RESUMO
To date, the NM_002472.2(MYH8):c.2021G>A (p.Arg674Gln) missense variant in the MYH8 gene is the only known genetic change in individuals with autosomal dominant trismus-pseudocamptodactyly syndrome with unknown molecular mechanism. Next-generation sequencing (NGS), including targeted gene panels and whole-exome sequencing, is routinely performed in many clinical diagnostic laboratories as standard-of-care testing aimed at identifying disease-causing genomic variants. Whole-exome sequencing has revealed loss-of-function variants in the MYH8 gene. To properly classify the MYH8 loss-of-function variants, we either retrieved them from public databases or retrospectively collected them from individuals genetically tested by custom NGS panels or by whole-exome sequencing and confirmed using Sanger sequencing. We further evaluated the respective clinical presentations of these individuals with the MYH8 loss-of-function variants. Heterozygous loss-of-function variants in the MYH8 gene were detected in 16 individuals without trismus-pseudocamptodactyly syndrome. Four of these 16 individuals had a pathogenic or likely pathogenic variant detected in another gene that could explain their clinical presentation. Moreover, there are â¼100 MYH8 heterozygous protein-truncating and splice site variants in the ExAC database in different populations. Our results, combined with the population data, indicate that loss-of-function variants in the MYH8 gene do not cause autosomal dominant trismus-pseudocamptodactyly syndrome, and the clinical significance of these variants remains unknown at present. This result highlights the importance of considering the molecular mechanism of disease, variants published in the medical literature, and population genomic data for the correct interpretation of loss-of-function variants in genes associated with autosomal dominant diseases.
Assuntos
Anormalidades Múltiplas/genética , Artrogripose/genética , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Trismo/genética , Anormalidades Múltiplas/diagnóstico , Artrogripose/diagnóstico , Exoma , Testes Genéticos/normas , Heterozigoto , Humanos , Splicing de RNA , Trismo/diagnósticoAssuntos
Centrômero/genética , Duplicação Cromossômica/genética , Cromossomos Humanos Par 11/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Impressão Genômica , Síndrome de Silver-Russell/genética , Criança , Hibridização Genômica Comparativa , Feminino , Humanos , Masculino , Prognóstico , Síndrome de Silver-Russell/patologiaRESUMO
Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.
Assuntos
Instabilidade Cromossômica , Síndromes de Imunodeficiência/genética , Verrugas/genética , Animais , Cromossomos Humanos , Modelos Animais de Doenças , Haploinsuficiência , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mosaicismo , Mutação , Células Mieloides/metabolismo , Doenças da Imunodeficiência Primária , Receptores CXCR4/genética , Remissão EspontâneaRESUMO
Haploinsufficiency of HDAC4 gene has been reported to result in brachydactyly-"mental retardation" syndrome (BDMR), a condition with significant intellectual impairment, brachydactyly type E, and typical facial features. Presented here are three individuals with haploinsufficiency of HDAC4 who have brachydactyly type E, non-dysmorphic facial features, and normal intelligence. This is in contradistinction to previous reports that haploinsufficiency of HDAC4 is sufficient to cause BDMR.
Assuntos
Estudos de Associação Genética , Haploinsuficiência , Histona Desacetilases/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Proteínas Repressoras/genética , Adulto , Deleção Cromossômica , Cromossomos Humanos Par 2 , Hibridização Genômica Comparativa , Feminino , Deformidades Congênitas do Pé , Deformidades Congênitas da Mão , Humanos , Lactente , Masculino , FenótipoRESUMO
BACKGROUND: Carney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating mutations. One-third of the patients, however, have no detectable PRKAR1A coding sequence defects. Small deletions of the gene were previously reported in few patients, but large deletions of the chromosomal PRKAR1A locus have not been studied systematically in a large cohort of patients with CNC. SETTING: A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. METHODS: Methods included genome-wide array analysis followed by fluorescent in situ hybridization, mRNA, and other studies as well as a retrospective analysis of clinical information and phenotype-genotype correlation. RESULTS: We detected 17q24.2-q24.3 deletions of varying size that included the PRKAR1A gene in 11 CNC patients (of 51 tested). Quantitative PCR showed that these patients had significantly lower PRKAR1A mRNA levels. Phenotype varied but was generally severe and included manifestations that are not commonly associated with CNC, presumably due to haploinsufficiency of other genes in addition to PRKAR1A. CONCLUSIONS: A significant number (21.6%) of patients with CNC that are negative in currently available testing may have PRKAR1A haploinsufficiency due to genomic defects that are not detected by Sanger sequencing. Array-based studies are necessary for diagnostic confirmation of these defects and should be done in patients with unusual and severe phenotypes who are PRKAR1A mutation-negative.
