RESUMEN
All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.
RESUMEN
All-trans-retinoic acid (ATRA) is a physiologically active metabolite of vitamin A. Its antitumour activities have been extensively studied in a variety of model systems and clinical trials; however, to date the only malignancy responsive to ATRA treatment is acute promyelocytic leukaemia (APL) where it induces complete remission in the majority of cases when administered in combination with light chemotherapy and/or arsenic trioxide. After decades of studies, the efficacy of ATRA to treat other acute myeloid leukaemia (AML) subtypes and solid tumours remains poor. Recent studies directed to improve ATRA responsiveness in non-APL AML seem to indicate that the lack of effective ATRA response in these tumours may be primarily due to aberrant epigenetics, which negatively affect ATRA-regulated gene expression and its antileukaemic activity. Epigenetic reprogramming could potentially restore therapeutic effects of ATRA in all AML subtypes. This review discusses the current progresses in the understanding how ATRA can be utilised in the therapy of non-APL AML and other cancers.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Tretinoina/uso terapéutico , Animales , Humanos , Receptores de Ácido Retinoico/fisiología , Tretinoina/farmacologíaRESUMEN
Translocations of the retinoic acid receptor-alpha (RARalpha) locus with the promyelocytic leukemia zinc-finger (PLZF) or PML genes lead to expression of oncogenic PLZF-RARalpha or PML-RARalpha fusion proteins, respectively. These fusion oncoproteins constitutively repress RARalpha target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARalpha have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here, we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (poxvirus and zinc-finger, POZ) and DNA-binding (zinc-finger) domains. In addition, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction of Rb with PML or E2F1, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. These data are supported by chromatin immunoprecipitation analysis, which indicates that PLZF associates with the promoter region of CDC6, a known E2F/Rb target gene. Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F/Rb target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to function in stem cells and taken together these data suggest that interactions between PLZF and Rb could be important in stem cell biology.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Inmunoprecipitación de Cromatina , Humanos , Fosforilación , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Unión Proteica , Transcripción GenéticaRESUMEN
In recent years the study of chemical modifications to chromatin and their effects on cellular processes has become increasingly important in the field of cancer research. Disruptions to the normal epigenetic pattern of the cell can serve as biomarkers and are important determinants of cancer progression. Accordingly, drugs that inhibit the enzymes responsible for modulating these epigenetic markers, in particular histone deacetylases, are the focus of intense research and development. In this chapter we provide an overview of class I and II histone deacetylases as well as a guide to the diverse types of histone deacetylase inhibitors and their activities in the context of APL. We also discuss the rationale for the use of histone deacetylase inhibitors in combination therapy for the treatment of cancer and the current status of clinical trials.
Asunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Leucemia Promielocítica Aguda/tratamiento farmacológico , Animales , Línea Celular Tumoral , Histona Desacetilasas/metabolismo , Humanos , RatonesRESUMEN
Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/genética , Estrógenos/metabolismo , Terapia Genética/métodos , Proteínas Recombinantes de Fusión/uso terapéutico , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel , Luciferasas/genética , Ratones , Ratones Desnudos , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Factores de Transcripción/metabolismo , Transfección/métodos , beta-Galactosidasa/genéticaRESUMEN
AP-1060 is a newly established acute promyelocytic leukemia (APL) cell line from a multiple-relapse patient clinically resistant to both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). The line was initially derived as a granulocyte colony-stimulating factor-dependent strain that underwent replicative senescence and, following ethylnitrosourea treatment, as a phenotypically similar immortalized line. Immortalization was associated with broadened cytokine sensitivity but not growth autonomy, in contrast to three previously derived APL lines. Both the AP-1060 strain and line had shortened telomeres and low telomerase activity, while the line had higher expression of many genes associated with macromolecular synthesis. The karyotype was 46,XY,t(3;14)(p21.1;q11.2),t(15;17)(q22;q11)[100%]; the unique t(3;14) was observed in 4/9 t(15;17)-positive metaphase cells at previous relapse on ATRA therapy. The PML-RARalpha mRNA harbored a missense mutation in the RARalpha-region ligand-binding domain (Pro900Ser). This was associated with a right-shift and sharpening of the ATRA-induced maturation response compared to ATRA-sensitive NB4 cells, which corresponded to the transcriptional activation by PML-RARalphaPro900Ser of a cotransfected ATRA-targeted reporter vector in COS-1 cells. AP-1060 also manifested relative resistance to ATO-induced apoptosis at >/=1 microM, while 0.25 microM ATO stimulated limited atypical maturation. These findings suggest that AP-1060 will be useful for further assessing molecular elements involved in APL progression and drug response/resistance.
