RESUMO
Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.
Assuntos
Aberrações Cromossômicas , Proteínas do Citoesqueleto/fisiologia , Genes Supressores de Tumor , Proteína da Polipose Adenomatosa do Colo , Animais , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Células Tumorais CultivadasRESUMO
BACKGROUND: In metastatic renal cell cancer (mRCC), the Memorial Sloan-Kettering Cancer Center (MSKCC) risk model is widely used for clinical trial design and patient management. To improve prognostication, we applied proteomics to identify novel serological proteins associated with overall survival (OS). PATIENTS AND METHODS: Sera from 114 mRCC patients were screened by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Identified proteins were related to OS. Three proteins were subsequently validated with enzyme-linked immunosorbent assays and immunoturbidimetry. Prognostic models were statistically bootstrapped to correct for overestimation. RESULTS: SELDI-TOF MS detected 10 proteins associated with OS. Of these, apolipoprotein A2 (ApoA2), serum amyloid alpha (SAA) and transthyretin were validated for their association with OS (P = 5.5 x 10(-9), P = 1.1 x 10(-7) and P = 0.0004, respectively). Combining ApoA2 and SAA yielded a prognostic two-protein signature [Akaike's Information Criteria (AIC) = 732, P = 5.2 x 10(-7)]. Including previously identified prognostic factors, multivariable Cox regression analysis revealed ApoA2, SAA, lactate dehydrogenase, performance status and number of metastasis sites as independent factors for survival. Using these five factors, categorization of patients into three risk groups generated a novel protein-based model predicting patient prognosis (AIC = 713, P = 4.3 x 10(-11)) more robustly than the MSKCC model (AIC = 729, P = 1.3 x 10(-7)). Applying this protein-based model instead of the MSKCC model would have changed the risk group in 38% of the patients. CONCLUSIONS: Proteomics and subsequent validation yielded two novel prognostic markers and survival models which improved prediction of OS in mRCC patients over commonly used risk models. Implementation of these models has the potential to improve current risk stratification, although prospective validation will still be necessary.
Assuntos
Apolipoproteína A-II/sangue , Biomarcadores/sangue , Carcinoma de Células Renais/sangue , Neoplasias Renais/sangue , Proteína Amiloide A Sérica/metabolismo , Idoso , Carcinoma de Células Renais/patologia , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Proteômica , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taxa de SobrevidaRESUMO
Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.
Assuntos
Adenocarcinoma/etiologia , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/etiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição TCF/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Linhagem Celular , Colo/citologia , Colo/metabolismo , Colo/patologia , Pólipos do Colo/genética , Pólipos do Colo/metabolismo , Pólipos do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Células Epiteliais/metabolismo , Eritropoetina/genética , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Rim , Células L , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição TCF/deficiência , Fatores de Transcrição TCF/genética , Fator de Transcrição 4RESUMO
CBP and its homolog p300 are large nuclear molecules that coordinate a variety of transcriptional pathways with chromatin remodeling. They interact with transcriptional activators as well as repressors, direct chromatin-mediated transcription, function in TP53-mediated apoptosis, and participate in terminal differentiation of certain tissue types. Recent evidence suggests that the demand for CBP/p300 is greater than the supply, and that competition for CBP/p300 might play an important role in cell growth regulation. Alterations of the human CBP gene have been implicated in hematological malignancies as well as in congenital malformation and mental retardation. Likewise, the p300 gene has been recently implicated in leukemia and mutations in both alleles have been observed in gastric and colorectal carcinomas. The role of these proteins in human disease coupled with biochemical evidence suggests that CBP and p300 are tumor suppressor proteins essential in cell-cycle control, cellular differentiation and human development.
