações , Erros Inatos do Metabolismo Lipídico/complicações , Masculino , Doenças Mitocondriais/complicações , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Doenças Musculares/complicações , Fenótipo , Estabilidade Proteica , Estrutura Quaternária de Proteína , Deleção de Sequência , Sequenciamento do Exoma
8.
Cell Mol Biol (Noisy-le-grand) ; 65(4): 79-82, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31078156

RESUMO

Effect of ginkgetin on proliferation of human cervical cancer (HeLa) cells and the underlying mechanism   were investigated. Human cervical cancer (HeLa) cells were cultured at 37 °C in 10 % fetal bovine serum (FBS) supplemented RPMI 1640 medium in a humidified incubator containing 5 % CO2. Cell proliferation was determined using MTT assay, while real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the levels of expression of interleukin 1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8). The expressions of p38 mitogen-activated protein kinases (p38 MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) were determined using Western blotting. Treatment of HeLa cells with ginkgetin significantly and time- and dose-dependently inhibited their proliferation (p < 0.05). The invasion of the cells were also significantly and dose-dependently decreased, when compared with control cells (p < 0.05). The expressions of p-p38 and p-NF-κB were significantly and dose-dependently down-regulated, relative to control group (p < 0.05). However, the expressions of p38 and NF-κB in ginkgetin-treated cells were not significantly different from those of control group (p > 0.05). The results of qRT-PCR and ELISA showed that the levels of expression of TNF-α, IL-1ß and IL-8 mRNAs were significantly and dose-dependently reduced in HeLa cells after 48 h of treatment with ginkgetin, when compared with the control group (p < 0.05). The anti-proliferative effect of ginkgetin on HeLa cells is exerted via a mechanism involving the p38/NF-κB pathway.


Assuntos
Biflavonoides/farmacologia , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Biflavonoides/química , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
Mitochondrion ; 38: 6-16, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28750948

RESUMO

Mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) share extensive clinical overlap but are considered to have distinct cellular pathophysiology. Here, we demonstrate that an essential physiologic connection exists between cellular N-linked deglycosylation capacity and mitochondrial function. Following identification of altered muscle and liver mitochondrial amount and function in two children with a CDG subtype caused by NGLY1 deficiency, we evaluated mitochondrial physiology in NGLY1 disease human fibroblasts, and in NGLY1-knockout mouse embryonic fibroblasts and C. elegans. Across these distinct evolutionary models of cytosolic NGLY1 deficiency, a consistent disruption of mitochondrial physiology was present involving modestly reduced mitochondrial content with more pronounced impairment of mitochondrial membrane potential, increased mitochondrial matrix oxidant burden, and reduced cellular respiratory capacity. Lentiviral rescue restored NGLY1 expression and mitochondrial physiology in human and mouse fibroblasts, confirming that NGLY1 directly influences mitochondrial function. Overall, cellular deglycosylation capacity is shown to be a significant factor in mitochondrial RC disease pathogenesis across divergent evolutionary species.


Assuntos
Defeitos Congênitos da Glicosilação/patologia , Defeitos Congênitos da Glicosilação/fisiopatologia , Fibroblastos/patologia , Fibroblastos/fisiologia , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Animais , Caenorhabditis elegans , Respiração Celular , Células Cultivadas , Pré-Escolar , Transporte de Elétrons , Feminino , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Masculino , Potencial da Membrana Mitocondrial , Camundongos Knockout
10.
JAMA Oncol ; 2(10): 1333-1339, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27467440

RESUMO

IMPORTANCE: Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. OBJECTIVE: To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. DESIGN, SETTING, AND PARTICIPANTS: We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. MAIN OUTCOMES AND MEASURES: Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. RESULTS: A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. CONCLUSIONS AND RELEVANCE: This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma.