Asunto(s)
Línea Celular Tumoral , Resistencia a Antineoplásicos , Leucemia Promielocítica Aguda/patología , Trióxido de Arsénico , Arsenicales/farmacología , Técnicas de Cultivo de Célula/métodos , Citocinas/farmacología , Humanos , Cariotipificación , Mutación Missense , Óxidos/farmacología , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Telomerasa/metabolismo , Telómero/ultraestructura , Tretinoina/farmacologíaRESUMEN
Acute promyelocytic leukemia (APL) has been recognized as a distinct clinical entity for over 40 years. Although relatively rare among hematopoietic malignancies (approximately 10% of AML cases), this disease has attracted a particularly good share of attention by becoming the first human cancer in which all-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, was able to induce complete remission (CR). ATRA induced remission is not associated with rapid cell death, as in the case of conventional chemotherapy, but with a restoration of the 'normal' granulocytic differentiation pathway. With this remarkable medical success story APL has overnight become a paradigm for the differentiation therapy of cancer. A few years later, excitement with APL was further enhanced by the discovery that a cytogenetic marker for this disease, the t(15:17) reciprocal chromosomal translocation, involves a fusion between the retinoic acid receptor alpha (RARalpha) gene and a previously unknown locus named promyelocytic leukemia (PML). Consequence of this gene rearrangement is expression of the PML-RARalpha chimeric oncoprotein, which is responsible for the cellular transformation as well as ATRA response that is observed in APL. Since this initial discovery, a number of different translocation partner genes of RARalpha have been reported in rarer cases of APL, strongly suggesting that disruption of RARalpha underlies its pathogenesis. This article reviews various rearrangements of the RARalpha gene that have so far been described in literature, functions of the proteins encoded by the different RARalpha partner loci, and implications that these may have for the molecular pathogenesis of APL.
Asunto(s)
Leucemia Promielocítica Aguda/genética , Proteínas de la Leche , Receptores de Ácido Retinoico/genética , Translocación Genética/genética , Antígenos Nucleares , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Proteínas de Neoplasias/genética , Proteínas Asociadas a Matriz Nuclear , Proteínas Nucleares/genética , Nucleofosmina , Proteínas de Fusión Oncogénica/genética , Receptor alfa de Ácido Retinoico , Factor de Transcripción STAT5 , Transactivadores/genética , TretinoinaRESUMEN
To better understand the role of retinoids in myelopoiesis, expression of the retinoid receptor genes (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) were examined during differentiation of factor-dependent cell-Paterson (FDCP)-mixA4 murine progenitor cells. The major receptor expressed in undifferentiated A4 cells was RARalpha (primarily the RARalpha1 isoform). Following induction of myelomonocytic differentiation with granulocyte and granulocyte-macrophage colony-stimulating factors, a dramatic increase in RARalpha expression (particularly the RARalpha2 isoform) was seen. In contrast, expression of both RARalpha isoforms was rapidly extinguished upon induction of erythroid differentiation with erythropoeitin (EPO). A modest induction of RXRalpha expression was seen, particularly during differentiation in the myelomonocytic lineage. Low expression levels of RARgamma2 and RXRbeta remained unchanged, irrespective of differentiation pathway. Consistent with the gene expression patterns, RARalpha agonists and antagonists stimulated myelomonocytic and erythroid differentiation of FDCP-mixA4 cells, respectively. Taken together, these results suggest that erythropoiesis and granulopoiesis require diminished and enhanced RARalpha activities, respectively, which at physiological all-trans-retinoic acid (RA) concentrations may be accomplished by reciprocal effects of EPO and myelomonocytic growth factors on its expression. This hypothesis is corroborated by data showing that RA, which positively regulates RARalpha2 expression, can exert inhibitory effects on erythroid differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Regulación de la Expresión Génica , Receptores de Ácido Retinoico/genética , Médula Ósea/fisiología , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Células Cultivadas , Cartilla de ADN , Eritropoyetina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células HL-60 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Leucemia , Modelos Biológicos , Receptores de Ácido Retinoico/efectos de los fármacos , Receptor alfa de Ácido Retinoico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacología , Células Tumorales CultivadasAsunto(s)
Antineoplásicos/uso terapéutico , Apoptosis , Leucemia Promielocítica Aguda/tratamiento farmacológico , Glicoproteínas de Membrana/uso terapéutico , Tretinoina/uso terapéutico , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas Reguladoras de la Apoptosis , Trióxido de Arsénico , Arsenicales/uso terapéutico , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Ligandos , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Óxidos/uso terapéutico , Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Leucemia Promielocítica Aguda/fisiopatología , Proteínas Proto-Oncogénicas , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras/metabolismo , Animales , Núcleo Celular/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico , Hematopoyesis/genética , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/etiología , Leucemia Promielocítica Aguda/genética , Ratones , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Receptores de Ácido Retinoico/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
This meeting formed part of a series of bi-annual conferences, which had its beginnings in Sardinia in 1985, with the goal of promoting the concept of differentiation induction in cancer therapy. As in the past, the organizers of this meeting aimed to bring together both basic and clinical investigators to promote their interaction. Therapeutic successes of all trans-retinoic acid (ATRA), arsenic trioxide and STI-571 have proven the importance of oncogene-targeted therapies in cancer treatment, and underlie the usefulness of such interactions for effective and rational design of therapeutic approaches to combat malignant diseases. Consequently, discussions devoted to the clinical use of the above drugs, as well as to the molecular pathogeneses of the diseases in which these drugs are effective, occupied a considerable part of the congress. Although the clinical application of differentiation therapy still remains confined to acute promyelocytic leukemia (APL), some interesting data have began to emerge in other cancers where agents, such as arsenic trioxide, retinoids and ligands for other nuclear receptors, are showing some promise.
