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1.
Nature ; 576(7785): 16-17, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31796901
2.
Cell Death Dis ; 10(11): 795, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641109

RESUMO

The DNA mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. Inhibition of a number of mitochondrial genes, including POLG and PINK1 can induce synthetic lethality in MLH1-deficient cells. Here we demonstrate for the first time that loss of MLH1 is associated with a deregulated mitochondrial metabolism, with reduced basal oxygen consumption rate and reduced spare respiratory capacity. Furthermore, MLH1-deficient cells display a significant reduction in activity of the respiratory chain Complex I. As a functional consequence of this perturbed mitochondrial metabolism, MLH1-deficient cells have a reduced anti-oxidant response and show increased sensitivity to reactive oxidative species (ROS)-inducing drugs. Taken together, our results provide evidence for an intrinsic mitochondrial dysfunction in MLH1-deficient cells and a requirement for MLH1 in the regulation of mitochondrial function.

3.
Nature ; 570(7762): 545, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31239570
7.
Gut ; 68(6): 985-995, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-29991641

RESUMO

OBJECTIVE: IBD confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of CA-CRC using multiregion sequencing. DESIGN: Exome sequencing was performed on fresh-frozen multiple regions of carcinoma, adjacent non-cancerous mucosa and blood from 12 patients with CA-CRC (n=55 exomes), and key variants were validated with orthogonal methods. Genome-wide copy number profiling was performed using single nucleotide polymorphism arrays and low-pass whole genome sequencing on archival non-dysplastic mucosa (n=9), low-grade dysplasia (LGD; n=30), high-grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=19). Phylogenetic trees were reconstructed, and evolutionary analysis used to reveal the temporal sequence of events leading to CA-CRC. RESULTS: 10/12 tumours were microsatellite stable with a median mutation burden of 3.0 single nucleotide alterations (SNA) per Mb, ~20% higher than S-CRC (2.5 SNAs/Mb), and consistent with elevated ageing-associated mutational processes. Non-dysplastic mucosa had considerable mutation burden (median 47 SNAs), including mutations shared with the neighbouring CA-CRC, indicating a precancer mutational field. CA-CRCs were often near triploid (40%) or near tetraploid (20%) and phylogenetic analysis revealed that copy number alterations (CNAs) began to accrue in non-dysplastic bowel, but the LGD/HGD transition often involved a punctuated 'catastrophic' CNA increase. CONCLUSIONS: Evolutionary genomic analysis revealed precancer clones bearing extensive SNAs and CNAs, with progression to cancer involving a dramatic accrual of CNAs at HGD. Detection of the cancerised field is an encouraging prospect for surveillance, but punctuated evolution may limit the window for early detection.


Assuntos
Transformação Celular Neoplásica/patologia , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transformação Celular Neoplásica/genética , Colonoscopia/métodos , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Medição de Risco , Índice de Gravidade de Doença
9.
J Cell Mol Med ; 22(11): 5617-5628, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30188001

RESUMO

Intestinal mesenchymal cells deposit extracellular matrix in fibrotic Crohn's disease (CD). The contribution of epithelial to mesenchymal transition (EMT) to the mesenchymal cell pool in CD fibrosis remains obscure. The miR-200 family regulates fibrosis-related EMT in organs other than the gut. E-cadherin, cytokeratin-18 and vimentin expression was assessed using immunohistochemistry on paired strictured (SCD) and non-strictured (NSCD) ileal CD resections and correlated with fibrosis grade. MiR-200 expression was measured in paired SCD and NSCD tissue compartments using laser capture microdissection and RT-qPCR. Serum miR-200 expression was also measured in healthy controls and CD patients with stricturing and non-stricturing phenotypes. Extra-epithelial cytokeratin-18 staining and vimentin-positive epithelial staining were significantly greater in SCD samples (P = 0.04 and P = 0.03, respectively). Cytokeratin-18 staining correlated positively with subserosal fibrosis (P < 0.001). Four miR-200 family members were down-regulated in fresh SCD samples (miR-141, P = 0.002; miR-200a, P = 0.002; miR-200c, P = 0.001; miR-429; P = 0.004); miR-200 down-regulation in SCD tissue was localised to the epithelium (P = 0.001-0.015). The miR-200 target ZEB1 was up-regulated in SCD samples (P = 0.035). No difference in serum expression between patient groups was observed. Together, these observations suggest the presence of EMT in CD strictures and implicate the miR-200 family as regulators. Functional studies to prove this relationship are now warranted.