Assuntos
Antígenos de Neoplasias/genética , Esôfago de Barrett/genética , Glicoproteínas de Membrana/genética , Adenocarcinoma/genética , Adulto , Idoso , Sequência de Aminoácidos , Células Cultivadas , Células Epiteliais/fisiologia , Neoplasias Esofágicas/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
11.
Mol Carcinog ; 55(11): 1526-1541, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26373456

RESUMO

Autophagy is a highly conserved mechanism that is activated during cellular stress. We hypothesized that autophagy may be induced by acid reflux, which causes injury, and inflammation, and therefore, contributes to the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Currently, the role of autophagy in BE and EAC is poorly studied. We quantitatively define autophagy levels in human BE cell lines, a transgenic mouse model of BE, and human BE, and EAC biopsies. Human non-dysplastic BE had the highest basal number of autophagic vesicles (AVs), while AVs were reduced in normal squamous cells and dysplastic BE cells, and nearly absent in EAC. To demonstrate a functional role for autophagy in BE pathogenesis, normal squamous (STR), non-dysplastic BE (CPA), dysplastic BE (CPD), and EAC (OE19) cell lines were exposed to an acid pulse (pH 3.5) followed by incubation in the presence or absence of chloroquine, an autophagy inhibitor. Acid exposure increased reactive oxygen species (ROS) levels in STR and CPA cells. Chloroquine alone had a small impact on intracellular ROS or cell survival. However, combination of chloroquine with the acid pulse resulted in a significant increase in ROS levels at 6 h in STR and CPA cells, and increased cell death in all cell lines. These findings establish increased numbers of AVs in human BE compared to normal squamous or EAC, and suggest that autophagy functions to improve cell survival after acid reflux injury. Autophagy may thus play a critical role in BE pathogenesis and progression. © 2015 Wiley Periodicals, Inc.


Assuntos
Ácidos/efeitos adversos , Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Neoplasias Esofágicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Adenocarcinoma/metabolismo , Animais , Autofagia/efeitos dos fármacos , Esôfago de Barrett/metabolismo , Linhagem Celular , Sobrevivência Celular , Cloroquina/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Humanos , Camundongos , Estresse Oxidativo
12.
Oncotarget ; 6(32): 32980-3005, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26460825

RESUMO

Cdx2, an intestine specific transcription factor, is expressed in Barrett's esophagus (BE). We sought to determine if esophageal Cdx2 expression would accelerate the onset of metaplasia in the L2-IL-1ß transgenic mouse model for Barrett's-like metaplasia. The K14-Cdx2::L2-IL-1ß double transgenic mice had half as many metaplastic nodules as control L2-IL-1ß mice. This effect was not due to a reduction in esophageal IL-1ß mRNA levels nor diminished systemic inflammation. The diminished metaplasia was due to an increase in apoptosis in the K14-Cdx2::L2-IL-1ß mice. Fluorescence activated cell sorting of immune cells infiltrating the metaplasia identified a population of CD11b+Gr-1+ cells that are significantly reduced in K14-Cdx2::L2-IL-1ß mice. These cells have features of immature granulocytes and have immune-suppressing capacity. We demonstrate that the apoptosis in K14-Cdx2::L2-IL-1ß mice is CD8+ T cell dependent, which CD11b+Gr-1+ cells are known to inhibit. Lastly, we show that key regulators of CD11b+Gr-1+ cell development, IL-17 and S100A9, are significantly diminished in the esophagus of K14-Cdx2::L2-IL-1ß double transgenic mice. We conclude that metaplasia development in this mouse model for Barrett's-like metaplasia requires suppression of CD8+ cell dependent apoptosis, likely mediated by immune-suppressing CD11b+Gr-1+ immature myeloid cells.


Assuntos
Esôfago de Barrett/patologia , Neoplasias Esofágicas/patologia , Animais , Apoptose/fisiologia , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Fator de Transcrição CDX2 , Modelos Animais de Doenças , Progressão da Doença , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Esofagite/genética , Esofagite/metabolismo , Esofagite/patologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Células Mieloides/patologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
13.
Dig Dis Sci ; 57(4): 845-57, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22147253

