RESUMO
Mitosis is the most dramatic--and potentially dangerous--event in the cell cycle, as sister chromatids are irreversibly segregated to daughter cells. Defects in the checkpoints that normally maintain the fidelity of this process can lead to chromosomal instability (CIN) and cancer. However, CIN--a driving force of tumorigenesis--could be the cancer cell's ultimate vulnerability. An important goal is to identify novel anticancer compounds that directly target the mitotic errors at the heart of CIN.
Assuntos
Segregação de Cromossomos/fisiologia , Neoplasias/genética , Proteínas Repressoras , Aneuploidia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação ao Cálcio/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Proteínas Cromossômicas não Histona , Aberrações Cromossômicas , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cromossomos Humanos/genética , Cromossomos Humanos/ultraestrutura , Neoplasias Colorretais/genética , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , Genes APC , Genes cdc , Inibidores do Crescimento/farmacologia , Humanos , Cariotipagem , Proteínas Mad2 , Camundongos , Camundongos Knockout , Mitose/efeitos dos fármacos , Mitose/fisiologia , Modelos Biológicos , Neoplasias/tratamento farmacológico , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Fuso Acromático/ultraestruturaRESUMO
Alterations of oxidative phosphorylation in tumour cells were originally believed to have a causative role in cancerous growth. More recently, mitochondria have again received attention with regards to neoplasia, largely because of their role in apoptosis and other aspects of tumour biology. The mitochondrial genome is particularly susceptible to mutations because of the high level of reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA repair. However, no detailed analysis of mitochondrial DNA in human tumours has yet been reported. In this study, we analysed the complete mtDNA genome of ten human colorectal cancer cell lines by sequencing and found mutations in seven (70%). The majority of mutations were transitions at purines, consistent with an ROS-related derivation. The mutations were somatic, and those evaluated occurred in the primary tumour from which the cell line was derived. Most of the mutations were homoplasmic, indicating that the mutant genome was dominant at the intracellular and intercellular levels. We showed that mitochondria can rapidly become homogeneous in colorectal cancer cells using cell fusions. These findings provide the first examples of homoplasmic mutations in the mtDNA of tumour cells and have potential implications for the abnormal metabolic and apoptotic processes in cancer.
Assuntos
Neoplasias Colorretais/genética , DNA Mitocondrial/genética , DNA de Neoplasias/genética , Genoma Humano , Mutação , Sequência de Bases , Fusão Celular , Neoplasias Colorretais/metabolismo , Dano ao DNA , Humanos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.
Assuntos
Cromossomos Humanos Par 18 , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Transativadores , Proteínas Supressoras de Tumor , Alelos , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Mapeamento Cromossômico , Receptor DCC , Análise Mutacional de DNA , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Receptores de Superfície Celular , Proteína Smad4 , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.
Assuntos
Neoplasias/genética , Animais , Humanos , Modelos GenéticosRESUMO
To gain insights into the molecular basis for metastasis, we compared the global gene expression profile of metastatic colorectal cancer with that of primary cancers, benign colorectal tumors, and normal colorectal epithelium. Among the genes identified, the PRL-3 protein tyrosine phosphatase gene was of particular interest. It was expressed at high levels in each of 18 cancer metastases studied but at lower levels in nonmetastatic tumors and normal colorectal epithelium. In 3 of 12 metastases examined, multiple copies of the PRL-3 gene were found within a small amplicon located at chromosome 8q24.3. These data suggest that the PRL-3 gene is important for colorectal cancer metastasis and provide a new therapeutic target for these intractable lesions.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Proteínas Imediatamente Precoces/genética , Metástase Neoplásica/genética , Proteínas Tirosina Fosfatases/genética , Adenoma/enzimologia , Adenoma/genética , Adenoma/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 8 , Colo/enzimologia , Neoplasias Colorretais/patologia , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mucosa Intestinal/enzimologia , Proteínas de Neoplasias , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases/metabolismo , Reto/enzimologiaRESUMO
To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.
Assuntos
Colo/irrigação sanguínea , Neoplasias Colorretais/irrigação sanguínea , Endotélio Vascular/metabolismo , Perfilação da Expressão Gênica , Neovascularização Patológica/genética , Reto/irrigação sanguínea , Biomarcadores Tumorais , Separação Celular , Células Cultivadas , Colo/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Proteínas da Matriz Extracelular/genética , Feminino , Expressão Gênica , Humanos , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reto/metabolismo , Células Tumorais CultivadasRESUMO
The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.
Assuntos
Neoplasias Colorretais/genética , Reparo do DNA/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Mutação da Fase de Leitura , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 2 , Códon , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Satélite/genética , Marcadores Genéticos , Mutação em Linhagem Germinativa , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, gamma radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.
Assuntos
Ciclinas/fisiologia , Dano ao DNA , Fase G2 , Proteína Supressora de Tumor p53/fisiologia , Apoptose , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Ciclina B/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , DNA/análise , Fase G2/efeitos dos fármacos , Raios gama , Regulação da Expressão Gênica , Genes p53 , Humanos , Mitose , Mutação , Nocodazol/farmacologia , Células Tumorais CultivadasRESUMO
We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.
