RESUMO
It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).
Assuntos
Cromossomos Humanos Par 17/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Sequência de Aminoácidos , Clonagem Molecular/métodos , DNA Complementar/genética , Efeito Fundador , Ligação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , UtahRESUMO
There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602-1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.
Assuntos
Proteína BRCA1/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sequência de Bases , Genes Reporter , Mutação em Linhagem Germinativa , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A model system has been developed to explore the relationship between cell cycle arrest and chemotherapeutic toxicity. An isopropyl-1-thio-beta-D-galactopyranoside-inducible P16 construct was introduced stably into a melanoma cell line and used to promote G0-G1 arrest in the recipient cells. The state of arrest was reversible and did not compromise cell viability over a period of at least 7 days. Isopropyl-1-thio-beta-D-galactopyranoside-treated, arrested cells were significantly more resistant to the chemotherapeutic agents methotrexate (approximately 50 times), vinblastine (>100 times), and cisplatin (approximately 10 times) compared to controls. This strategy of protection from chemotherapy exploits one of the basic genotypic differences between normal cells and tumor cells: the integrity of genetic pathways that regulate growth.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Transporte/farmacologia , Ciclo Celular , Antineoplásicos/efeitos adversos , Ciclo Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Inibidor p16 de Quinase Dependente de Ciclina , Esquema de Medicação , Humanos , Metotrexato/administração & dosagem , Células Tumorais Cultivadas , Vimblastina/administração & dosagemRESUMO
Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Hominidae/genética , Camundongos/genética , Alinhamento de Sequência , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Inibidores Enzimáticos , Evolução Molecular , Humanos , Dados de Sequência Molecular , Inibidores de Proteínas Quinases , Análise de SequênciaRESUMO
The P15 gene (MTS2) encodes a cyclin-dependent kinase (CDK) inhibitor with considerable sequence identity and biochemical similarity to the CDK inhibitor p16. It is closely linked to the P16 gene (MTS1) and is homozygously deleted in many tumor cell lines. These features suggest that p15 may be a tumor suppressor. We have determined the genomic structure of P15 and examined its pattern of mRNA expression. In addition, we have shown that ectopic expression of p15 inhibits growth of tumor-derived cell lines. We have also searched for P15 mutations in tumor cell lines and in 9p21-linked melanoma kindreds. Other than the previously described homozygous deletions, no mutations of P15 were found. Collectively, these observations suggest a role for p15 in growth regulation, but a limited role for p15 in tumor progression.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Inibidores de Proteínas Quinases , Proteínas Supressoras de Tumor , Sequência de Bases , Proteínas de Transporte/fisiologia , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Humanos , Melanoma/genética , Dados de Sequência Molecular , Mutação , RNA Mensageiro/análiseRESUMO
A critical step in positional cloning is the identification of candidate genes from a large, genetically defined region. Candidate gene isolation by hybrid selection, genomic sequencing, and direct cDNA library screening identified 45 candidate gene fragments (CGFs) from a 600 kb genomic region that contains the BRCA1 gene. These CGFs define a minimum of 15 genes, six of which are newly localized to the BRCA1 region. We present an analysis of the efficiency and the sequences generated for each of these methods. We also compare our CGF set to those reported for the BRCA1 region by three other groups, revealing a surprising lack of overlap among the sets.
Assuntos
Neoplasias da Mama/genética , Clonagem Molecular/métodos , Oncogenes , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , DNA Complementar/genética , Feminino , Genoma Humano , Humanos , Dados de Sequência MolecularRESUMO
The p16 gene (P16, MTS1, CDKN2) encodes a negative regulator of the cell cycle. Molecular genetic techniques have been used to explore the role of p16 in normal development and cancer. Two transcripts derived from the p16 gene with distinct protein coding potentials are described. The previously undescribed transcript form has the same exons 2 and 3 as the p16-encoding mRNA but contains a different exon 1. The human p16 transcripts are detected in various tissues, and the ratio of the transcripts is regulated in both a tissue-specific and cell cycle-specific manner. The P16-derived mRNAs are probably generated from separate promoters, and transcription from one of the promoters appears to be regulated, at least in part, by the retinoblastoma gene product.
