RESUMO
Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Antissenso/farmacologia , Cafeína/farmacologia , Códon sem Sentido/análise , Neoplasias do Colo/patologia , Análise Mutacional de DNA , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)(8) mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação da Fase de Leitura , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/genética , Animais , Apoptose/genética , Diferenciação Celular , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.
Assuntos
Apoptose , Neoplasias da Mama/patologia , Proteínas de Neoplasias/biossíntese , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Caspases/biossíntese , Caspases/genética , Proteínas de Ligação a DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genes bcl-2 , Humanos , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Gravidez , Prognóstico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição , Células Tumorais Cultivadas , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Cancers of the microsatellite mutator phenotype (MMP) show exaggerated genomic instability at simple repeat sequences. More than 50 percent (21 out of 41) of human MMP+ colon adenocarcinomas examined were found to have frameshift mutations in a tract of eight deoxyguanosines [(G)8] within BAX, a gene that promotes apoptosis. These mutations were absent in MMP- tumors and were significantly less frequent in (G)8 repeats from other genes. Frameshift mutations were present in both BAX alleles in some MMP+ colon tumor cell lines and in primary tumors. These results suggest that inactivating BAX mutations are selected for during the progression of colorectal MMP+ tumors and that the wild-type BAX gene plays a suppressor role in a p53-independent pathway for colorectal carcinogenesis.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Mutação da Fase de Leitura , Genes Supressores de Tumor , Repetições de Microssatélites/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/genética , Alelos , Apoptose , Sequência de Bases , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Deleção de Sequência , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
A new mutator mechanism for tumorigenesis, characterized by somatic genomic instability (SGI) at simple repeated sequences (SRS) or microsatellites, underlies hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic tumors of the colon and other types. To determine whether the microsatellite mutator phenotype (MMP) is dominant or recessive, we generate somatic cell hybrids between a tumor cell line without SGI at SRS (D98OR) and colon carcinoma cell lines with relative low (HCT-15) and high (LS174-T) SGI at SRS. The normal fidelity of replication of these unstable sequences was observed in each of these cell hybrids. Fusion of HCT-15/DLD-1 low instability cells, with LS174-T, HCT116 and LoVo cell lines, all exhibiting relative high instability, also restored the replication fidelity of SRS in all of the hybrids. Hybrids between the high instability cell lines did not grow possibly because of senescence or apoptosis. These results indicate that, in the cell lines analysed, the characterized mutator phenotype of the mismatch repair system resulting in high SGI at SRS, and the uncharacterized mutator phenotype underlying low SGI at SRS, are both recessive. The results also suggest that different tumor cells of the MMP harbor distinct altered growth-related genes.
Assuntos
Neoplasias do Colo/genética , DNA Satélite/genética , Genes Recessivos , Teste de Complementação Genética , Células Híbridas , Mutação , Fenótipo , Células Tumorais CultivadasRESUMO
RNA fingerprinting by RAP-PCR is a powerful tool for the temporal and spatial analysis of differential gene expression. Many biological situations exist where differential gene expression results in distinguishable phenotypes, including, for example, tissue and cell types, responses to hormones, growth factors, stress, and the heterologous expression of certain genes. There are several methods for detecting differential gene expression and cloning differentially expressed genes that do not rely on a biological assay of phenotype. Most of these methods fall into two general categories: subtractive hybridization and differential screening. RAP-PCR offers numerous advantages over these methods, including its simplicity and its ability to compare the fluctuations in gene expression between multiple samples simultaneously using minute amounts of RNA. In addition, RAP-PCR can yield information on the overall patterns of gene expression between different cell types or between different physiological conditions of the same cell type. Comparison of the RAP-PCR fingerprints from these different experimental groups permits one to draw inferences regarding the overall cellular states of gene expression and the interrelation between gene transcripts belonging to the same or different regulatory pathways. Hypotheses regarding signal transduction pathways can be obtained using this information. RAP-PCR offers applications in cancer research in the detection of tumor-specific alterations in gene expression, providing a bountiful source of tumor markers. The pleiotropic impact of oncogene activation, tumor suppressor gene inactivation, and mutator mutations, in gene regulation, can be readily assessed by RAP-PCR in model systems both in vitro and in vivo.
Assuntos
Impressões Digitais de DNA/métodos , DNA de Neoplasias/química , Técnicas Genéticas , Neoplasias/genética , Proto-Oncogenes , RNA Neoplásico/química , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sequência de Bases , Southern Blotting/métodos , Clonagem Molecular/métodos , DNA/química , Primers do DNA , DNA Polimerase Dirigida por DNA , Eletroforese/métodos , Genes ras , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular , Mutagênese , RNA/química , Valores de Referência , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Taq PolimeraseRESUMO
A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.
Assuntos
Neoplasias Colorretais/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Satélite/genética , Neoplasias do Endométrio/genética , Linhagem Celular , Feminino , Teste de Complementação Genética , Células HeLa , Humanos , Células Tumorais CultivadasRESUMO
Genomic instability at simple repeated sequences (SRS) is a landmark for some sporadic and hereditary cancers of the colon. We have identified several human tumour cell lines with up to 1,000-fold increases in mutation rates for endogenous microsatellite sequences, relative to normal cells or tumour cells without the mutator phenotype and show that they are very early events in tumorigenesis. Our in vivo and in vitro results show that the genomic instability persists after transformation and that microsatellite mutations accumulate as consecutive somatic slippage events of a single or a few repeated units. This mechanism may account for the repeat expansions in triplet hereditary diseases and the same defect in replication fidelity in non-polyposis colon cancer could also contribute to the non-mendelian anticipation in these diseases.
Assuntos
Neoplasias Colorretais/genética , Sequências Repetitivas de Ácido Nucleico , Transformação Celular Neoplásica/genética , DNA de Neoplasias/genética , DNA Satélite/genética , Feminino , Humanos , Masculino , Mutação , Oligodesoxirribonucleotídeos/genética , Fenótipo , Células Tumorais CultivadasAssuntos
Replicação do DNA/genética , Mutação , Neoplasias/etiologia , Sequências Repetitivas de Ácido Nucleico , Neoplasias Colorretais/genética , Impressões Digitais de DNA , DNA de Neoplasias/genética , Genes Recessivos , Humanos , Repetições de Microssatélites , Modelos Genéticos , Neoplasias/genética , Oncogenes , Reação em Cadeia da PolimeraseRESUMO
Spontaneous errors in DNA replication have been suggested to play a significant role in neoplastic transformation and to explain the chromosomal alterations seen in cancer cells. A defective replication factor could increase the mutation rate in clonal variants arising during tumour progression, but despite intensive efforts, increases in tumour cell mutation rates have not been unambiguously shown. Here we use an unbiased genomic fingerprinting technique to show that 12 per cent of colorectal carcinomas carry somatic deletions in poly(dA.dT) sequences and other simple repeats. We estimate that cells from these tumours can carry more than 100,000 such mutations. Only tumours with affected poly(dA.dT) sequences carry mutations in the other simple repeats examined, and such mutations can be found in all neoplastic regions of multiple tumours from the same patient, including adenomas. Tumours with these mutations show distinctive genotypic and phenotypic features. We conclude that these mutations reflect a previously undescribed form of carcinogenesis in the colon (predisposition to which may be inherited) mediated by a mutation in a DNA replication factor resulting in reduced fidelity for replication or repair (a 'mutator mutation').