Assuntos
Antineoplásicos/farmacologia , Genes p53/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias do Colo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/deficiência , Ciclinas/genética , Dano ao DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Deleção de Genes , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo/patologia , Células Tumorais Cultivadas/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Chromosomal instability is believed to be a common feature of most human tumors, but the stage at which such instability originates has not been defined. At the molecular level, chromosomal instability is characterized by allelic imbalance (AI), representing losses or gains of defined chromosomal regions. We have assessed AI in early colorectal tumors using newly developed methods for assessing AI in complex cell populations. A total of 32 adenomas of average size (2 mm; range, 1-3 mm) were studied. AI of chromosome 5q markers occurred in 55% of tumors analyzed, consistent with a gatekeeping role of the adenomatous polyposis coli tumor suppressor gene located at chromosomal position 5q21. AI was also detected in each of the other four chromosomes tested. The fractions of adenomas with AI of chromosomes 1p, 8p, 15q, and 18q were 10,19, 28, and 28%, respectively. Over 90% of the tumors exhibited AI of at least one chromosome, and 67% had allelic imbalance of a chromosome other than 5q. These findings demonstrate that AI is a common event, even in very small tumors, and suggest that chromosomal instability occurs very early during colorectal neoplasia.
Assuntos
Adenoma/genética , Desequilíbrio Alélico , Neoplasias Colorretais/genética , Adenoma/patologia , Neoplasias Colorretais/patologia , Primers do DNA , DNA de Neoplasias/genética , Marcadores Genéticos/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , ProbabilidadeRESUMO
A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.
Assuntos
Deleção Cromossômica , Cromossomos Fúngicos , Cromossomos Humanos , Biblioteca Gênica , Leucemia Mieloide/genética , Leucemia Promielocítica Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes/genética , Translocação Genética , Leveduras/genética , Sequência de Bases , Doença Crônica , Feminino , Humanos , Interfase , Masculino , Metáfase , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
The detailed definition of karyotype changes associated with hyperdiploid acute lymphoblastic leukemia (ALL) is a precondition for their exploitation in minimal residual disease studies with fluorescence in situ hybridization analysis (FISH). In addition, certain karyotype patterns may have different prognostic implications. We have therefore used comparative genomic hybridization (CGH) to analyze the quantitative karyotype abnormalities in 14 cases of hyperdiploid ALL and correlated the results with those obtained by flow cytometry and conventional cytogenetic analyses. Despite an overall good agreement between the karyotypes obtained by classical banding techniques and CGH, we came across at least one karyotype discrepancy per case. Clarification of the discordant findings with fluorescence in situ hybridization (FISH) showed that all stem lines had been correctly defined by CGH. In eight cases, however, cytogenetic analyses revealed structural abnormalities that were undetectable by CGH. The other discrepancies were mainly due to a cytogenetic misinterpretation of similar sized and shaped chromosomes. Based on these findings we present a new diagnostic strategy for childhood ALL that includes flow cytometry and classical cytogenetics as well as CGH for the analysis of aneuploid cases and FISH to resolve the unavoidable discrepancies.
Assuntos
Diploide , Hibridização de Ácido Nucleico/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Criança , Pré-Escolar , Bandeamento Cromossômico , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem , MasculinoRESUMO
The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.
Assuntos
Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Deleção Cromossômica , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Clonagem Molecular/métodos , Mapeamento de Sequências Contíguas , Anormalidades Craniofaciais/genética , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Interphase cytogenetics has been used to detect tumor cells in the presence of a large excess of normal cells. Probes for fluorescence in situ hybridization were chosen to reveal a specific hybridization pattern in tumor cell nuclei as well as to provide an internal control for the assessment of the hybridization results. By enumerating mixtures of cytogenetically normal cells and tumor cells from a Burkitt lymphoma cell line, we were able to detect tumor cells at a frequency of one in 500. Normal cells could be differentiated from Burkitt lymphoma cells with a specificity of approximately 99.9%.
Assuntos
Linfoma de Burkitt/genética , Aberrações Cromossômicas , Hibridização in Situ Fluorescente , Estudos de Avaliação como Assunto , Feminino , Humanos , Interfase , Células Tumorais CultivadasRESUMO
A patient with acute nonlymphocytic leukemia (ANLL), M5b according to French-American-British (FAB) classification, showed monosomy 16, an extra 1p-, and a 21q+. These derivative chromosomes could not be defined by GTG-banding. For better characterization, we performed two-color fluorescence in situ hybridization (FISH) experiments applying DNA libraries from sorted human chromosomes, chromosome-specific repetitive probes, and a band-specific YAC-clone. With these FISH studies the karyotype could be characterized as 46,XY, +der(1)t(1;21)(p11;?), -16,der(21)t(16;21) (p11.1;q22).
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 21 , Leucemia Mieloide Aguda/genética , Adulto , Bandeamento Cromossômico , Cromossomos Artificiais de Levedura , Sondas de DNA , Biblioteca Gênica , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Monossomia , Translocação GenéticaAssuntos
Sondas de DNA , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Cromossomos Humanos , Sequência Conservada , Sondas de DNA/genética , DNA Satélite/genética , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoAssuntos
Cromossomos Artificiais de Levedura/genética , Hibridização in Situ Fluorescente/métodos , Neoplasias/genética , Sequência de Bases , Aberrações Cromossômicas , Clonagem Molecular , Citogenética , DNA/genética , Primers do DNA/genética , Sondas de DNA , Amplificação de Genes , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodosRESUMO
Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence - polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.
Assuntos
Cromossomos Humanos , Sondas de DNA , Sequências Repetitivas de Ácido Nucleico , Animais , Linhagem Celular , Cricetinae , Humanos , Células Híbridas , Cariotipagem , Camundongos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Coloração e RotulagemRESUMO
S phase entry depends on cyclin-dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits. We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication. The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1. The former is due to Swi5, while the latter is due to MBF or SBF transcription factors. Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly. Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents. This 'reductional' anaphase occurs with almost wild-type kinetics and depends on the activity of G2 cyclins. Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects. We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the 'reductional' anaphase.