RESUMEN
Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.
Asunto(s)
Transformación Celular Neoplásica/genética , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Translocación Genética/genética , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Humanos , Factores de Transcripción de Tipo Kruppel , Leucemia Promielocítica Aguda/patología , Ratones , Ratones Transgénicos , Mutación/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/patología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transgenes/genética , Tretinoina/farmacologíaRESUMEN
The t(12;21)(p13;q22) chromosomal translocation is the most frequent illegitimate gene recombination in a pediatric cancer and occurs in approximately 25% of common acute lymphoblastic leukemia (cALL) cases. This rearrangement results in the in frame fusion of the 5'-region of the ETS-related gene, TEL (ETV6), to almost the entire acute myeloid leukemia 1 (AML1) (also called CBFA2 or PEBP2AB1) locus and expression of the TEL-AML1 chimeric protein. Although AML1 stimulates transcription, TEL-AML1 functions as a repressor of some AML1 target genes. In contrast to the wild type AML1 protein, both TEL and TEL-AML1 interact with N-CoR, a component of the nuclear receptor corepressor complex with histone deacetylase activity. The interaction between TEL and N-CoR requires the central region of TEL, which is retained in TEL-AML1, and TEL lacking this domain is impaired in transcriptional repression. Taken together, our results suggest that TEL-AML1 may contribute to leukemogenesis by recruiting N-CoR to AML1 target genes and thus imposing an altered pattern of their expression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas , Proteínas Represoras/metabolismo , Factores de Transcripción/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Técnicas de Inmunoadsorción , Proteínas Nucleares/genética , Co-Represor 1 de Receptor Nuclear , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-ets , Proteínas Recombinantes , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Factores de Transcripción/genética , Transfección , Proteína ETS de Variante de Translocación 6RESUMEN
Nuclear receptors (NRs), which include those for steroid and thyroid hormones as well as retinoids, are encoded by a large gene superfamily that has evolved to regulate nearly every facet of metazoan life, from development to basic metabolism. This article reviews the conservation in structure and function of distinct receptors across different species and attempts to draw conclusions as to the evolution of this gene superfamily. Although sequences related to NRs can be found in plants and yeast, gene sequence analyses suggest that the NR ancestor(s) first appeared in the early metazoans and subsequently diversified into the six receptor sub-families, which were already recognisable at the time of the Arthropoda-Chordata split over 700 million years ago. At the time when a primitive NR emerged, the basic components of the transcription regulatory machinery, which are conserved from yeast to vertebrates, were already in place and the ancestral NR must have evolved with the ability to communicate with them. The first such NRs likely acted as monomers and in a ligand-independent fashion. As members of the NR superfamily acquired the ability to hetero- and homodimerise, and to bind and be regulated by ligands, the functional complexity of the NR superfamily increased. This exponentially increasing complexity subsequently provided a potential driving force for evolution of higher organisms by supplying a sophisticated regulatory gene network that could control complex physiological processes during development and in adult organisms.
