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1.
Nat Cell Biol ; 23(10): 1055-1057, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34616021
2.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
3.
Cancers (Basel) ; 13(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809306

RESUMO

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.

4.
iScience ; 24(2): 102021, 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33426509

RESUMO

The unparalleled global effort to combat the continuing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic over the last year has resulted in promising prophylactic measures. However, a need still exists for cheap, effective therapeutics, and targeting multiple points in the viral life cycle could help tackle the current, as well as future, coronaviruses. Here, we leverage our recently developed, ultra-large-scale in silico screening platform, VirtualFlow, to search for inhibitors that target SARS-CoV-2. In this unprecedented structure-based virtual campaign, we screened roughly 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets. In addition to targeting the active sites of viral enzymes, we also targeted critical auxiliary sites such as functionally important protein-protein interactions.

5.
ChemRxiv ; 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-33200116

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019 novel coronavirus (2019-nCoV), has spread rapidly across the globe, creating an unparalleled global health burden and spurring a deepening economic crisis. As of July 7th, 2020, almost seven months into the outbreak, there are no approved vaccines and few treatments available. Developing drugs that target multiple points in the viral life cycle could serve as a strategy to tackle the current as well as future coronavirus pandemics. Here we leverage the power of our recently developed in silico screening platform, VirtualFlow, to identify inhibitors that target SARS-CoV-2. VirtualFlow is able to efficiently harness the power of computing clusters and cloud-based computing platforms to carry out ultra-large scale virtual screens. In this unprecedented structure-based multi-target virtual screening campaign, we have used VirtualFlow to screen an average of approximately 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets in the cloud. In addition to targeting the active sites of viral enzymes, we also target critical auxiliary sites such as functionally important protein-protein interaction interfaces. This multi-target approach not only increases the likelihood of finding a potent inhibitor, but could also help identify a collection of anti-coronavirus drugs that would retain efficacy in the face of viral mutation. Drugs belonging to different regimen classes could be combined to develop possible combination therapies, and top hits that bind at highly conserved sites would be potential candidates for further development as coronavirus drugs. Here, we present the top 200 in silico hits for each target site. While in-house experimental validation of some of these compounds is currently underway, we want to make this array of potential inhibitor candidates available to researchers worldwide in consideration of the pressing need for fast-tracked drug development.

7.
Cell Res ; 30(11): 997-1008, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32561860

RESUMO

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

8.
Cancer Res ; 79(7): 1297-1298, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936075

RESUMO

DNA replication stress is prevalent in human cancers, but absent in normal cells, suggesting that proteins involved in the cellular response to DNA replication stress could be potential therapeutic targets. SMARCAL1 and ZRANB3 are annealing helicases that mediate the repair of collapsed DNA replication forks. In a study in this issue of Cancer Research, Puccetti and colleagues report that mice lacking either SMARCAL1 or ZRANB3 activity have delayed development of MYC-induced B-cell lymphomas. Thus, inhibiting the response to DNA replication stress could benefit patients with cancer.See related article by Puccetti et al., p. 1612.


Assuntos
DNA Helicases/genética , Replicação do DNA , Animais , Humanos , Camundongos
9.
Nat Protoc ; 14(1): 51-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30487655

RESUMO

A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5' end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2'-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks. Using EdUseq-HU, the spatiotemporal program of DNA replication in human cell lines can be interrogated in <2 weeks. The protocol requires basic cell culture and molecular biology skills, as well as familiarity with the Perl programming language and the Linux operating system.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Química Click/métodos , Replicação do DNA , DNA/genética , Técnicas de Sonda Molecular , Biotina/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Genoma Humano , Células HeLa , Humanos , Hidroxiureia/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Origem de Replicação , Software , Estreptavidina/química
10.
Nature ; 555(7694): 112-116, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29466339

RESUMO

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.


Assuntos
Replicação do DNA , Fase G1/genética , Instabilidade Genômica/genética , Neoplasias/genética , Oncogenes/genética , Origem de Replicação/genética , Fase S/genética , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Estudos de Coortes , Ciclina E/genética , Ciclina E/metabolismo , DNA/biossíntese , DNA/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Humanos , Proteínas Oncogênicas/genética , Transcrição Genética/genética , Translocação Genética/genética
11.
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
12.
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
13.
Front Physiol ; 8: 75, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28239357

RESUMO

Tooth eruption, the process by which teeth emerge from within the alveolar bone into the oral cavity, is poorly understood. The post-emergent phase of tooth eruption continues throughout life, in particular, if teeth are not opposed by antagonists. The aim of the present study was to better understand the molecular processes underlying post-emergent tooth eruption. Toward this goal, we removed the crowns of the maxillary molars on one side of the mouth of 14 young rats and examined gene expression patterns in the periodontal ligaments (PDLs) of the ipsilateral and contralateral mandibular molars, 3 and 15 days later. Nine untreated rats served as controls. Expression of six genes, Adamts18, Ostn, P4ha3, Panx3, Pth1r, and Tnmd, was upregulated in unopposed molars relative to molars with antagonists. These genes function in osteoblast differentiation and proliferation, cell adhesion and collagen metabolism. Proliferation of PDL cells also increased following loss of the antagonist teeth. Interestingly, mutations in PTH1R have been linked to defects in the post-emergent phase of tooth eruption in humans. We conclude that post-emergent eruption of unopposed teeth is associated with gene expression patterns conducive to alveolar bone formation and PDL remodeling.

