RESUMEN
FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Predisposición Genética a la Enfermedad , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Transgenes , Animales , Secuencia de Bases , Western Blotting , Carcinógenos/toxicidad , Metilación de ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Cartilla de ADN , Femenino , Inhibidores de Histona Desacetilasas , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hemizygous deletions of the fragile histidine triad (FHIT) gene at human chromosome band 3p14.2 and down-regulation of its gene product are found in the majority of renal cell carcinomas (RCCs). Functional tumor suppressive activity of Fhit in renal cancer cells previously was observed in RCC cell line RC48, which lacks endogenous Fhit expression. To further investigate the potential role of FHIT as a tumor suppressor gene in RCC, we transfected FHIT cDNA expression constructs into RCC cell lines RCC-1 and SN12C, which show low-level expression of endogenous Fhit and reveal an intact von Hippel-Lindau (VHL) gene. Stable transfectants of both cell lines showed no alterations of cell morphology, proliferation kinetics, or cell cycle parameters in vitro. The FHIT gene transfer rate, however, was significantly lower in RCC-1 cells compared with SN12C cells, suggesting a selection against exogenous Fhit expression. In addition, in nude mouse assays, a significant delay of tumor formation was observed for FHIT-transfected RCC-1 cell lines, with outgrowing tumors demonstrating loss of Fhit expression in the majority of cells. In contrast, tumorigenicity of FHIT-transfected SN12C cell clones was not suppressed, despite stable transgene expression. In conclusion, our results demonstrate a selective tumor suppressive activity of Fhit in RCC cells in vivo and suggest that the susceptibility to suppression is not restricted to cancer cells with complete loss of Fhit expression.
Asunto(s)
Ácido Anhídrido Hidrolasas , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteínas de Neoplasias , Proteínas/genética , Proteínas/metabolismo , Supresión Genética , Animales , Western Blotting , Ciclo Celular/genética , Cromosomas Humanos Par 3 , ADN Complementario/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transfección , Células Tumorales CultivadasRESUMEN
To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.
Asunto(s)
Ácido Anhídrido Hidrolasas , Neoplasias Primarias Múltiples/genética , Proteínas/genética , Adenoma/genética , Animales , Carcinógenos , Dimetilnitrosamina/análogos & derivados , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Neoplasias Primarias Múltiples/inducido químicamente , Neoplasias Primarias Múltiples/patología , Papiloma/genética , Proteínas/metabolismo , Mapeo Restrictivo , Neoplasias de las Glándulas Sebáceas/genética , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , SíndromeRESUMEN
The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.
Asunto(s)
Ácido Anhídrido Hidrolasas , Aminohidrolasas/genética , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Proteínas de Neoplasias , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.
Asunto(s)
Ácido Anhídrido Hidrolasas , Adenocarcinoma de Células Claras/metabolismo , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patología , Adulto , Anciano , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Ratones , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas/genéticaRESUMEN
Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.
Asunto(s)
Ácido Anhídrido Hidrolasas , Proteínas de Neoplasias , Proteínas/química , Animales , Cristalografía por Rayos X , Dimerización , Fosfatos de Dinucleósidos/metabolismo , Humanos , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , Electricidad EstáticaRESUMEN
The FHIT gene, which encodes a 1-kb message and a 16.8-kDa protein that hydrolyses diadenosine triphosphate (ApppA) to ADP and AMP in vitro, covers a megabase genomic region at chromosome band 3p14.2. The gene encompasses the most active of the common human chromosomal fragile regions, FRA3B. Over the years, it has been suggested that fragile sites might be especially susceptible to carcinogen damage and that chromosomal regions of nonrandom alterations in cancer cells may coincide with defined fragile sites. Within the FRA3B region, the characteristic induced chromosome gaps can occur across the entire region, but 60% of the gaps are centered on a 300-kb region flanking FHIT exon 5, the first protein-coding exon. Numerous hemizygous and homozygous deletions, translocations and DNA insertions occur within FHIT in cancer cell lines, uncultured tumors, and even in preneoplastic lesions, especially in tissues such as lung that are targets of carcinogens. This supports the proposed cancer-fragile site connection and suggests that the FHIT gene, expression of which is frequently altered in cells showing FHIT locus damage, is a tumor suppressor gene whose inactivation may drive clonal expansion of preneoplastic and neoplastic cells. Replacement of Fhit expression in Fhit-negative cancer cells abrogates their tumorigenicity in nude mice. Analysis of the approximately 300-kb DNA sequence encompassing FHIT exon 5 in the FRA3B epicenter has provided clues to the mechanism of repair of the fragile site double strand breaks. The mechanism involves recombination between LINE 1 elements with deletion of the intervening sequence, often including FHIT exons. These studies have also shown that FHIT alterations generally entail independent deletion of both FHIT alleles. Future studies will focus on two objectives: study of (1) the in vivo function of the Fhit protein and (2) mechanisms of break and repair in the FRA3B fragile region.
