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1.
Genes Dev ; 34(3-4): 179-193, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31879358

RESUMO

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.


Assuntos
Neoplasias da Mama/fisiopatologia , Carcinogênese/genética , Diferenciação Celular/genética , Células Epiteliais/citologia , Proteínas Repressoras/metabolismo , Animais , Neoplasias da Mama/genética , Caderinas/genética , Sobrevivência Celular/genética , Cromatina/genética , Cromatina/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Ligação Proteica/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética
2.
Proc Natl Acad Sci U S A ; 120(4): e2216055120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669105

RESUMO

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.


Assuntos
Dano ao DNA , Animais , Reparo do DNA , Replicação do DNA , Células-Tronco Hematopoéticas/metabolismo , Mamíferos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitinação
3.
EMBO J ; 39(5): e102169, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31930530

RESUMO

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.


Assuntos
Neoplasias da Mama/genética , Sistemas CRISPR-Cas , Edição de Genes , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Mutação
4.
Blood ; 139(16): 2483-2498, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35020836

RESUMO

NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/genética , Humanos , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética
5.
Genes Dev ; 30(12): 1470-80, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27340177

RESUMO

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinoma Lobular/genética , Carcinoma Lobular/fisiopatologia , Edição de Genes , Glândulas Mamárias Humanas/fisiopatologia , Animais , Sistemas CRISPR-Cas , Caderinas/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Genes Supressores de Tumor , Humanos , Camundongos
6.
Br J Cancer ; 118(12): 1586-1595, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29736010

RESUMO

BACKGROUND: Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. METHODS: To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1-/-;TP53-/- mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. RESULTS: The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. CONCLUSIONS: Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Docetaxel/farmacologia , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Docetaxel/administração & dosagem , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Camundongos , Mitose/efeitos dos fármacos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Paclitaxel/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nucleic Acids Res ; 44(10): 4734-44, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26926109

RESUMO

PrimPol is a DNA damage tolerant polymerase displaying both translesion synthesis (TLS) and (re)-priming properties. This led us to study the consequences of a PrimPol deficiency in tolerating mutagenic lesions induced by members of the APOBEC/AID family of cytosine deaminases. Interestingly, during somatic hypermutation, PrimPol counteracts the generation of C>G transversions on the leading strand. Independently, mutation analyses in human invasive breast cancer confirmed a pro-mutagenic activity of APOBEC3B and revealed a genome-wide anti-mutagenic activity of PRIMPOL as well as most Y-family TLS polymerases. PRIMPOL especially prevents APOBEC3B targeted cytosine mutations within TpC dinucleotides. As C transversions induced by APOBEC/AID family members depend on the formation of AP-sites, we propose that PrimPol reprimes preferentially downstream of AP-sites on the leading strand, to prohibit error-prone TLS and simultaneously stimulate error-free homology directed repair. These in vivo studies are the first demonstrating a critical anti-mutagenic activity of PrimPol in genome maintenance.


Assuntos
Citidina Desaminase/metabolismo , DNA Primase/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Antígenos de Histocompatibilidade Menor/metabolismo , Enzimas Multifuncionais/fisiologia , Mutagênese , Animais , Linfócitos B/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Sistemas CRISPR-Cas , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Citidina Desaminase/antagonistas & inibidores , DNA/metabolismo , Replicação do DNA , Feminino , Humanos , Switching de Imunoglobulina , Camundongos Endogâmicos C57BL , Hipermutação Somática de Imunoglobulina , Linfócitos T/enzimologia , Raios Ultravioleta
8.
Proc Natl Acad Sci U S A ; 112(27): 8409-14, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26100884

RESUMO

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.