Assuntos
Proteínas Nucleares/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação a CREB , Divisão Celular , Humanos , Neoplasias/metabolismo , Transcrição GênicaRESUMO
The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16 , Leucemia Mieloide/genética , Doença Aguda , Sequência de Bases , Clonagem Molecular , Subunidade beta de Fator de Ligação ao Core , DNA Complementar , Proteínas de Ligação a DNA/genética , Humanos , Íntrons , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição AP-2 , Fatores de Transcrição/genéticaRESUMO
The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16 , DNA de Neoplasias/genética , Rearranjo Gênico , Leucemia Mielomonocítica Aguda/genética , Cadeias Pesadas de Miosina/genética , Humanos , Cadeias Pesadas de Miosina/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Translocação GenéticaRESUMO
The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 8 , Rearranjo Gênico , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores , Fatores de Transcrição/genética , Translocação Genética , Acetiltransferases/genética , Southern Blotting , Proteína de Ligação a CREB , Histona Acetiltransferases , Humanos , Hibridização in Situ FluorescenteRESUMO
Functional loss of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), which is part of an E3-ubiquitin ligase complex, initiates most inherited and sporadic clear-cell renal cell carcinomas (ccRCC). Genetic inactivation of the TP53 gene in ccRCC is rare, suggesting that an alternate mechanism alleviates the selective pressure for TP53 mutations in ccRCC. Here we use a zebrafish model to describe the functional consequences of pVHL loss on the p53/Mdm2 pathway. We show that p53 is stabilized in the absence of pVHL and becomes hyperstabilized upon DNA damage, which we propose is because of a novel in vivo interaction revealed between human pVHL and a negative regulator of Mdm2, the programmed cell death 5 (PDCD5) protein. PDCD5 is normally localized at the plasma membrane and in the cytoplasm. However, upon hypoxia or loss of pVHL, PDCD5 relocalizes to the nucleus, an event that is coupled to the degradation of Mdm2. Despite the subsequent hyperstabilization and normal transcriptional activity of p53, we find that zebrafish vhl(-/-) cells are still as highly resistant to DNA damage-induced cell cycle arrest and apoptosis as human ccRCC cells. We suggest this is because of a marked increase in expression of birc5a, the zebrafish homolog of Survivin. Accordingly, when we knock down Survivin in human ccRCC cells we are able to restore caspase activity in response to DNA damage. Taken together, our study describes a new mechanism for p53 stabilization through PDCD5 upon hypoxia or pVHL loss, and reveals new clinical potential for the treatment of pathobiological disorders linked to hypoxic stress.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma de Células Renais/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma de Células Renais/patologia , Núcleo Celular/genética , Dano ao DNA/genética , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas de Neoplasias/metabolismo , Proteólise , Survivina , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/antagonistas & inibidores , Proteína Supressora de Tumor Von Hippel-Lindau/biossíntese , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
Rubinstein-Taybi syndrome (RTS) is a multiple congenital anomalies and mental retardation syndrome characterized by facial abnormalities, broad thumbs, and broad big toes. We have shown previously that disruption of the human CREB-binding protein (CBP) gene, either by gross chromosomal rearrangements or by point mutations, leads to RTS. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (approximately 10%). Breakpoints of six translocations and inversions in RTS patients described thus far were found clustered in a 13-kb intronic region at the 5' end of the CBP gene and could theoretically only result in proteins containing the extreme N-terminal region of CBP. In contrast, in one patient with a translocation t(2;16)(q36.3;p13.3) we show by using fiber FISH and Southern blot analysis that the chromosome 16 breakpoint lies about 100 kb downstream of this breakpoint cluster. In this patient, Western blot analysis of extracts prepared from lymphoblasts showed both a normal and an abnormal shorter protein lacking the C-terminal domain, indicating expression of both the normal and the mutant allele. The results suggest that the loss of C-terminal domains of CBP is sufficient to cause RTS. Furthermore, these data indicate the potential utility of Western blot analysis as an inexpensive and fast approach for screening RTS mutations.
Assuntos
Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 2/genética , Síndrome de Rubinstein-Taybi/genética , Translocação Genética , Proteína de Ligação a CREB , Pré-Escolar , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Proteínas Nucleares/genética , Transativadores/genéticaRESUMO
Rabies virus, the gray fox, and the Alleghenian Biotic Region form a pathobiocenose; rabies in Virginia exists in a diffuse nidus, the Alleghenian Biotic Region. Physiographic features may serve as barriers or as passageways for epidemic spread of rabies. Epidemic spread is characterized by a radial pattern and a linear northeast-southwest pattern reflecting physiographic features. Incidence of rabies in the fox varies according to seasons. Present control efforts are generally ineffective; under certain conditions, population reduction for control of rabies may be detrimental instead of beneficial.
Assuntos
Raiva/veterinária , Animais , Quirópteros , Ecologia , Raposas , Geografia , Mephitidae , Raiva/epidemiologia , VirginiaAssuntos
Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação a CREB , Proteína p300 Associada a E1A , Feminino , Viabilidade Fetal/genética , Camundongos , Camundongos Transgênicos , GravidezRESUMO
Inactivation of the tumor suppressor adenomatous polyposis coli (APC) protein is a critical early step in the development of familial and sporadic colon cancer. Close examination of the function of APC has shown that it is a multifunctional protein involved in a wide variety of processes, including regulation of cell proliferation, cell migration, cell adhesion, cytoskeletal reorganization, and chromosomal stability. Tantalizing clues to the different functions of APC have been provided by the identification of proteins interacting with several discrete motifs within APC. Each of these putative functions could link APC inactivation with tumorigenesis. Here, we will summarize recent findings regarding the diverse role of APC. We will emphasize the interaction of APC with different binding partners, the role of these complex interactions for normal functioning of the cell, and how disruption of these interactions may play a role in tumor development. The rapid progress made recently shows the many faces of APC, leading to a constant reappreciation of this multitasking tumor suppressor protein.