Assuntos
Antígenos CD/genética , Caderinas/genética , Doença de Crohn/genética , Fibrose/genética , MicroRNAs/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Adulto , Doença de Crohn/patologia , Doença de Crohn/cirurgia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Fibrose/patologia , Fibrose/cirurgia , Regulação da Expressão Gênica/genética , Humanos , Íleo/patologia , Íleo/ultraestrutura , Queratina-18/genética , Masculino , Vimentina/genética
13.
Undersea Hyperb Med ; 45(2): 157-164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29734567

RESUMO

Purpose: Hyperbaric oxygen (HBO2) therapy is used to improve the survival of compromised flaps. Compromised flaps are complications encountered postsurgically, or in traumatic degloving or avulsion injuries. Failed flaps lead to persistence of the defect, requirement of another donor site, and psychosocial sequelae. Although evidence of the benefit of HBO2 therapy is significant, there is no consensus on the optimal treatment regimen. The purpose of this study is to examine whether twice-daily treatments (BID HBO2) provide additional benefit compared to daily treatments (QD HBO2) in a rat compromised random flap model. Methods: A rat random flap model was used with subjects divided into three groups: 1) control group; 2) QD HBO2; and 3) BID HBO2, where HBO2 was performed with 100% oxygen at 2.5 atmospheres absolute/ATA (253 kPa) for 90 minutes. After 10 days, areas of flap necrosis were measured and biopsies were taken for histologic analysis. Statistical analysis was performed using ANOVA and paired t-tests. A P-value ⟨0.05 was considered significant. Result: Both treatment groups had significantly increased mean flap survival compared to controls (P⟨0.05). There was no significant difference in flap survival between the QD and BID groups. Capillary proliferation in the QD group was increased compared with controls. Conclusion: Both QD and BID HBO2 protocols can significantly decrease random flap necrosis. However, the results of this study suggest there is no additional benefit gained with BID treatments. Clinical studies are warranted to confirm these findings and assist in formalization of protocols for the use of HBO2in treating compromised random flaps.


Assuntos
Sobrevivência de Enxerto , Oxigenação Hiperbárica/métodos , Análise de Variância , Animais , Capilares/anatomia & histologia , Masculino , Necrose/patologia , Ratos , Ratos Sprague-Dawley , Retalhos Cirúrgicos/irrigação sanguínea , Retalhos Cirúrgicos/patologia , Fatores de Tempo
14.
Inflamm Bowel Dis ; 24(1): 136-148, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29272487

RESUMO

Background: DUOX2 and DUOXA2 form the predominant H2O2-producing system in human colorectal mucosa. Inflammation, hypoxia, and 5-aminosalicylic acid increase H2O2 production, supporting innate defense and mucosal healing. Thiocyanate reacts with H2O2 in the presence of lactoperoxidase (LPO) to form hypothiocyanate (OSCN-), which acts as a biocide and H2O2 scavenging system to reduce damage during inflammation. We aimed to discover the organization of Duox2, Duoxa2, and Lpo expression in colonic crypts of Lieberkühn (intestinal glands) of mice and how distributions respond to dextran sodium sulfate (DSS)-induced colitis and subsequent mucosal regeneration. Methods: We studied tissue from DSS-exposed mice and human biopsies using in situ hybridization, reverse transcription quantitative polymerase chain reaction, and cDNA microarray analysis. Results: Duox2 mRNA expression was mostly in the upper crypt quintile while Duoxa2 was more apically focused. Most Lpo mRNA was in the basal quintile, where stem cells reside. Duox2 and Duoxa2 mRNA were increased during the induction and resolution of DSS colitis, while Lpo expression did not increase during the acute phase. Patterns of Lpo expression differed from Duox2 in normal, inflamed, and regenerative mouse crypts (P < 0.001). We found no evidence of LPO expression in the human gut. Conclusions: The spatial and temporal separation of H2O2-consuming and -producing enzymes enables a thiocyanate- H2O2 "scavenging" system in murine intestinal crypts to protect the stem/proliferative zones from DNA damage, while still supporting higher H2O2 concentrations apically to aid mucosal healing. The absence of LPO expression in the human gut suggests an alternative mechanism or less protection from DNA damage during H2O2-driven mucosal healing.


Assuntos
Colite/metabolismo , Oxidases Duais/metabolismo , Peróxido de Hidrogênio/metabolismo , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Lactoperoxidase/metabolismo , Cicatrização , Animais , Colite/induzido quimicamente , Colite/patologia , Oxidases Duais/genética , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/metabolismo , Humanos , Inflamação/patologia , Mucosa Intestinal/patologia , Lactoperoxidase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie
15.
Oncotarget ; 8(48): 84258-84275, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-29137421

RESUMO

The estrogen receptor ERß is the predominant ER subtype expressed in normal well-differentiated colonic epithelium. However, ERß expression is lost under the hypoxic microenvironment as colorectal cancer (CRC) malignancy progresses. This raises questions about the role of signalling through other estrogen receptors such as ERα or G-protein coupled estrogen receptor (GPER, GPR30) by the estrogen 17ß-estradiol (E2) under hypoxic conditions after ERß is lost in CRC progression. We tested the hypothesis that E2 or hypoxia can act via GPER to contribute to the altered phenotype of CRC cells. GPER expression was found to be up-regulated by hypoxia and E2 in a panel of CRC cell lines. The E2-modulated gene, Ataxia telangiectasia mutated (ATM), was repressed in hypoxia via GPER signalling. E2 treatment enhanced hypoxia-induced expression of HIF1-α and VEGFA, but repressed HIF1-α and VEGFA expression under normoxic conditions. The expression and repression of VEGFA by E2 were mediated by a GPER-dependent mechanism. E2 treatment potentiated hypoxia-induced CRC cell migration and proliferation, whereas in normoxia, cell migration and proliferation were suppressed by E2 treatment. The effects of E2 on these cellular responses in normoxia and hypoxia were mediated by GPER. In a cohort of 566 CRC patient tumor samples, GPER expression significantly associated with poor survival in CRC Stages 3-4 females but not in the stage-matched male population. Our findings support a potentially pro-tumorigenic role for E2 in ERß-negative CRC under hypoxic conditions transduced via GPER and suggest a novel route of therapeutic intervention through GPER antagonism.