Asunto(s)
Ácido Anhídrido Hidrolasas , Pérdida de Heterocigocidad , Proteínas de Neoplasias , Neoplasias/etiología , Proteínas/genética , Secuencia de Aminoácidos , Animales , Sitios Frágiles del Cromosoma , Fragilidad Cromosómica , Humanos , Ratones , Datos de Secuencia Molecular , Neoplasias/genéticaRESUMEN
The FHIT gene, encoded by 10 exons in a 1.1-kb transcript, encompasses approximately 1 Mb of genomic DNA, which includes the hereditary RCC t(3;8) translocation break at 3p14.2, the FRA3B common fragile region, and homozygous deletions in various cancer-derived cell lines. Because some of these genetic landmarks (e.g., the t(3;8) break between untranslated FHIT exons 3 and 4, a major fragile region that includes a viral integration site between exons 4 and 5, and cancer cell homozygous deletions in intron 5) do not necessarily affect coding exons and yet apparently affect expression of the gene product, we examined the FHIT locus and its expression in detail in more than 10 tumor-derived cell lines to clarify mechanisms underlying aberrant expression. We observed some cell lines with apparently continuous large homozygous deletions, which included one or more coding exons; cell lines with discontinuous deletions, some of which included or excluded coding exons; and cell lines that exhibited heterozygous and/or homozygous deletions, by Southern blot analysis for the presence of specific exons. Most of the cell lines that exhibited genomic alterations showed alteration of FHIT transcripts and absence or diminution of Fhit protein.
Asunto(s)
Ácido Anhídrido Hidrolasas , Proteínas de Neoplasias , Neoplasias/genética , Proteínas/genética , Secuencia de Bases , Southern Blotting , Exones , Eliminación de Gen , Expresión Génica , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proteínas/análisis , Células Tumorales CultivadasRESUMEN
The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5',5"'-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase "dead" Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5',5"'-P1, P3-triphosphate hydrolysis is not required for tumor suppression.
Asunto(s)
Ácido Anhídrido Hidrolasas , Genes Supresores de Tumor , Proteínas de Neoplasias , Proteínas/genética , Proteínas/metabolismo , Animales , División Celular/genética , División Celular/fisiología , Sitios Frágiles del Cromosoma , Fragilidad Cromosómica , Cromosomas Humanos Par 3/genética , Fosfatos de Dinucleósidos/metabolismo , Humanos , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Fenotipo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.
Asunto(s)
Ácido Anhídrido Hidrolasas , Fosfatos de Dinucleósidos/metabolismo , Proteínas de Neoplasias , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas/metabolismo , Secuencia de Bases , Cartilla de ADN , Escherichia coli/genética , Genes Supresores de Tumor , Histidina/genética , Humanos , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Hidrolasas Diéster Fosfóricas/genética , Reacción en Cadena de la Polimerasa , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.
Asunto(s)
Ácido Anhídrido Hidrolasas , Fragilidad Cromosómica , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 8 , Neoplasias Gastrointestinales/genética , Genes Relacionados con las Neoplasias , Hidrolasas , Neoplasias Renales/genética , Proteínas de Neoplasias , Proteínas/genética , Translocación Genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios Frágiles del Cromosoma , Mapeo Cromosómico , Neoplasias del Colon/genética , Cósmidos/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Neoplasias Esofágicas/genética , Exones/genética , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Neoplasias Gástricas/genética , Células Tumorales Cultivadas/fisiologíaAsunto(s)
Cisteína/metabolismo , Escherichia coli/enzimología , Uridina Fosforilasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Uridina Fosforilasa/metabolismoRESUMEN
Using site-directed mutagenesis, mutant genes of the E.coli UDPase that coded proteins with point substitutions of histidine residues (i.e., H8N, H47N, H101N, H122N, H152N, H179N, and H240N) were constructed. Study of the enzymatic activity of mutant UDPases showed that histidine-asparagine substitutions at the positions 47, 101, 152, 179, and 240 do not affect protein functioning. Whereas H122N and H8N substitutions inhibit the activity of UDPase by 60 and 100%, respectively. This evidences the important functional role of the His122 and His8 residues for the formation of the active site fo the enzyme.
Asunto(s)
Escherichia coli/enzimología , Histidina/metabolismo , Ingeniería de Proteínas , Uridina Fosforilasa/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mutación Puntual , Uridina Fosforilasa/química , Uridina Fosforilasa/genéticaAsunto(s)
Escherichia coli/enzimología , Uridina Fosforilasa/química , Uridina Fosforilasa/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Uridina Difosfato/biosíntesis , Uridina Difosfato/genética , Uridina Difosfato/metabolismo , Uridina Fosforilasa/genéticaRESUMEN
Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.