RESUMO
Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids.
Assuntos
Divisão Celular/fisiologia , Microscopia/métodos , Organoides/fisiologia , Células-Tronco/fisiologia , Animais , Técnicas de Cultura de Células/métodos , HumanosRESUMO
Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Mutantes/antagonistas & inibidores , Organoides/efeitos dos fármacos , Proteínas ras/antagonistas & inibidores , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Recombinação GenéticaRESUMO
Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Intestinos/patologia , Mutação/genética , Organoides/metabolismo , Organoides/patologia , Células-Tronco/patologia , Aneuploidia , Animais , Sistemas CRISPR-Cas , Criança , Pré-Escolar , Neoplasias Colorretais/metabolismo , Feminino , Genes APC , Genes p53/genética , Xenoenxertos , Humanos , Imidazóis , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transplante de Neoplasias , Piperazinas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Smad4/deficiência , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismoRESUMO
A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates. These intermediates could lead to recombinogenic events and trigger a persistent ATR-dependent checkpoint signaling. It is now evident that DNA damage signaling is not limited to NER proficient cells. ATR-dependent checkpoint activation also occurs in UV-exposed non-cycling repair deficient cells coinciding with the formation of endonuclease APE1-mediated DNA strand breaks. In addition, the encounter of elongating RNA polymerase II (RNAPIIo) with DNA damage lesions and its persistent stalling provides a strong DNA damage signaling leading to cell cycle arrest, apoptosis and increased mutagenesis. The mechanism underlying the strong and strand specific induction of UV-induced mutations in NER deficient cells has been recently resolved by the finding that gene transcription itself increases UV-induced mutagenesis in a strand specific manner via increased deamination of cytosines. The cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER) without displacement of the DNA damage stalled RNAPIIo. Deficiency in TC-NER associates with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). CSB functions as a repair coupling factor to attract NER proteins, chromatin remodelers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo; CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The molecular mechanisms by which these proteins bring about efficient TC-NER and trigger signaling after transcription arrest remain elusive; particularly the role of chromatin remodeling in TC-NER needs to be clarified in the context of anticipated structural changes that allow repair and transcription restart.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/genética , Instabilidade Genômica , Transcrição Gênica , Montagem e Desmontagem da Cromatina , DNA/metabolismo , DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Distúrbios no Reparo do DNA/genética , Distúrbios no Reparo do DNA/metabolismo , Replicação do DNA , Genoma Humano , Humanos , Mutagênese , Mutação , Fosforilação , Transdução de Sinais , Raios UltravioletaRESUMO
Single-stranded DNA gaps that might arise by futile repair processes can lead to mutagenic events and challenge genome integrity. Nucleotide excision repair (NER) is an evolutionarily conserved repair mechanism, essential for removal of helix-distorting DNA lesions. In the currently prevailing model, NER operates through coordinated assembly of repair factors into pre- and post-incision complexes; however, its regulation in vivo is poorly understood. Notably, the transition from dual incision to repair synthesis should be rigidly synchronized as it might lead to accumulation of unprocessed repair intermediates. We monitored NER regulatory events in vivo using sequential UV irradiations. Under conditions that allow incision yet prevent completion of repair synthesis or ligation, preincision factors can reassociate with new damage sites. In contrast, replication protein A remains at the incomplete NER sites and regulates a feedback loop from completion of DNA repair synthesis to subsequent damage recognition, independently of ATR signaling. Our data reveal an important function for replication protein A in averting further generation of DNA strand breaks that could lead to mutagenic and recombinogenic events.
Assuntos
Reparo do DNA , Proteína de Replicação A/fisiologia , Células Cultivadas , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Fibroblastos/metabolismo , Imunofluorescência , Genoma Humano , Humanos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Histonas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Simples , Dano ao DNA/efeitos da radiação , DNA de Cadeia Simples/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Humanos , Fase de Repouso do Ciclo Celular/fisiologia , Transdução de Sinais/fisiologia , Raios UltravioletaRESUMO
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.
Assuntos
DNA Polimerase III/metabolismo , Reparo do DNA/fisiologia , Proteína de Replicação C/metabolismo , Sítios de Ligação , Linhagem Celular , Citarabina/farmacologia , Dano ao DNA , Replicação do DNA , Recuperação de Fluorescência Após Fotodegradação , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hidroxiureia/farmacologia , Cinética , Modelos Biológicos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação C/antagonistas & inibidores , Proteína de Replicação C/genética , Raios UltravioletaRESUMO
Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Fibroblastos/enzimologia , ATPases Associadas a Diversas Atividades Celulares , Proteínas de Transporte/metabolismo , Linhagem Celular , Senescência Celular , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Fibroblastos/efeitos da radiação , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação C/metabolismo , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases , Ubiquitinação , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2alpha. Similar to HIF-1alpha, HIF-2alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2alpha during hypoxia but did not affect the total level of HIF-2alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2alpha, indicating that sumoylated HIF-2alpha is degraded via SUMO-targeted ubiquitin ligases.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Sequência Consenso , Cisteína Endopeptidases , Endopeptidases/fisiologia , Células HeLa , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Ubiquitina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologiaRESUMO
The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.
Assuntos
Linhagem Celular , Instabilidade Cromossômica , Códon sem Sentido , Cricetulus/genética , Genes BRCA2 , Alelos , Sequência de Aminoácidos , Animais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Centrossomo/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Códon de Terminação , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , Feminino , Heterozigoto , Modelos Genéticos , Dados de Sequência Molecular , Fenótipo , Rad51 Recombinase/metabolismo , Troca de Cromátide IrmãRESUMO
The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability. Four ATP-dependent ligases, designated DNA ligases I-IV, have been identified in higher eukaryotes, and each one has distinct functions. In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining. Recently, an Arabidopsis thaliana orthologue of the yeast and mammalian DNA ligase IV gene was found and termed AtLIG4. Here we describe the isolation and functional characterisation of a plant line with a T-DNA insertion in the AtLIG4 gene. Plants homozygous for the T-DNA insertion did not display any growth or developmental defects and were fertile. However, mutant seedlings were hypersensitive to the DNA-damaging agents methyl methanesulfonate and X-rays, demonstrating that AtLIG4 is required for the repair of DNA damage. Recently, we showed that a yeast lig4 mutant is deficient in Agrobacterium T-DNA integration. However, using tumorigenesis and germline transformation assays, we found that the plant AtLIG4 mutant is not impaired in T-DNA integration. Thus, in contrast to yeast, DNA ligase IV is not required for T-DNA integration in plants.