RESUMO

BACKGROUND: Esophageal intestinal metaplasia, also known as Barrett's esophagus, is the replacement of the normal epithelium with one that resembles the intestine morphologically. Generally, this includes intestinal mucin-secreting goblet cells. Barrett's esophagus is an important risk factor for adenocarcinoma development. In-vitro models for Barrett's esophagus have not, to date, focused on the induction of goblet cells in Barrett's epithelium. AIMS: To explore the contribution of Math1/Atoh1 to induction of Barrett's esophagus and intestinal mucin-secreting goblet cells from normal human esophageal epithelium. METHODS: We explored the level and pattern of Math1/Atoh1 mRNA and protein expression in human Barrett's esophagus. Then, using retroviral-mediated gene expression, we induced Math1 mRNA and protein expression in a human esophageal keratinocyte cell line. We evaluated the effects of this ectopic Math1 expression on cell proliferation and gene expression patterns in cells cultured under two-dimensional and three-dimensional tissue-engineering conditions. RESULTS: Math1/Atoh1 mRNA and protein are detected in human Barrett's esophagus specimens, but the mRNA levels vary substantially. In the keratinocyte expression studies, we observed that Math1/Atoh1 ectopic expression significantly reduced cell proliferation and altered cell morphology. Moreover, Math1/Atoh1 expression is associated with a more intestinalized gene expression pattern that is distinct from that reported in after studies using other intestinal transcription factors. Most significantly, we observe the induction of the Barrett's esophagus markers Mucin-2 and Keratin-20 with Math1/Atoh1 expression. CONCLUSIONS: We conclude that ectopic Math1/Atoh1 expression makes unique contributions to intestinalization of the esophageal epithelium in Barrett's esophagus.


Assuntos
Esôfago de Barrett/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Esôfago/patologia , Queratina-20/metabolismo , Queratinócitos/metabolismo , Mucina-2/metabolismo , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cultura , Expressão Gênica , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Queratina-20/genética , Mucina-2/genética , RNA Mensageiro/metabolismo
14.
Neoplasia ; 13(9): 792-805, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21969813

RESUMO

The incidence of esophageal adenocarcinoma (EAC) is rising in the United States. An important risk factor for EAC is the presence of Barrett esophagus (BE). BE is the replacement of normal squamous esophageal epithelium with a specialized columnar epithelium in response to chronic acid and bile reflux. However, the emergence of BE from squamous keratinocytes has not yet been demonstrated. Our research has focused on this. Wnt and cyclooxygenase 2 (Cox2) are two pathways whose activation has been associated with BE and progression to EAC, but their role has not been tested experimentally. To explore their contribution, we engineered a human esophageal keratinocyte cell line to express either a dominant-active Wnt effector CatCLef or a Cox2 complementary DNA. In a two-dimensional culture environment, Cox2 expression increases cell proliferation and migration, but neither transgene induces known BE markers. In contrast, when these cells were placed into three-dimensional organotypic culture conditions, we observed more profound effects. CatCLef-expressing cells were more proliferative, developed a thicker epithelium, and upregulated Notch signaling and several BE markers including NHE2. Cox2 expression also increased cell proliferation and induced a thicker epithelium. More importantly, we observed cysts form within the epithelium, filled with intestinal mucins including Muc5B and Muc17. This suggests that Cox2 expression in a three-dimensional culture environment induces a lineage of mucin-secreting cells and supports an important causal role for Cox2 in BE pathogenesis. We conclude that in vitro modeling of BE pathogenesis can be improved by enhancing Wnt signaling and Cox2 activity and using three-dimensional organotypic culture conditions.


Assuntos
Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Ciclo-Oxigenase 2/metabolismo , Neoplasias Esofágicas/patologia , Esôfago/patologia , Fatores de Transcrição TCF/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Adenocarcinoma , Esôfago de Barrett/genética , Linhagem Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Ciclo-Oxigenase 2/biossíntese , Humanos , Queratinócitos , Mucina-5B/metabolismo , Mucinas/metabolismo , Técnicas de Cultura de Órgãos , Receptores Notch/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de Transcrição TCF/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/metabolismo , beta Catenina/genética
15.
Ann N Y Acad Sci ; 1232: 381-91, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21950830

RESUMO

The following on molecular mechanisms of Barrett's esophagus and adenocarcinoma contains commentaries on the mechanism of bile and gastric acid induced damage; the roles of BMP-4 and CDX-2 in the development of intestinal metaplasia; the transcription factors driving intestinalization in Barrett's esophagus; the contribution of bone marrow to metaplasia and adenocarcinoma; activation and inactivation of transcription factors; and a novel study design targeting molecular pathways in Barrett's esophagus.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Adenocarcinoma/fisiopatologia , Esôfago de Barrett/fisiopatologia , Ácidos e Sais Biliares/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Fator de Transcrição CDX2 , Dano ao DNA , Neoplasias Esofágicas/fisiopatologia , Proteínas de Homeodomínio/fisiologia , Humanos , Transativadores/fisiologia
16.
PLoS One ; 6(4): e18280, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21494671

RESUMO

Barrett's esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm(2) ±343.5 to 508 Ohm*cm(2)±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barrett's epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled "Distinctive" cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5'-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE.