Asunto(s)
Evolución Molecular , Receptores Citoplasmáticos y Nucleares/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Secuencia Conservada , Amplificación de Genes , Humanos , Ligandos , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Estructura Terciaria de Proteína/genética , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)). PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties. PLZF encodes a transcription factor belonging to the POZ/domain and Krüppel zinc finger (POK) family which interacts directly with PML. PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases. PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex. Given these similarities, we tested whether both proteins could be targeted by each other. Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots. Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other. The colocalization and the reciprocal 'rescue' is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments. In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and 'cross' contacts between the zinc finger region and the POZ domain of each partner. Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast. Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells. These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Linfoma no Hodgkin/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Células CHO , Células COS , Cricetinae , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Humanos , Factores de Transcripción de Tipo Kruppel , Leucemia Promielocítica Aguda/genética , Linfoma no Hodgkin/genética , Sustancias Macromoleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteína de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Proto-Oncogénicas c-bcl-6 , Eliminación de Secuencia , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos HíbridosRESUMEN
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Proteínas Nucleares , Proteínas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Chromosomal translocations are frequently linked to multiple hematological malignancies. The study of the resulting abnormal gene products has led to fundamental advances in the understanding of cancer biology. This is the first report of t(2;15)(p23;q22) and t(2;17)(p23;q21) translocations in human malignancy. Patient 1, a 73-year-old male, was diagnosed with myeloblastic (FAB M1 sub-type) AML. Cytogenetic analysis showed a 47,XY,t(2;15)(p23;q22),+13 karyotype. Fluorescent in situ hybridization (FISH) showed that the PML gene was transferred intact to the short arm of chromosome 2 while the ALK gene on chromosome 2p23 was passively transferred to the long arm of chromosome 15. Patient 2 was a 60-year-old male diagnosed with monocytic (FAB M4-type) AML. Cytogenetic analysis showed 46,XY,t(2;17)(p23;q21) karyotype. FISH analysis showed that neither RARalpha nor ALK were disrupted by the translocation. None of the coding region of the three genes studied were translocated in these patients. This raises the possibilities that other neighboring genes could be involved or that noncoding regulatory sequences of the studied genes could be put in contact and deregulate expression of other genes. Alternatively, displacement of ALK, RARalpha and PML to novel positions could lead to loss of their normal regulation
Asunto(s)
Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 2 , Reordenamiento Génico , Leucemia Mieloide/genética , Enfermedad Aguda , Anciano , Quinasa de Linfoma Anaplásico , Mapeo Cromosómico , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas Receptoras , Translocación GenéticaRESUMEN
The promyelocytic leukemia zinc finger gene (PLZF) is involved in chromosomal translocation t(11;17) associated with acute promyelocytic leukemia. In this work, a 201-kilobase genomic DNA region containing the entire PLZF gene was sequenced. Repeated elements account for 19.83%, and no obvious coding information other than PLZF is present over this region. PLZF contains six exons and five introns, and the exon organization corresponds well with protein domains. There are at least four alternative splicings (AS-I, -II, -III, and -IV) within exon 1. AS-I could be detected in most tissues tested whereas AS-II, -III, and -IV were present in the stomach, testis, and heart, respectively. Although splicing donor and acceptor signals at exon-intron boundaries for AS-I and exons 1-6 were classical (gt-ag), AS-II, -III, and -IV had atypical splicing sites. These alternative splicings, nevertheless, maintained the ORF and may encode isoforms with absence of important functional domains. In mRNA species without AS-I, there is a relatively long 5' UTR of 6.0 kilobases. A TATA box and several transcription factor binding sites were found in the putative promoter region upstream of the transcription start site. PLZF is a well conserved gene from Caenorhabditis elegans to human. PLZF paralogous sequences are found in human genome. The presence of two MLL/PLZF-like alignments on human chromosome 11q23 and 19 suggests a syntenic replication during evolution. The chromosomal breakpoints and joining sites in the index acute promyelocytic leukemia case with t(11;17) also were characterized, which suggests the involvement of DNA damage-repair mechanism.
Asunto(s)
Proteínas de Unión al ADN/genética , Reordenamiento Génico , Factores de Transcripción/genética , Dedos de Zinc/genética , Empalme Alternativo , Secuencia de Bases , Exones , Genes Reguladores , Humanos , Intrones , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Receptores de Ácido Retinoico/genética , Recombinación Genética , Receptor alfa de Ácido Retinoico , TATA BoxRESUMEN
The human PLZF (promyelocytic leukaemia zinc finger) gene encodes a Krüppel-like zinc finger protein, which was identified via the reciprocal translocation t(11;17)(q23;q21) fusing it to the retinoic acid receptor alpha (RARalpha) gene in promyelocytic leukaemia. To determine its complete genomic organisation, we constructed a cosmid-map fully containing the hPLZF gene. The gene has seven exons, including a novel 5' untranslated exon, varying in size from 87 to 1358bp and spans at least 120kb. Flanking intronic sequences were identified and all splice acceptor and donor sites conformed to the gt/ag rule. Five polymorphic markers could be fine located in its vicinity. These data will facilitate mutation analysis of hPLZF in t(11;17) leukaemia cases, as well as assist mapping and loss-of-heterozygosity analysis. Here we have tested hPLZF as a possible candidate for the PGL1 locus involved in hereditary head and neck paragangliomas. However, mutation analysis revealed no aberration in 12 paraganglioma patients from different families.