16.
Nature ; 550(7674): 143-144, 2017 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-28980652
17.
Cell Rep ; 20(13): 3135-3148, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28954230

RESUMO

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic ß cells (Fh1ßKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1ßKO mice led to dysregulated metabolism in ß cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1ßKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fumarato Hidratase/deficiência , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Camundongos
18.
Oxid Med Cell Longev ; 2017: 9303158, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28751935

RESUMO

Dysregulation of c-Jun N-terminal kinase (JNK) activation promoted DNA damage response bypass and tumorigenesis in our model of hydrogen peroxide-associated ulcerative colitis (UC) and in patients with quiescent UC (QUC), UC-related dysplasia, and UC-related carcinoma (UC-CRC), thereby adapting to oxidative stress. In the UC model, we have observed features of oncogenic transformation: increased proliferation, undetected DNA damage, and apoptosis resistance. Here, we show that Chk1 was downregulated but activated in the acute and quiescent chronic phases. In both phases, Chk1 was linked to DNA damage response bypass by suppressing JNK activation following oxidative stress, promoting cell cycle progression despite DNA damage. Simultaneously, activated Chk1 was bound to chromatin. This triggered histone acetylation and the binding of histone acetyltransferases and transcription factors to chromatin. Thus, chromatin-immobilized activated Chk1 executed a dual function by suppressing DNA damage response and simultaneously inducing chromatin modulation. This caused undetected DNA damage and increased cellular proliferation through failure to transmit the appropriate DNA damage signal. Findings in vitro were corroborated by chromatin accumulation of activated Chk1, Ac-H3, Ac-H4, and c-Jun in active UC (AUC) in vivo. Targeting chromatin-bound Chk1, GCN5, PCAF, and p300/CBP could be a novel therapeutic strategy to prevent UC-related tumor progression.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/metabolismo , Colite Ulcerativa/metabolismo , Dano ao DNA , Peróxido de Hidrogênio/efeitos adversos , MAP Quinase Quinase 4/metabolismo , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/genética , Cromatina/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , MAP Quinase Quinase 4/genética
20.
Carcinogenesis ; 38(5): 559-569, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28383667

RESUMO

We recently reported that dysregulated c-Jun N-terminal kinases (JNK) activity causes defective cell cycle checkpoint control, inducing neoplastic transformation in a cellular ulcerative colitis (UC) model. In the quiescent chronic phase of UC, p-p54 JNK was down-regulated and p-p46 JNK was up-regulated. Both were up-regulated in the acute phase. Consequently, increased p21WAF1 and γ-H2AX, two JNK-regulated proteins, induced cell cycle arrest. Their down-regulation led to checkpoint override, causing increased proliferation and undetected DNA damage in quiescent chronic phase, all characteristics of tumorigenesis. We investigated expression of p-JNK2, p-JNK1-3, p21WAF1, γ-H2AX and Ki67 by immunohistochemistry in cases of quiescent UC (QUC), active UC (AUC), UC-dysplasia and UC-related colorectal carcinoma (UC-CRC). Comparison was made to normal healthy colorectal mucosa, sporadic adenoma and colorectal carcinoma (CRC), diverticulitis and Crohns disease (CD). We found p-JNK2 up-regulation in AUC and its early down-regulation in UC-CRC and CRC carcinogenesis. With down-regulated p-JNK2, p21WAF1 was also decreased. Ki67 was inversely expressed, showing increased proliferation early in UC-CRC and CRC carcinogenesis. p-JNK1-3 was increased in AUC and QUC. Less increased γ-H2AX in UC-CRC compared to CRC gave evidence that colitis-triggered inflammation masks DNA damage, thus contributing to neoplastic transformation. We hypothesize that JNK-dependent cell cycle arrest is important in AUC, while chronic inflammation causes dysregulated JNK activity in quiescent phase that may contribute to checkpoint override, promoting UC carcinogenesis. We suggest restoring p-JNK2 expression as a novel therapeutic strategy to early prevent the development of UC-related cancer.


Assuntos
Transformação Celular Neoplásica/genética , Colite/complicações , Colite/genética , Neoplasias Colorretais/etiologia , Proteína Quinase 9 Ativada por Mitógeno/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Transformação Celular Neoplásica/metabolismo , Colite/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Expressão Gênica , Estudos de Associação Genética , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Adulto Jovem
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