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1.
Blood Adv ; 3(7): 1039-1046, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30940639

RESUMO

Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

2.
Nat Genet ; 51(2): 296-307, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643249

RESUMO

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.


Assuntos
Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Doença Aguda , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Cromossomos/genética , Feminino , Rearranjo Gênico/genética , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação/genética , Transcriptoma/genética , Adulto Jovem
3.
Cancer Cell ; 33(5): 937-948.e8, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29681510

RESUMO

Somatic genetic alterations of IKZF1, which encodes the lymphoid transcription factor IKAROS, are common in high-risk B-progenitor acute lymphoblastic leukemia (ALL) and are associated with poor prognosis. Such alterations result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding germline IKZF1 variation in familial childhood ALL and 0.9% of presumed sporadic B-ALL, identifying 28 unique variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.


Assuntos
Mutação em Linhagem Germinativa , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animais , Criança , Feminino , Mutação da Fase de Leitura , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Linhagem , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Análise de Sequência de DNA
4.
Clin Cancer Res ; 23(24): 7558-7568, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28974549

RESUMO

Purpose: BCR-ABL+ B-ALL leukemic cells are highly dependent on the expression of endogenous antiapoptotic MCL-1 to promote viability and are resistant to BH3-mimetic agents such as navitoclax (ABT-263) that target BCL-2, BCL-XL, and BCL-W. However, the survival of most normal blood cells and other cell types is also dependent on Mcl-1 Despite the requirement for MCL-1 in these cell types, initial reports of MCL-1-specific BH3-mimetics have not described any overt toxicities associated with single-agent use, but these agents are still early in clinical development. Therefore, we sought to identify approved drugs that could sensitize leukemic cells to ABT-263.Experimental Design: A screen identified dihydroartemisinin (DHA), a water-soluble metabolite of the antimalarial artemisinin. Using mouse and human leukemic cell lines, and primary patient-derived xenografts, the effect of DHA on survival was tested, and mechanistic studies were carried out to discover how DHA functions. We further tested in vitro and in vivo whether combining DHA with ABT-263 could enhance the response of leukemic cells to combination therapy.Results: DHA causes the downmodulation of MCL-1 expression by triggering a cellular stress response that represses translation. The repression of MCL-1 renders leukemic cells highly sensitive to synergistic cell death induced by ABT-263 in a mouse model of BCR-ABL+ B-ALL both in vitro and in vivo Furthermore, DHA synergizes with ABT-263 in human Ph+ ALL cell lines, and primary patient-derived xenografts of Ph+ ALL in culture.Conclusions: Our findings suggest that combining DHA with ABT-263 can improve therapeutic response in BCR-ABL+ B-ALL. Clin Cancer Res; 23(24); 7558-68. ©2017 AACR.


Assuntos
Compostos de Anilina/administração & dosagem , Proteínas de Fusão bcr-abl/genética , Leucemia de Células B/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Sulfonamidas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Artemisininas/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Exp Hematol ; 46: 1-8, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27865806

