RESUMO
Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of >50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/farmacologia , Proteínas de Drosophila/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Animais , Transformação Celular Neoplásica , Modelos Animais de Doenças , Drosophila/genética , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Camundongos , Proteína de Leucina Linfoide-Mieloide , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Dedos de ZincoRESUMO
The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Análise por Conglomerados , Regulação para Baixo , Histona-Lisina N-Metiltransferase , Humanos , Proteína de Leucina Linfoide-Mieloide , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica , Regulação para CimaRESUMO
Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators, which is required to maintain active states of Hox gene expression during development. We have purified from Drosophila embryos a trithorax acetylation complex (TAC1) that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Fatores de Transcrição , Acetilação , Acetiltransferases/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Proteína de Ligação a CREB , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Cromossomos/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Histona Acetiltransferases , Histonas/metabolismo , Mutação , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Elementos de Resposta , Transativadores/genética , TransgenesRESUMO
Rearrangements of the human ALL-1 gene are frequently encountered in acute lymphocytic leukemias (ALL) and acute myeloid leukemias (AML). These rearrangements are mostly due to chromosome translocations and result in production of chimeric proteins composed of the N-terminal fragment of ALL-1 and the C-terminal segments of the partner proteins. The most common chromosome translocation involving ALL-1 is the t(4 : 11) associated with ALL. ALL-1 is the human homologue of Drosophila trithorax and directly activates transcription of multiple Hox genes. A preliminary DNA microarray screen indicated that the Meis1, HoxA9 and AC133 genes were overexpressed in ALLs with t(4 : 11), compared to ALLs with very similar phenotype but without the chromosomal abnormality. These genes, as well as additional five Hox genes, were subjected to comprehensive semi-quantitative or quantitative RT-PCR analysis in 57 primary ALL and AML tumors. Meis1 and HoxA9 were found expressed in 13/14 of ALLs with the t(4 : 11) and in 8/8 of AMLs with ALL-1 rearrangements. The two genes were not consistently transcribed in other types of ALL. AC133 was transcribed in 13/14 of ALLs with t(4 : 11), but in only 4/8 of AMLs with ALL-1 rearrangements. HoxA10 was expressed in most leukemias with ALL-1 alterations, but was also transcribed in PrePreB CD10(-) ALLs lacking the t(4 : 11). Expression of HoxA5, HoxA7, HoxC8 and HoxC10 did not correlate with ALL-1 rearrangements. Coexpression of Meis1 and HoxA9, overexpression of HoxA10, and overexpression or fusion of HoxA9 were previously implicated in certain acute myeloid leukemias in mice and humans. The present work suggests that upregulation of Meis1, HoxA9, and possibly HoxA10 might also play a role in pathogenesis of acute lymphocytic and acute myeloid leukemias associated with ALL-1 fusions.
Assuntos
Aberrações Cromossômicas/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Transtornos Cromossômicos , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Leucemia Mieloide/genética , Proteína Meis1 , Proteína de Leucina Linfoide-Mieloide , Regulação para CimaRESUMO
During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an approximately 10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.
Assuntos
Núcleo Celular/genética , Proteínas de Ligação a DNA , Proteínas de Drosophila , Junções Íntimas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Drosophila , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Junções Íntimas/metabolismo , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax. The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex. Here we apply yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques to show self association of the SET motifs of ALL-1, TRITHORAX and ASH1 proteins (Drosophila ASH1 is encoded by a trithorax-group gene). Point mutations in evolutionary conserved residues of TRITHORAX SET, abolish the interaction. SET-SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila , Proto-Oncogenes , Fatores de Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cromatina/química , Sequência Conservada , Drosophila , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Mutação Puntual , Testes de PrecipitinaRESUMO
Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila/genética , Drosophila/metabolismo , Genes de Insetos , Proteínas de Homeodomínio/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Drosophila/crescimento & desenvolvimento , Genes Homeobox , Hibridização in Situ Fluorescente , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.
