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1.
Cell Rep ; 19(11): 2185-2192, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28614706

RESUMO

The most prevalent single-nucleotide substitution (SNS) found in cancers is a C-to-T substitution in the CpG motif. It has been proposed that many of these SNSs arise during organismal aging, prior to transformation of a normal cell into a precancerous/cancer cell. Here, we isolated single intestinal crypts derived from normal tissue or from adenomas of Apcmin/+ mice, expanded them minimally in vitro as organoids, and performed exome sequencing to identify point mutations that had been acquired in vivo at the single-cell level. SNSs, most of them being CpG-to-TpG substitutions, were at least ten times more frequent in adenoma than normal cells. Thus, contrary to the view that substitutions of this type are present due to normal-cell aging, the acquisition of point mutations increases upon transformation of a normal intestinal cell into a precancerous cell.


Assuntos
Adenoma/metabolismo , Mucosa Intestinal/metabolismo , Mutação Puntual/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
2.
DNA Repair (Amst) ; 56: 129-134, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28623093

RESUMO

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Telômero/metabolismo , Animais , Reparo do DNA , Humanos
3.
Mol Cell ; 64(6): 1127-1134, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27984746

RESUMO

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Ciclina E/genética , Quebras de DNA de Cadeia Dupla , DNA/genética , Osteossarcoma/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação , Proteína da Polipose Adenomatosa do Colo/deficiência , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina E/metabolismo , DNA/metabolismo , Fase G1 , Expressão Gênica , Instabilidade Genômica , Humanos , Camundongos , Camundongos Knockout , Nocodazol/farmacologia , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Osteossarcoma/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fase S , Estresse Fisiológico , Análise de Sobrevida
4.
Mol Cell ; 63(5): 877-83, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27524497

RESUMO

The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3(+/-) mice were born at sub-Mendelian ratios, and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by the expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.


Assuntos
DNA Polimerase III/deficiência , Replicação do DNA , Haploinsuficiência , Hidrocefalia/genética , Longevidade/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , DNA Polimerase III/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Homozigoto , Hidrocefalia/metabolismo , Hidrocefalia/mortalidade , Hidrocefalia/patologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Fosforilação , Análise de Sobrevida
5.
EMBO Rep ; 17(5): 769-79, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26993089

RESUMO

Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R-spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5-positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non-regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R-spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder-derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride-treated mice. Human gallbladders were also a source of organoid-forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.


Assuntos
Proteínas de Transporte/metabolismo , Vesícula Biliar/citologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Animais , Biomarcadores , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Diferenciação Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Fígado/citologia , Camundongos , Camundongos Transgênicos , Organoides , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Células-Tronco/efeitos dos fármacos , Trombospondinas/genética , Trombospondinas/farmacologia , Transcriptoma
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