RESUMO

Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high-risk B-progenitor acute lymphoblastic leukemia (ALL), such as BCR-ABL1-positive (Ph+) and Ph-like ALL, and are associated with poor outcome even in the era of contemporary chemotherapy incorporating tyrosine kinase inhibitors. Recent experimental mouse modeling of B-progenitor ALL has shown that IKZF1 alterations have multiple effects, including arresting differentiation, skewing lineage of leukemia from myeloid to lymphoid, and, in Ph+ leukemia, conferring resistance to tyrosine kinase inhibitor (TKI) therapy without abrogating ABL1 inhibition. These effects are in part mediated by acquisition of an aberrant hematopoietic stem cell-like program accompanied by induction of cell surface expression of stem cell and adhesion molecules that mediate extravascular invasion and residence in the niche and activation of integrin signaling pathways. These effects can be exploited therapeutically using several approaches. IKZF1 alterations also result in upregulation of RXRA that encodes part of the heterodimeric retinoic acid X receptor. Rexinoids, a synthetic class of retinoids that bind specifically to retinoid "X" receptors such as bexarotene potently reverse aberrant adhesion and niche mislocalization in vivo and induce differentiation and cell cycle arrest. Focal adhesion kinase inhibitors block the downstream integrin-mediated signaling, reverse adhesion, and niche mislocalization. Both agents act synergistically with TKIs to prolong survival of Ph+ ALL in mouse and human xenograft model, with long-term remission induced by focal adhesion kinase inhibitors. Therefore, these findings provide important new conceptual insights into the mechanisms by which IKZF1 alterations result in drug resistance and indicate that therapeutic strategies directed against the pathways deregulated by mutation, rather than attempting to restore IKZF1 expression directly, represent promising therapeutic approaches in this disease.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Fator de Transcrição Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Nicho de Células-Tronco , Animais , Adesão Celular , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Leucêmica da Expressão Gênica , Variação Genética , Humanos , Fator de Transcrição Ikaros/antagonistas & inibidores , Fator de Transcrição Ikaros/química , Fator de Transcrição Ikaros/genética , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Transdução de Sinais , Mutações Sintéticas Letais
6.
Nat Genet ; 48(12): 1481-1489, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27776115

RESUMO

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.


Assuntos
Transformação Celular Neoplásica/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Isoformas de Proteínas , Regulador Transcricional ERG/genética , Adulto Jovem
7.
JCI Insight ; 1(4)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27123491

RESUMO

BCR-ABL1+ B progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is an aggressive disease that frequently responds poorly to currently available therapies. Alterations in IKZF1, which encodes the lymphoid transcription factor Ikaros, are present in over 80% of Ph+ ALL and are associated with a stem cell-like phenotype, aberrant adhesion molecule expression and signaling, leukemic cell adhesion to the bone marrow stem cell niche, and poor outcome. Here, we show that FAK1 is upregulated in Ph+ B-ALL with further overexpression in IKZF1-altered cells and that the FAK inhibitor VS-4718 potently inhibits aberrant FAK signaling and leukemic cell adhesion, potentiating responsiveness to tyrosine kinase inhibitors, inducing cure in vivo. Thus, targeting FAK with VS-4718 is an attractive approach to overcome the deleterious effects of FAK overexpression in Ph+ B-ALL, particularly in abrogating the adhesive phenotype induced by Ikaros alterations, and warrants evaluation in clinical trials for Ph+ B-ALL, regardless of IKZF1 status.

8.
Plant Cell ; 27(11): 3065-80, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26546445

RESUMO

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.


Assuntos
Sequência Conservada , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Embriófitas/metabolismo , Família Multigênica , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Fenômenos Biomecânicos , Morte Celular , Proliferação de Células , Embriófitas/genética , Endorreduplicação , Técnicas de Inativação de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Filogenia , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Proteínas de Plantas/genética , Ligação Proteica , Protoplastos/metabolismo , Tricomas/citologia , Tricomas/metabolismo , Tricomas/ultraestrutura
9.
Cancer Cell ; 28(3): 343-56, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26321221

RESUMO

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.


Assuntos
Proteínas de Fusão bcr-abl/genética , Fator de Transcrição Ikaros/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Retinoides/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/genética , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores do Ácido Retinoico/metabolismo
10.
J Exp Med ; 211(4): 701-13, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24687960

RESUMO

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) exhibits lymphoid, myeloid, and stem cell features and is associated with a poor prognosis. Whole genome sequencing of human ETP-ALL cases has identified recurrent mutations in signaling, histone modification, and hematopoietic development genes but it remains to be determined which of these abnormalities are sufficient to initiate leukemia. We show that activating mutations in the interleukin-7 receptor identified in human pediatric ETP-ALL cases are sufficient to generate ETP-ALL in mice transplanted with primitive transduced thymocytes from p19(Arf-/-) mice. The cellular mechanism by which these mutant receptors induce ETP-ALL is the block of thymocyte differentiation at the double negative 2 stage at which myeloid lineage and T lymphocyte developmental potential coexist. Analyses of samples from pediatric ETP-ALL cases and our murine ETP-ALL model show uniformly high levels of LMO2 expression, very low to undetectable levels of BCL11B expression, and a relative lack of activating NOTCH1 mutations. We report that pharmacological blockade of Jak-Stat signaling with ruxolitinib has significant antileukemic activity in this ETP-ALL model. This new murine model recapitulates several important cellular and molecular features of ETP-ALL and should be useful to further define novel therapeutic approaches for this aggressive leukemia.