Assuntos
Encéfalo/metabolismo , Cinesinas/metabolismo , Miosinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Transformada , Membrana Celular/metabolismo , Cães , Hipocampo/metabolismo , Humanos , Junções Intercelulares , Cinesinas/genética , Miosinas/genética , Testes de Precipitina , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor EphA7 , Receptor EphB2 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos , Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células da Medula Óssea/metabolismo , Mapeamento Cromossômico , Proteínas de Ligação a DNA/biossíntese , Desoxirribonucleases , Desenvolvimento Embrionário e Fetal , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/química , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Fatores de Transcrição Box Pareados , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Transcrição Gênica , Proteína Homeobox PITX2RESUMO
The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proto-Oncogenes , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Evolução Biológica , Linhagem Celular , Proteínas Cromossômicas não Histona , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Leucemia , Proteína de Leucina Linfoide-Mieloide , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína SMARCB1 , Transfecção , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
Assuntos
Células Epiteliais/fisiologia , Cinesinas/fisiologia , Proteínas de Membrana/fisiologia , Miosinas/fisiologia , Fosfoproteínas/fisiologia , Junções Íntimas/fisiologia , Proteínas ras/fisiologia , Animais , Cálcio/fisiologia , Bovinos , Comunicação Celular , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais/metabolismo , Fibroblastos , Intestinos , Rim , Cinesinas/genética , Cinesinas/metabolismo , Cinesinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Microscopia Imunoeletrônica , Miosinas/genética , Miosinas/metabolismo , Miosinas/ultraestrutura , Ocludina , Células PC12 , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestrutura , Ratos , Proteínas Recombinantes/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1 , alfa Catenina , Proteínas ras/metabolismoRESUMO
The ALL1 gene (also called MLL, HRX, or Htrx1) at the cytogenetic band 11q23 is consistently altered by chromosome rearrangements in acute leukemias (ALs) of early infancy, in ALs developed after exposure to topoisomerase (topo) II-inhibitory drugs, and in a small subset of de novo ALs in children and adults. Because exposure to natural or medicinal substances blocking topo II during pregnancy have been proposed as etiological agents for infant leukemia, we have compared the distribution of ALL1 gene breakpoints in infant leukemias with an altered ALL1 gene configuration to those in secondary leukemia associated with prior exposure to topo II targeting drugs and in reference to the major topo consensus binding site in exon 9. ALL1 gene breakpoint distribution was determined by Southern blot hybridization and/or reverse transcription-PCR of the ALL1/AF4 fusion cDNA in 70 patients. Using restriction enzyme analysis, the 8.3-kb ALL1 breakpoint cluster region was divided in a centromeric portion of 3.5 kb (region A) and telomeric portion of a 4.8 kb (region B). ALL1 breakpoint were located in region A in 8 of 28 (28.5%) cases of infant ALs, 16 of 24 (66%) cases of de novo ALs, and 0 of 5 cases of therapy-related (TR) ALs. Conversely, ALL1 breakpoints in region B were detected in 20 of 28 (71.5%) cases of infant AL, 8 of 24 (33%) cases of de novo AL, and 5 of 5 (100%) cases of TR AL (P = 0.002). These results were confirmed by direct sequencing of the ALL1/AF4 fusion transcript in 30 cases (19 infants and 11 child and adult de novo cases). The analysis of ALL1/AF4 junction types showed that children and adults with de novo leukemia had ALL1 breakpoints in intron 6 (9 cases) or intron 7 (2 cases), whereas breakpoints in infant cases were mainly located in intron 8 (14 cases) and less frequently in intron 6 (4 cases) and intron 7 (1 case). The difference in ALL1 breakpoint location between infant and noninfant AL patients with ALL1/AF4 fusion was statistically significant (P = 0.00005). These data demonstrated that infant and TR ALs share a similar biased clustering of ALL1 gene breakpoints, which supports the possibility that topo II inhibitors may also operate in utero and play a crucial role in the etiology of infant leukemia.