Assuntos
Complexo de Carney/genética , Cromossomos Humanos Par 17/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Loci Gênicos/genética , Deleção de Sequência , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Estudos de Associação Genética , Testes Genéticos/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK. RESULTS: In this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5' end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3' end of the ALK signal. Two patients showed a deletion of the 3'ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested. CONCLUSIONS: In a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.
RESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive (AR) neuromuscular disease that is one of the most common lethal genetic disorders in children, with carrier frequencies as high as â¼1 in 35 in US Whites. As part of our genetic studies in the Hutterites from South Dakota, we identified a large 22 Mb run of homozygosity, spanning the SMA locus in an affected child, of which 10 Mb was also homozygous in three affected Hutterites from Montana, supporting a single founder origin for the mutation. We developed a haplotype-based method for identifying carriers of the SMN1 deletion that leveraged existing genome-wide SNP genotype data for â¼1400 Hutterites. In combination with two direct PCR-based assays, we identified 176 carriers of the SMN1 deletion, one asymptomatic homozygous adult and three carriers of a de novo deletion. This corresponds to a carrier frequency of one in eight (12.5%) in the South Dakota Hutterites, representing the highest carrier frequency reported to date for SMA and for an AR disease in the Hutterite population. Lastly, we show that 26 SNPs can be used to predict SMA carrier status in the Hutterites, with 99.86% specificity and 99.71% sensitivity.
Assuntos
Efeito Fundador , Triagem de Portadores Genéticos/métodos , Haplótipos , Atrofia Muscular Espinal/genética , Deleção de Sequência , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Genótipo , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Montana/etnologia , Atrofia Muscular Espinal/epidemiologia , Atrofia Muscular Espinal/etnologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Sensibilidade e Especificidade , South Dakota/etnologia , População Branca/genética , Adulto JovemRESUMO
BACKGROUND: Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy. METHODOLOGY/PRINCIPAL FINDINGS: Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent. CONCLUSIONS/SIGNIFICANCE: Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.
Assuntos
Flavonoides/farmacocinética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Piperidinas/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Animais , Linhagem Celular , Feminino , Flavonoides/administração & dosagem , Flavonoides/metabolismo , Genótipo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Modelos Biológicos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Piperidinas/administração & dosagem , Piperidinas/metabolismo , Polimorfismo de Nucleotídeo Único , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Distribuição Tecidual , UDP-Glucuronosiltransferase 1ARESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is on the rise worldwide. HCC responds poorly to chemotherapy. Lapatinib is an inhibitor of epidermal growth factor receptor and HER2/NEU both implicated in hepatocarcinogenesis. This trial was designed to determine the safety and efficacy of lapatinib in HCC. METHODS: A Fleming phase II design with a single stage of 25 patients with a 90% power to exclude a true response rate of <10% and detect a true response rate of > or =30% was used. The dose of lapatinib was 1,500 mg/day administered orally in 28-day cycles. Tumor and blood specimens were analyzed for expression of HER2/NEU/CEP17 and status of downstream signal pathway proteins. RESULTS: Twenty-six patients with HCC enrolled on this study. Nineteen percent had one prior therapy. Most common toxicities were diarrhea (73%), nausea (54%), and rash (42%). No objective responses were observed. Ten (40%) patients had stable disease as their best response including six (23%) with stable disease lasting >120 days. Median progression-free survival was 1.9 months and median overall survival was 12.6 months. Patients who developed a rash had a borderline statistically significant longer survival. Tissue and blood specimens were available on >90% of patients. No somatic mutations in EGFR (exons 18-21) were found. In contrast to our previous findings, we did not find evidence of HER2/NEU somatic mutations. PTEN, P-AKT, and P70S6K expression did not correlate with survival. CONCLUSIONS: Lapatinib is well-tolerated but seems to benefit only a subgroup of patients for whom predictive molecular or clinical characteristics are not yet fully defined.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Quinazolinas/efeitos adversos , Quinazolinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Humanos , Lapatinib , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Quinazolinas/administração & dosagem , Taxa de SobrevidaRESUMO
BACKGROUND: Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms - mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here 'structural RNA SNPs' (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have used AEI to discover and characterize regulatory polymorphisms in OPRM1, TPH2, MDR1, DRD2, and VKORC1. The objective of this study was to use AEI to determine the extent of cis-regulatory factors in pharmacogenetic genes. METHODS: We applied a rapid and accurate AEI methodology for testing 42 genes implicated in cardiovascular and central nervous system diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. RESULTS: Substantial AEI was observed in approximately 55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in ACE and SOD2 mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that approximately 90% of srSNPs affect mRNA folding, and hence potentially function. CONCLUSION: Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.