14.
PLoS One ; 12(1): e0169126, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28046023

RESUMO

DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle.


Assuntos
Sistemas CRISPR-Cas/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Telômero/ultraestrutura , Linhagem Celular Tumoral , Separação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Desoxirribonucleases/metabolismo , Citometria de Fluxo , Fase G1 , Humanos , Plasmídeos/metabolismo
15.
EMBO Mol Med ; 9(1): 46-60, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27940445

RESUMO

The liver has an intrinsic capacity to regenerate in response to injury or surgical resection. Nevertheless, circumstances in which hepatocytes are unresponsive to proliferative signals result in impaired regeneration and hepatic failure. As the Hippo pathway has a canonical role in the maintenance of liver size, we investigated whether it could serve as a therapeutic target to support regeneration. Using a standard two-thirds partial hepatectomy (PH) model in young and aged mice, we demonstrate that the Hippo pathway is modulated across the phases of liver regeneration. The activity of the core kinases MST1 and LATS1 increased during the early hypertrophic phase and returned to steady state levels in the proliferative phase, coinciding with activation of YAP1 target genes and hepatocyte proliferation. Moreover, following PH in aged mice, we demonstrate that Hippo signaling is anomalous in non-regenerating livers. We provide pre-clinical evidence that silencing the Hippo core kinases MST1 and MST2 with siRNA provokes hepatocyte proliferation in quiescent livers and rescues liver regeneration in aged mice following PH. Our data suggest that targeting the Hippo core kinases MST1/2 has therapeutic potential to improve regeneration in non-regenerative disorders.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Regeneração Hepática , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Envelhecimento , Animais , Perfilação da Expressão Gênica , Hepatectomia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Camundongos , Modelos Animais , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores
16.
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
17.
EMBO Rep ; 17(12): 1731-1737, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27760777

RESUMO

Human malignancies overcome replicative senescence either by activating the reverse-transcriptase telomerase or by utilizing a homologous recombination-based mechanism, referred to as alternative lengthening of telomeres (ALT). In budding yeast, ALT exhibits features of break-induced replication (BIR), a repair pathway for one-ended DNA double-strand breaks (DSBs) that requires the non-essential subunit Pol32 of DNA polymerase delta and leads to conservative DNA replication. Here, we examined whether ALT in human cancers also exhibits features of BIR A telomeric fluorescence in situ hybridization protocol involving three consecutive staining steps revealed the presence of conservatively replicated telomeric DNA in telomerase-negative cancer cells. Furthermore, depletion of PolD3 or PolD4, two subunits of human DNA polymerase delta that are essential for BIR, reduced the frequency of conservatively replicated telomeric DNA ends and led to shorter telomeres and chromosome end-to-end fusions. Taken together, these results suggest that BIR is associated with conservative DNA replication in human cells and mediates ALT in cancer.


Assuntos
Reparo do DNA , Replicação do DNA , Neoplasias/genética , Homeostase do Telômero , Quebras de DNA de Cadeia Dupla , DNA Polimerase III/deficiência , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Recombinação Homóloga/genética , Humanos , Hibridização in Situ Fluorescente , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Leveduras/genética , Leveduras/fisiologia
18.
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
19.
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
20.
Cell Cycle ; 14(11): 1748-58, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25695757

RESUMO

The mammalian E3 ubiquitin ligases RNF8 and RNF168 facilitate recruitment of the DNA damage response protein 53BP1 to sites of DNA double-strand breaks (DSBs). The mechanism involves recruitment of RNF8, followed by recruitment of RNF168, which ubiquitinates histones H2A/H2AX on K15. 53BP1 then binds to nucleosomes at sites of DNA DSBs by recognizing, in addition to methyl marks, histone H2A/H2AX ubiquitinated on K15. We report here that expressing H2AX fusion proteins with N-terminal bulky moieties can rescue 53BP1 recruitment to sites of DNA DSBs in cells lacking RNF8 or RNF168 or in cells treated with proteasome inhibitors, in which histone ubiquitination at sites of DNA DSBs is compromised. The rescue required S139 at the C-terminus of the H2AX fusion protein and was occasionally accompanied by partial rescue of ubiquitination at sites of DNA DSBs. We conclude that recruitment of 53BP1 to sites of DNA DSBs is possible in the absence of RNF8 or RNF168, but still dependent on chromatin ubiquitination.


Assuntos
Dano ao DNA/genética , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Ubiquitina/metabolismo , Linhagem Celular , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Immunoblotting , Proteínas Recombinantes de Fusão/metabolismo , Corantes de Rosanilina , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismo
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