Assuntos
Proteína BRCA1/deficiência , Neoplasias Mamárias Experimentais/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Proteína BRCA1/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinossarcoma/tratamento farmacológico , Carcinossarcoma/genética , Carcinossarcoma/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/metabolismo , Proto-Oncogene Mas , Análise de Sobrevida
9.
Blood ; 120(7): 1516-27, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22740442

RESUMO

Blood vessel networks form in a 2-step process of sprouting angiogenesis followed by selective branch regression and stabilization of remaining vessels. Pericytes are known to function in stabilizing blood vessels, but their role in vascular sprouting and selective vessel regression is poorly understood. The endosialin (CD248) receptor is expressed by pericytes associated with newly forming but not stable quiescent vessels. In the present study, we used the Endosialin(-/-) mouse as a means to uncover novel roles for pericytes during the process of vascular network formation. We demonstrate in a postnatal retina model that Endosialin(-/-) mice have normal vascular sprouting but are defective in selective vessel regression, leading to increased vessel density. Examination of the Endosialin(-/-) mouse tumor vasculature revealed an equivalent phenotype, indicating that pericytes perform a hitherto unidentified function to promote vessel destabilization and regression in vivo in both physiologic and pathologic angiogenesis. Mechanistically, Endosialin(-/-) mice have no defect in pericyte recruitment. Rather, endosialin binding to an endothelial associated, but not a pericyte associated, basement membrane component induces endothelial cell apoptosis and detachment. The results of the present study advance our understanding of pericyte biology and pericyte/endothelial cell cooperation during vascular patterning and have implications for the design of both pro- and antiangiogenic therapies.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/patologia , Padronização Corporal , Neovascularização Fisiológica , Pericitos/patologia , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Aorta/crescimento & desenvolvimento , Aorta/patologia , Apoptose , Membrana Basal/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Pericitos/metabolismo , Ratos , Retina/metabolismo , Retina/patologia , Vasos Retinianos/crescimento & desenvolvimento , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Transgenic Res ; 23(4): 691-5, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24798251

RESUMO

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.


Assuntos
Coeficiente de Natalidade , Implantação do Embrião , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Gravidez/estatística & dados numéricos , Animais , Blastocisto , Feminino , Camundongos
11.
J Immunol ; 188(1): 111-21, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22140254

RESUMO

Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vß usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Tolerância Imunológica , Neoplasias/imunologia , Peptídeos/imunologia , Animais , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Epitopos/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Peptídeos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
12.
Bioessays ; 33(9): 701-10, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21735458

RESUMO

Recent technological advances have opened the door for the fast and cost-effective generation of genetically engineered mouse models (GEMMs) to study cancer. We describe here a conceptually novel approach for the generation of chimeric GEMMs based on the controlled introduction of various genetic elements in embryonic stem cells (ESCs) that are derived from existing mouse strains with a predisposition for cancer. The isolation of GEMM-derived ESC lines is greatly facilitated by the availability of the newly defined culture media containing inhibitors that effectively preserve ESC pluripotency. The feasibility of the GEMM-ESC approach is discussed in light of current literature and placed into the context of existing models. This approach will allow for fast and flexible validation of candidate cancer genes and drug targets and will result in a repository of GEMM-ESC lines and corresponding vector collections that enable easy distribution and use of preclinical models to the wider scientific community.


Assuntos
Quimera/genética , Modelos Animais de Doenças , Genes Neoplásicos , Camundongos , Neoplasias/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Animais Geneticamente Modificados , Quimera/metabolismo , Meios de Cultura , Sistemas de Liberação de Medicamentos , Células-Tronco Embrionárias/metabolismo , Humanos
13.
Sci Rep ; 13(1): 338, 2023 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611064

RESUMO

Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin binding protein with deubiquitinase (DUB) catalytic activity. Rare MYSM1 mutations in human patients result in an inherited bone marrow failure syndrome, highlighting the biomedical significance of MYSM1 in the hematopoietic system. We and others characterized Mysm1-knockout mice as a model of this disorder and established that MYSM1 regulates hematopoietic function and leukocyte development in such models through different mechanisms. It is, however, unknown whether the DUB catalytic activity of MYSM1 is universally required for its many functions and for the maintenance of hematopoiesis in vivo. To test this, here we generated a new mouse strain carrying a Mysm1D660N point mutation (Mysm1DN) and demonstrated that the mutation renders MYSM1 protein catalytically inactive. We characterized Mysm1DN/DN and Mysm1fl/DN CreERT2 mice, against appropriate controls, for constitutive and inducible loss of MYSM1 catalytic function. We report a profound similarity in the developmental, hematopoietic, and immune phenotypes resulting from the loss of MYSM1 catalytic function and the full loss of MYSM1 protein. Overall, our work for the first time establishes the critical role of MYSM1 DUB catalytic activity in vivo in hematopoiesis, leukocyte development, and other aspects of mammalian physiology.