Assuntos
Polipose Adenomatosa do Colo/genética , Proteínas do Citoesqueleto/fisiologia , Genes APC , Proteínas de Peixe-Zebra , Proteína da Polipose Adenomatosa do Colo , Adesão Celular , Ciclo Celular , Movimento Celular , Aberrações Cromossômicas , Proteínas do Citoesqueleto/química , Humanos , Modelos Biológicos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Proteínas WntRESUMO
The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two-colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p-arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p-arm breakpoint which is present in 20% of all cases.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16/genética , Hibridização in Situ Fluorescente/métodos , Leucemia Mieloide/diagnóstico , Doença Aguda , Cor , Humanos , Interfase , Leucemia Mieloide/genéticaRESUMO
The pericentric inversion of chromosome 16 and the t(16;16) are two recurrent aberrations in bone marrow of patients with acute nonlymphocytic leukemia subtype M4 Eo, characterized by abnormal eosinophilic granulation. We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread over the short arm of chromosome 16 and the detection, isolation and characterization of a 14Kb EcoRI fragment containing a cluster of breakpoints. First, cosmids were mapped to intervals defined by constitutional 16p rearrangements, second, the inv(16) and t(16;16) breakpoints were mapped to one of the intervals using FISH with the mapped cosmids and third, cosmids within this interval were ordered using two color interphase FISH. An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) breakpoints and one t(16;16) breakpoint analysed. In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint. Since this patient has the same phenotype, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo. This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to the 16q breakpoint. While four of the five YACs showed instability of the region around the inv(16) breakpoint, DNA halo analysis allowed us to identify one YAC which was co-linear with normal genomic DNA and has yielded the actual breakpoint sequences which could be subcloned into cosmids and fosmids.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16/ultraestrutura , Leucemia Mielomonocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Sequência de Bases , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Translocação Genética , Células Tumorais CultivadasRESUMO
In the interest of cloning and analyzing the genes responsible for two very different diseases, the Rubinstein-Taybi syndrome (RTS) and acute myeloid leukemia (AML) associated with the somatic translocation t(8;16)(p11;p13.3), we constructed a high-resolution restriction map of contiguous cosmids (contig) covering 1.2 Mb of chromosome 16p13.3. By fluorescence in situ hybridization and Southern blot analysis, we assigned all tested RTS and t(8;16) translocation breakpoints to a 100-kb region. We have previously reported exact physical locations of these 16p breakpoints, which all disrupt one gene we mapped to this interval: the CREB-binding protein (CBP or CREBBP) gene. Intriguingly, mutations in the CBP gene are responsible for RTS as well as the t(8;16)-associated AML. CBP functions as an integrator in the assembly of various multiprotein regulatory complexes and is thus necessary for transcription in a broad range of transduction pathways. We report here the cloning, physical mapping, characterization, and full cDNA nucleotide sequence of the human CBP gene.
Assuntos
Cromossomos Humanos Par 16/genética , Proteínas Nucleares/genética , Transativadores , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Proteína de Ligação a CREB , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 8 , Clonagem Molecular , Cosmídeos , Primers do DNA/genética , DNA Complementar/genética , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Síndrome de Rubinstein-Taybi/genética , Translocação GenéticaRESUMO
The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.
Assuntos
Proteínas Nucleares/genética , Mutação Puntual , Síndrome de Rubinstein-Taybi/genética , Transativadores , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Proteína de Ligação a CREB , Linhagem Celular Transformada , Passeio de Cromossomo , Cromossomos Humanos Par 16 , Cosmídeos , DNA , Feminino , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Deleção de Sequência , Translocação GenéticaRESUMO
As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/diagnóstico , Miosinas/genética , Proteínas de Neoplasias , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase , Splicing de RNA , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Fatores de Ligação ao Core , Proteínas de Ligação a DNA/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miosinas/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Biossíntese de Proteínas , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
Rubinstein-Taybi syndrome (RTS) is a malformation syndrome characterised by facial abnormalities, broad thumbs, broad big toes, and mental retardation. In a subset of RTS patients, microdeletions, translocations, and inversions involving chromosome band 16p13.3 can be detected. We have previously shown that disruption of the human CREB binding protein (CREBBP or CBP) gene, either by these gross chromosomal rearrangements or by point mutations, leads to RTS. CBP is a large nuclear protein involved in transcription regulation, chromatin remodelling, and the integration of several different signal transduction pathways. Here we report diagnostic analysis of CBP in 194 RTS patients, divided into several subsets. In one case the mother is also suspect of having RTS. Analyses of the entire CBP gene by the protein truncation test showed 4/37 truncating mutations. Two point mutations, one 11 bp deletion, and one mutation affecting the splicing of the second exon were detected by subsequent sequencing. Screening the CBP gene for larger deletions, by using different cosmid probes in FISH, showed 14/171 microdeletions. Using five cosmid probes that contain the entire gene, we found 8/89 microdeletions of which 4/8 were 5' or interstitial. This last subset of microdeletions would not have been detected using the commonly used 3' probe RT1, showing the necessity of using all five probes.