Assuntos
Desequilíbrio Alélico , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Transcrição Gênica , Autopsia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Farmacogenética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Variants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability can result from genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc), and proteins relevant to cell survival (e.g., cell cycle, DNA repair, and apoptosis). The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and -sensitivity, and treatment outcomes. METHODS: We have adopted SNPlex for genotyping 432 single nucleotide polymorphisms (SNPs) in 160 candidate genes implicated in response to anticancer chemotherapy. RESULTS: The genotyping panels were applied to 39 patients with chronic lymphocytic leukemia undergoing flavopiridol chemotherapy, and 90 patients with colorectal cancer. 408 SNPs (94%) produced successful genotyping results. Additional genotyping methods were established for polymorphisms undetectable by SNPlex, including multiplexed SNaPshot for CYP2D6 SNPs, and PCR amplification with fluorescently labeled primers for the UGT1A1 promoter (TA)nTAA repeat polymorphism. CONCLUSION: This genotyping panel is useful for supporting clinical anticancer drug trials to identify polymorphisms that contribute to interindividual variability in drug response. Availability of population genetic data across multiple studies has the potential to yield genetic biomarkers for optimizing anticancer therapy.
RESUMO
MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growth-inhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growth-inhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that approximately 30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy.
Assuntos
Antineoplásicos/farmacologia , MicroRNAs/genética , Neoplasias/genética , Neoplasias/patologia , Antineoplásicos/química , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estrutura Molecular , RNA Mensageiro/genética , Regulação para CimaRESUMO
BACKGROUND: Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning." RESULTS: We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation. CONCLUSION: The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation.
Assuntos
Ilhas de CpG/genética , Enzimas de Restrição do DNA/metabolismo , Genoma/genética , Genômica/métodos , Mapeamento por Restrição/métodos , Animais , Biologia Computacional , Metilação de DNA , Eletroforese em Gel Bidimensional , Genoma Humano/genética , Humanos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Especificidade de Órgãos/genéticaRESUMO
The cystine-glutamate transporter SLC7A11 has been implicated in chemoresistance, by supplying cystine to the cell for glutathione maintenance. In the NCI-60 cell panel, SLC7A11 expression shows negative correlation with growth inhibitory potency of geldanamycin but not with its analog 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), which differs in the C-17 substituent in that the the methoxy moiety of geldanamycin is replaced by an amino group. Structure and potency analysis classified 18 geldanamycin analogs into two subgroups, "17-O/H" (C-17 methoxy or unsubstituted) and "17-N" (C-17 amino), showing distinct SLC7A11 correlation. We used three 17-O/H analogs and four 17-N analogs to test the role of the 17-substituents in susceptibility to SLC7A11-mediated resistance. In A549 cells, which are resistant to geldanamycin and strongly express SLC7A11, inhibition of SLC7A11 by (S)-4-carboxyphenylglycine or small interfering RNA increased sensitivity to 17-O/H, but had no effect on 17-N analogs. Ectopic expression of SLC7A11 in HepG2 cells, which are sensitive to geldanamycin and express low SLC7A11, confers resistance to geldanamycin, but not to 17-AAG. Antioxidant N-acetylcysteine, a precursor for glutathione synthesis, completely suppressed cytotoxic effects of 17-O/H but had no effect on 17-N analogs, whereas the prooxidant ascorbic acid had the opposite effect. Compared with 17-AAG, geldanamycin led to significantly more intracellular reactive oxygen species (ROS) production, which was quenched by addition of N-acetylcysteine. We conclude that SLC7A11 confers resistance selectively to 17-O/H (e.g., geldanamycin) but not to 17-N (e.g., 17-AAG) analogs partly as a result of differential dependence on ROS for cytotoxicity. Distinct mechanisms could significantly affect antitumor response and organ toxicity of these compounds in vivo.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Benzoquinonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Lactamas Macrocíclicas/farmacologia , Benzoquinonas/química , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Lactamas Macrocíclicas/química , Relação Estrutura-AtividadeRESUMO
Advances in the understanding of cancer cell biology and response to drug treatment have benefited from new molecular technologies and methods for integrating information from multiple sources. The NCI-60, a panel of 60 diverse human cancer cell lines, has been used by the National Cancer Institute to screen >100,000 chemical compounds and natural product extracts for anticancer activity. The NCI-60 has also been profiled for mRNA and protein expression, mutational status, chromosomal aberrations, and DNA copy number, generating an unparalleled public resource for integrated chemogenomic studies. Recently, microRNAs have been shown to target particular sets of mRNAs, thereby preventing translation or accelerating mRNA turnover. To complement the existing NCI-60 data sets, we have measured expression levels of microRNAs in the NCI-60 and incorporated the resulting data into the CellMiner program package for integrative analysis. Cell line groupings based on microRNA expression were generally consistent with tissue type and with cell line clustering based on mRNA expression. However, mRNA expression seemed to be somewhat more informative for discriminating among tissue types than was microRNA expression. In addition, we found that there does not seem to be a significant correlation between microRNA expression patterns and those of known target transcripts. Comparison of microRNA expression patterns and compound potency patterns showed significant correlations, suggesting that microRNAs may play a role in chemoresistance. Combined with gene expression and other biological data using multivariate analysis, microRNA expression profiles may provide a critical link for understanding mechanisms involved in chemosensitivity and chemoresistance.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Neoplasias/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Análise por Conglomerados , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , RNA Mensageiro/metabolismoRESUMO