Assuntos
Antineoplásicos/efeitos adversos , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/efeitos adversos , Leucemia/genética , Segunda Neoplasia Primária/genética , Proto-Oncogenes , Inibidores da Topoisomerase II , Fatores de Transcrição , Doença Aguda , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-MieloideRESUMO
The ALL-1 gene is involved in human acute leukemia through chromosome translocations or internal rearrangements. ALL-1 is the human homologue of Drosophila trithorax. The latter is a member of the trithorax group (trx-G) genes which together with the Polycomb group (Pc-G) genes act as positive and negative regulators, respectively, to determine the body structure of Drosophila. We have cloned a novel human gene, ALR, which encodes a gigantic 5262 amino acid long protein containing a SET domain, five PHD fingers, potential zinc fingers, and a very long run of glutamines interrupted by hydrophobic residues, mostly leucine. The SET motif, PDH fingers, zinc fingers and two other regions are most similar to domains of ALL-1 and TRX. The first two motifs are also found in other trx-G and Pc-G proteins. The ALR gene was mapped to chromosome band 12q12-13, adjacent to the VDR gene. This region is involved in duplications and translocations associated with cancer. The analysis of ALR expression showed that its approximately 18 kb long mRNA is expressed, like ALL-1, in most adult tissues, including a variety of hematopoietic cells, with the exception of the liver. Whole mount in situ hybridization to early mouse embryos indicates expression in multiple tissues. Based on similarities in structure and expression pattern, ALR is likely to play a similar role to ALL-1 and trx, although its target genes have yet to be identified.
Assuntos
Cromossomos Humanos Par 12 , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Proteínas de Neoplasias , Proto-Oncogenes , Fatores de Transcrição , Fatores Etários , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase , Humanos , Hibridização In Situ/métodos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Análise de Sequência , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Dedos de ZincoRESUMO
The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.
Assuntos
Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proto-Oncogenes , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Células COS , Núcleo Celular/ultraestrutura , Células HeLa , Histona-Lisina N-Metiltransferase , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Análise de SequênciaRESUMO
In this study we investigated the presence of structural lesions in the ALL-1, p53 and p16 (cyclin-dependent kinase 4 inhibitor) genes in leukaemic cells obtained from 22 patients with infant acute leukaemia (aged < 18 months). Of these, 18 cases were classified as acute lymphoblastic leukaemia (ALL) and four as acute myeloid leukaemia (AML). Tumour DNAs were analysed by a combination of Southern blot. polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequence analyses. The results showed ALL-1 gene rearrangements in 15/22 (68%) cases, p53 gene mutations in 5/22 (26%), and a homozygous deletion of p16 in a single T-ALL case. p53 and p16 alterations were all found in the group of patients with ALL-1 gene rearrangements. p53 mutations were more often associated with a myeloid phenotype (3/5). In summary, multiple molecular alterations were found in 6/15 (40%) infant acute leukaemias with ALL-1 rearrangements. As to the clinical course, patients with additional lesions had similar clinical outcome with respect to patients with ALL-1 gene rearrangement as the sole genetic aberration. This may support the hypothesis that ALL-1 alterations are genetic events per se sufficient to confer a fully malignant phenotype to the leukaemic clone.
Assuntos
Proteínas de Transporte/genética , Genes p53/genética , Leucemia Linfoide/genética , Doença Aguda , Southern Blotting , Inibidor p16 de Quinase Dependente de Ciclina , Éxons , Feminino , Deleção de Genes , Rearranjo Gênico , Genótipo , Homozigoto , Humanos , Lactente , Cariotipagem , Leucemia Mieloide/genética , Masculino , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genéticaRESUMO
The ALL-1 gene is involved in human acute leukemia through chromosome translocations and fusion to partner genes, or through partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which transregulates the homeotic genes of the Antennapedia and bithorax complexes controlling body segment identity. ALL-1 encodes a very large protein of 3968 amino acids which presumably interacts with many proteins. Here we applied yeast two hybrid screening to identify proteins interacting with the N-terminal segment of ALL-1. One protein obtained in this way was the product of the unr gene. This protein consists of multiple repeats homologous to the cold shock domain (CSD), a motif common to some bacterial and eukaryotic nucleic acids-binding proteins. The minimal region on unr required for the interaction with ALL-1 included two CSD and two intervening polypeptides. The interaction was confirmed by in vitro binding studies, and by coimmunoprecipitation from COS cells overexpressing the relevant segments of the two proteins. These results suggest that unr is involved in an interaction of ALL-1 with DNA or RNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proto-Oncogenes , Proteínas de Ligação a RNA , Fatores de Transcrição , Animais , Linfócitos B , Sítios de Ligação , Células COS , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico/metabolismo , Hemaglutininas/genética , Hemaglutininas/imunologia , Histona-Lisina N-Metiltransferase , Humanos , Células Híbridas , Proteína de Leucina Linfoide-Mieloide , Plasmídeos/genética , Plasmídeos/imunologia , Testes de Precipitina , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transcrição Gênica , Transfecção , Leveduras/genéticaRESUMO
The ALL1 gene is found rearranged in approximately 10% of acute lymphoblastic leukemias and in over 5% of acute myeloid leukemias. The gene undergoes fusion with either a variety of partner genes located on different chromosomes or with itself. To further characterize the role of the ALL1 gene in the leukemogenic process, and possibly in solid malignancies, we defined its complete genomic structure. The gene, which spans a region on chromosome band 11q23 approximately 90 kb in length, consists of 36 exons, ranging in size from 65 bp to 4249 bp. The determination of intronic sequences flanking the exon boundaries will allow the determination of whether point mutations may be responsible for inactivation of the gene in solid tumors showing loss of heterozygosity at region 11q23.