Assuntos
Células-Tronco Multipotentes/citologia , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores de Interleucina-7/genética , Timócitos/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Criança , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Proteínas com Domínio LIM/metabolismo , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Transplante de Neoplasias , Fenótipo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Cancer Cell ; 22(2): 153-66, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22897847

RESUMO

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.


Assuntos
Predisposição Genética para Doença , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Quinases/genética , Receptores de Citocinas/genética , Transdução de Sinais/genética , Animais , Sequência de Bases , Transformação Celular Neoplásica , Análise Mutacional de DNA , Ativação Enzimática/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Cromossomo Filadélfia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Recidiva , Fatores de Risco , Deleção de Sequência/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética
12.
PLoS Genet ; 7(7): e1002157, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21811412

RESUMO

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19(Arf) is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19(Arf) in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Espermatogônias/metabolismo , Espermatozoides/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Imunofluorescência , Hormônio Foliculoestimulante/sangue , Histonas/metabolismo , Immunoblotting , Hormônio Luteinizante/sangue , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitose , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato , Rad51 Recombinase/metabolismo , Espermatogônias/citologia , Testículo/citologia , Testículo/metabolismo , Proteína Supressora de Tumor p53/metabolismo
13.
Proc Natl Acad Sci U S A ; 106(15): 6285-90, 2009 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-19339492

RESUMO

The Arf tumor suppressor is expressed transiently during mouse male germ cell and eye development. Its inactivation compromises spermatogenesis as mice age and leads to aberrant postnatal proliferation of cells in the vitreous of the eye, resulting in blindness. In the testis, expression of p19(Arf) is limited to spermatogonia but is extinguished completely in spermatocytes, suggesting that Arf plays a physiologic role in setting the balance between mitotic and meiotic germ cell division. A knock-in allele encoding Cre recombinase regulated by the mouse cellular Arf promoter was used to trace Arf gene induction in vivo. Interbreeding to a reporter strain that expresses Cre-dependent YFP provided proof-of-principle that the Arf-Cre allele was appropriately expressed in the male germ cell lineage. However, Cre expression resulted in male sterility, limiting germ line transmission of the knock-in allele to females. Arf-null mice fail to resorb the hyaloid vasculature within the ocular vitreous where pericyte-like cells that express the PDGF-beta receptor (Pdgfrbeta) proliferate aberrantly and destroy the retina and lens. Interbreeding of Arf-Cre females to males containing "floxed" (FL) Arf alleles yielded Arf(Cre/FL) progeny that exhibited variably penetrant defects in visual acuity ranging to total blindness. Crossing the Arf(Cre/FL) alleles onto a Pdgfrbeta(FL/FL) background normalized all histopathology and restored vision fully.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Olho/metabolismo , Genes Reporter/genética , Células Germinativas/metabolismo , Integrases/metabolismo , Animais , Animais Geneticamente Modificados , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Genótipo , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
14.
J Biol Chem ; 282(35): 25588-96, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17599908

RESUMO

The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.


Assuntos
Ciclo Celular/fisiologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Desidratação/metabolismo , Oryza/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Temperatura Baixa , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Desidratação/genética , Mutação de Sentido Incorreto , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Oryza/genética , Propionatos/farmacologia , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido
15.
Plant Cell ; 18(11): 3145-57, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17098811

RESUMO

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Sequência de Aminoácidos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Núcleo Celular/metabolismo , Tamanho Celular , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , DNA de Plantas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/ultraestrutura , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
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