Assuntos
Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Esôfago/metabolismo , Esôfago/patologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/fisiopatologia , Fator de Transcrição CDX2 , Proliferação de Células , Forma Celular , Desmocolinas , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Epitélio/metabolismo , Epitélio/patologia , Epitélio/fisiopatologia , Epitélio/ultraestrutura , Esôfago/fisiopatologia , Esôfago/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Queratina-14/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Glicoproteínas de Membrana/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Transgenes/genética
17.
Gastroenterology ; 140(2): 517-528.e8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21081128

RESUMO

BACKGROUND & AIMS: Caudal-related homeobox protein 2 (Cdx2) is an intestine-specific transcription factor that is important for intestinal development and intestine-specific gene expression. Cdx2 regulates intestinal cell-cell adhesion, proliferation, and the transcriptional activities of Wnt and ß-catenin in cell culture systems. We generated transgenic mice that overexpress Cdx2 in the small intestinal and colonic epithelium to investigate the role of Cdx2 in differentiation and function of the intestinal epithelium. METHODS: We established 4 different lines of villin-Cdx2 transgenic mice. Intestines were collected from infant, 3-month old, and wild-type mice. Genes of interest and cell lineage markers were examined by polymerase chain reaction and immunohistochemistry. RESULTS: Villin-Cdx2 transgenic mice had complex phenotypes that were associated with transgene expression levels. The 2 lines that had the greatest levels of transgene expression had significant, preweaning failure to grow and death; these were the result of early epithelial maturation and alterations in nutrient digestion and absorption. Fat malabsorption was a prominent feature. Other effects associated with the transgene expression included loss of Paneth cell markers, increases in goblet cells, and migration of proliferating, EphB2-expressing cells to the crypt base. Loss of Paneth cell markers was associated with reduced nuclear localization of ß-catenin but not homeotic posteriorization of the epithelium by Cdx2. CONCLUSIONS: Overexpression of Cdx2 in the small intestine is associated with reduced post-natal growth, early epithelial maturation, alterations in crypt base organization, and changes in Paneth and goblet cell lineages. Cdx2 is a critical regulator not only of intestine-specific genes, but also processes that determine epithelial maturity and function.


Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/fisiologia , Celulas de Paneth/fisiologia , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/análise , Fator de Transcrição CDX2 , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Células Caliciformes/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Síndromes de Malabsorção/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Mosaicismo , Celulas de Paneth/metabolismo , Receptor EphB2/análise , Fatores de Transcrição/genética , beta Catenina/análise
18.
Prog Mol Biol Transl Sci ; 96: 231-70, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21075347

RESUMO

Intestinal metaplasia (IM) is a biologically interesting and clinically relevant condition in which one differentiated type of epithelium is replaced by another that is morphologically similar to normal intestinal epithelium. Two classic examples of this are gastric IM and Barrett's esophagus (BE). In both, a chronic inflammatory microenvironment, provoked either by Helicobacter pylori infection of the stomach or acid and bile reflux into the esophagus, precedes the metaplasia. The Caudal-related homeodomain transcription factors Cdx1 and Cdx2 are critical regulators of the normal intestinal epithelial cell phenotype. Ectopic expression of Cdx1 and Cdx2 occurs in both gastric IM as well as in BE. This expression precedes the onset of the metaplasia and implies a causal role for these factors in this process. We review the observations regarding the role of chronic inflammation and the Cdx transcription factors in the pathogenesis of gastric IM and BE.