Assuntos
Endopeptidases , Proteases Específicas de Ubiquitina , Humanos , Camundongos , Animais , Endopeptidases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Diferenciação Celular , Hematopoese/genética , Mutação , Células-Tronco Hematopoéticas/metabolismo , Camundongos Knockout , Mamíferos/metabolismo , Transativadores/metabolismo
14.
NPJ Parkinsons Dis ; 9(1): 6, 2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681683

RESUMO

Glucose metabolism is dysregulated in Parkinson's disease (PD) causing a shift toward the metabolism of lipids. Carnitine palmitoyl-transferase 1A (CPT1A) regulates the key step in the metabolism of long-chain fatty acids. The aim of this study is to evaluate the effect of downregulating CPT1, either genetically with a Cpt1a P479L mutation or medicinally on PD using chronic rotenone mouse models using C57Bl/6J and Park2 knockout mice. We show that Cpt1a P479L mutant mice are resistant to rotenone-induced PD, and that inhibition of CPT1 is capable of restoring neurological function, normal glucose metabolism, and alleviate markers of PD in the midbrain. Furthermore, we show that downregulation of lipid metabolism via CPT1 alleviates pathological motor and non-motor behavior, oxidative stress, and disrupted glucose homeostasis in Park2 knockout mice. Finally, we confirm that rotenone induces gut dysbiosis in C57Bl/6J and, for the first time, in Park2 knockout mice. We show that this dysbiosis is alleviated by the downregulation of the lipid metabolism via CPT1.

15.
PLoS Genet ; 5(9): e1000666, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19779552

RESUMO

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed.


Assuntos
Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/genética , Bovinos/genética , Surtos de Doenças , Mutação da Fase de Leitura/genética , Glicoproteínas de Membrana/genética , Seleção Genética , Animais , Pareamento de Bases/genética , Sequência de Bases , Bélgica/epidemiologia , Códon sem Sentido/genética , Simulação por Computador , Regulação da Expressão Gênica , Heterozigoto , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Tamanho do Órgão , Especificidade de Órgãos , Penetrância , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismo , Deleção de Sequência
16.
Sci Rep ; 12(1): 9606, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35688932

RESUMO

Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Edição de Genes/métodos , Camundongos , RNA Guia de Cinetoplastídeos/genética
17.
Cancer Res Commun ; 2(10): 1266-1281, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36467895

RESUMO

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.


Assuntos
Cisplatino , Platina , Camundongos , Animais , Cisplatino/farmacologia , Carboplatina/farmacologia , Platina/metabolismo , Proteína Supressora de Tumor p53/genética , Estudos Prospectivos , Proteínas de Membrana/genética , Ânions/metabolismo
18.
Cancer Res ; 81(24): 6171-6182, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34548335

RESUMO

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.


Assuntos
Proteína BRCA1/fisiologia , Proteína do Grupo de Complementação N da Anemia de Fanconi/fisiologia , Regulação Neoplásica da Expressão Gênica , Recombinação Homóloga , Neoplasias Mamárias Animais/patologia , Reparo de DNA por Recombinação , Animais , Apoptose , Proteína BRCA2/fisiologia , Proliferação de Células , Feminino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia
19.
Commun Biol ; 3(1): 273, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472011

RESUMO

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant 'Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8+ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Genes Reporter/imunologia , Transgenes/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos
20.
Cell Rep ; 33(13): 108533, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33378683

RESUMO

Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.


Assuntos
Enzimas Desubiquitinantes/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Leucemia/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteases Específicas de Ubiquitina/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Dano ao DNA , Reparo do DNA , Hematopoese , Células-Tronco Hematopoéticas/enzimologia , Humanos , Células K562 , Leucemia/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitinação
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