Assuntos
Proteínas de Ligação a DNA/genética , Éxons , Leucemia Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Primers do DNA , DNA Complementar , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Mapeamento por Restrição , Dedos de ZincoRESUMO
In this study we used reverse transcriptase-polymerase chain reaction (RT-PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with All-1/AF-4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults. Eleven patients were treated with high-dose intensive induction and consolidation chemotherapy without bone marrow transplantation and one received conservative treatment due to poor performance status. Three had resistant disease, four relapsed within 12 months after achieving complete remission, and five are in continuous complete remission (CCR) at 32, 39, 52, 53 and 61 months from diagnosis, respectively. The sequential analysis of the ALL-1/AF-4 hybrid transcript showed a persistently negative RT-PCR in the five CCR long-term survivors. The PCR analysis resulted persistently positive in the remaining seven cases, including the four cases who relapsed after the achievement of clinical CR. These data emphasize the clinical relevance of PCR monitoring analysis in t(4;11) ALL patients and should be considered in order to better determine variable post-remission treatment according to risk prediction.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas Nucleares/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Fatores de Transcrição , Adulto , Antineoplásicos/uso terapêutico , Sequência de Bases , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Neoplasia Residual , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , DNA Polimerase Dirigida por RNA , Proteínas Recombinantes de Fusão/análise , Estudos Retrospectivos , Fatores de Elongação da Transcrição , Translocação GenéticaRESUMO
The ALL-1 gene is involved in translocations with many partner genes in different types of the acute leukemias, but it is not clear whether it acts as an oncogene or whether the fusion proteins resulting from the translocations have dominant negative effects. To distinguish between these two possibilities, we analyzed the ability of wild-type AB2.1 embryonal stem (ES) cells and of single or double ALL-1 gene knockout cells derived from them to differentiate along hematopoietic lineages after withdrawal of leukemia inhibitory factor, using in vitro colony formation assays. All-1 double knockout ES cells formed a significantly greater number of colonies with faster kinetics than wild-type and ALL-1 single knockout ES cells. Parental ES cells formed lineage-restricted colonies, whereas single and double knockout ES cells developed, at high frequency, immature and/or "biphenotypic" colonies, mimicking the aberrant hematopoiesis typical of leukemic patients. These data are consistent with the possibility that loss of function of the ALL-1 gene is important in leukemogenesis.
Assuntos
Genes Supressores de Tumor/fisiologia , Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Biomarcadores , Ensaio de Unidades Formadoras de Colônias , Genes Supressores de Tumor/genética , Marcadores Genéticos , Globinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Peroxidase/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Translocação Genética , Células Tumorais CultivadasRESUMO
Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of various cell functions such as gene expression and cell proliferation downstream from specific extracellular signals. Here, we partially purified a Ras-interacting protein with molecular mass of about 180 kDa (p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography. This protein bound to the GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).GST-Ha-Ras affinity column but not to those containing GDP.GST-Ha-Ras or GTP gamma S.GST-Ha-Ras with a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP gamma S.GST-Ha-Ras. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP gamma S.GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.