Assuntos
Proteínas de Homeodomínio/genética , Inflamação/genética , Intestinos/patologia , Animais , Dano ao DNA , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metaplasia
19.
Am J Physiol Gastrointest Liver Physiol ; 299(5): G1054-67, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20671195

RESUMO

Cdx2 is an intestine-specific transcription factor required for normal intestinal epithelium development. Cdx2 regulates the expression of intestine-specific genes and induces cell adhesion and columnar morphogenesis. Cdx2 also has tumor-suppressor properties, including the reduction of colon cancer cell proliferation and cell invasion, the latter due to its effects on cell adhesion. E-cadherin is a cell adhesion protein required for adherens junction formation and the establishment of intestinal cell polarity. The objective of this study was to elucidate the mechanism by which Cdx2 regulates E-cadherin function. Two colon cancer cell lines were identified in which Cdx2 expression was associated with increased cell-cell adhesion and diminished cell migration. In both cell lines, Cdx2 did not directly alter E-cadherin levels but increased its trafficking to the cell membrane compartment. Cdx2 enhanced this trafficking by altering receptor tyrosine kinase (RTK) activity. Cdx2 expression diminished phosphorylated Abl and phosphorylated Rac levels, which are downstream effectors of RTKs. Specific chemical inhibition or short interfering RNA (shRNA) knockdown of c-Abl kinase phenocopied Cdx2's cell-cell adhesion effects. In Colo 205 cells, Cdx2 reduced PDGF receptor and IGF-I receptor activation. This was mediated by caveolin-1, which was induced by Cdx2. Targeted shRNA knockdown of caveolin-1 restored PDGF receptor and reversed E-cadherin membrane trafficking, despite Cdx2 expression. We conclude that Cdx2 regulates E-cadherin function indirectly by disrupting RTK activity and enhancing E-cadherin trafficking to the cell membrane compartment. This novel mechanism advances Cdx2's prodifferentiation and antitumor properties and suggests that Cdx2 may broadly regulate RTK activity in normal intestinal epithelium by modulating membrane trafficking of proteins.


Assuntos
Caderinas/metabolismo , Membrana Celular/metabolismo , Colo/metabolismo , Proteínas de Homeodomínio/metabolismo , Análise de Variância , Western Blotting , Fator de Transcrição CDX2 , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Cultivadas , Imunofluorescência , Humanos , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Receptor IGF Tipo 1/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia
20.
Biochem Soc Trans ; 38(2): 321-6, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20298176

RESUMO

Barrett's oesophagus is the replacement of normal squamous oesophageal epithelium with an intestinalized columnar epithelium. Although some insight has been gained as to what Barrett's oesophagus is, how this columnar epithelium emerges from within a stratified squamous epithelium remains an unanswered question. We have sought to determine whether oesophageal keratinocytes can be trans-differentiated into Barrett's oesophagus cells. Using an Affymetrix microarray, we found unexpectedly that gene-expression patterns in the Barrett's oesophagus were only slightly more similar to the normal small intestine than they were to the normal oesophagus. Thus gene-expression patterns suggest significant molecular similarities remain between Barrett's oesophagus cells and normal squamous oesophageal epithelium, despite their histological resemblance with intestine. We next determined whether directed expression of intestine-specific transcription factors could induce intestinalization of keratinocytes. Retroviral-mediated Cdx2 (Caudal-type homeobox 2) expression in immortalized human oesophageal keratinocytes engineered with human telomerase reverse transcriptase (EPC2-hTERT cells) could be established transiently, but not maintained, and was associated with a reduction in cell proliferation. Co-expression of cyclin D1 rescued proliferation in the Cdx2-expressing cells, but co-expression of dominant-negative p53 did not. Cdx2 expression in the EPC2-hTERT.D1 cells did not induce intestinalization. However, when combined with treatments that induce chromatin remodelling, there was a significant induction of Barrett's oesophagus-associated genes. Studies are ongoing to determine whether other intestinal transcription factors, either alone or in combination, can provoke greater intestinalization of oesophageal keratinocytes. We conclude that, on the basis of gene-expression patterns, Barrett's oesophagus epithelial cells may represent an intermediate between oesophageal keratinocytes and intestinal epithelial cells. Moreover, our findings suggest that it may be possible to induce Barrett's oesophagus epithelial cells from oesophageal keratinocytes by altering the expression of certain critical genes.


Assuntos
Esôfago de Barrett/patologia , Modelos Teóricos , Animais , Esôfago de Barrett/genética , Técnicas de Cultura de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Modelos Biológicos , Fenótipo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia
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