PURPOSE: A prerequisite for geldanamycin (GA, NSC122750) to targeting heat shock protein 90 and inhibiting tumor growth is sufficient intracellular drug accumulation. We hypothesized that membrane transporters on tumor cells determine at least in part the response to GA analogues. MATERIALS AND METHODS: To facilitate a systematic study of chemosensitivity across a group of GA analogues with similar chemical structures, we correlated mRNA expression profiles of most known transporters with growth inhibitory potencies of compounds in 60 tumor cell lines (NCI-60). We subsequently validated the gene-drug correlations using cytotoxicity and transport assays. RESULTS: Geldanamycin analogues displayed a range of negative correlations coefficients with ABCB1 (MDR1, or P-glycoprotein) expression. Suppressing ABCB1 in multidrug resistant cells (NCI/ADR-RES and K562/DOX) and ABCB1-transfected cells (BC19) increased sensitivity to GA analogues, as expected for substrates. Moreover, ABCB1-mediated efflux of daunorubicin in K562/DOX cells could be blocked markedly by GA analogues in a dose-dependent fashion. The IC(50) values (half-maximum inhibition of daunorubicin efflux) were 5.5, 7.3 and 12 muM for macbecin II (NSC330500), 17-AAG (NSC330507) and GA, respectively. CONCLUSIONS: These observations demonstrate that GA analogues are substrates as well as inhibitors of ABCB1, suggesting that drug interactions between GA analogues and other agents that are ABCB1 substrates may occur via ABCB1 in normal or tumor cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antibióticos Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Lactamas Macrocíclicas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Células K562 , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Glutathione detoxification has been broadly implicated in resistance to chemotherapy. This study explores the relationship between chemical structure and GSH-mediated chemoresistance. System xc-, the heterodimeric cystine/glutamate exchanger composed of SLC7A11 and SLC3A2, plays a role in maintaining cellular glutathione (GSH) levels. Previous results show that SLC7A11 expression negatively correlates with drug potency across the National Cancer Institute's 60 cell lines for compounds susceptible to GSH-mediated chemoresistance. The number of significant SLC7A11-drug correlations was much greater than those of other genes tested, suggesting that SLC7A11 plays a critical role. Approximately 15% of a curated set of 3045 compounds yielded significant negative SLC7A11 correlations. These compounds tend to contain structural features amenable to GSH reactivity, such as Mannich bases. In cell lines strongly expressing SLC7A11, the potency of selected compounds, was enhanced by inhibition of SLC7A11. This system provides a rapid screen for detecting susceptibility of anticancer drugs to GSH-mediated resistance.
Assuntos
Sistema y+ de Transporte de Aminoácidos/biossíntese , Sistema y+ de Transporte de Aminoácidos/química , Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos , Glutationa/biossíntese , Sistema y+ de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Arsenicais/química , Arsenicais/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Butionina Sulfoximina/química , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Daunorrubicina/química , Daunorrubicina/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Glutationa/antagonistas & inibidores , Glicina/análogos & derivados , Glicina/química , Glicina/farmacologia , Humanos , Bases de Mannich/química , Bases de Mannich/farmacologia , Patulina/química , Patulina/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
PURPOSE: A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. METHODS: Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with 3H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. RESULTS: Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. CONCLUSION: Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.
Assuntos
Hipóxia Celular , Daunorrubicina/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Floretina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Glucose/metabolismo , Transportador de Glucose Tipo 1/análise , Transportador de Glucose Tipo 3/análise , Humanos , Neoplasias/metabolismoRESUMO
The chemotherapeutic agent camptothecin, 10-OH (CPT,10-OH), was shown to act synergistically with the epithelial growth factor receptor (EGFR) inhibitor (AG1478) against several transformed cell lines. To study the cellular response to these drugs, the non-small-cell lung carcinoma cell line, EKVX, was treated with these compounds either alone or in combination. We performed a proteomic analysis using capillary-HPLC coupled with electrospray ion trap mass spectrometry (capillary-LC-ESI/MS) of a tryptic digest to obtain a global protein profile of the EKVX cell line and identify changes in protein expression. The combination of AG1478 and CPT,10-OH showed synergistic cytotoxicity and also changed the expression of multiple proteins, while individual treatments showed a lesser effect on protein expression. Thus, the synergistic action of AG1478 and CPT,10-OH was reflected in altered protein profiles, showing that a proteomic analysis can serve to evaluate chemotherapeutic agents and their combinations.