RESUMEN
Several genetic epidemiological studies have provided data to support the hypothesis that there are genes on the X chromosome that may contribute to prostate cancer susceptibility. A recent linkage study of 360 prostate cancer families described evidence for a prostate cancer predisposition gene, termed HPCX, which maps to Xq27-28. To confirm the potential contribution of this locus to prostate cancer susceptibility in an independent dataset, we studied 153 unrelated families who are participants in the University of Michigan Prostate Cancer Genetics Project. Families selected for this analysis have at least two living family members with prostate cancer that are related in a way that they could potentially share a common ancestral copy of an X chromosome. DNA samples were genotyped using a panel of seven polymorphic markers spanning 30 cM and containing the HPCX candidate region. The resulting data were analyzed using both nonparametric and parametric linkage methods. Analysis of all 153 families using multipoint non-parametric linkage (NPL) methods resulted in positive NPL Z-scores across the entire candidate interval (NPL Z-scores of 0.23-1.06, with corresponding one-sided Ps of 0.41 and 0.15, respectively). The 11 African-American families had negative NPL Z-scores across the same 30-cM interval. Analysis of the 140 Caucasian families produced a maximal NPL Z-score of 1.20, with a corresponding one-sided P of 0.12 at marker DXS1113. The subset of families with no evidence of male-to-male disease transmission and with early-onset prostate cancer (average age at diagnosis within a family < or = 65 years) contributed disproportionately to the evidence for linkage for the entire dataset in the HPCX candidate region (near marker DXS1113). In conclusion, this study of 153 families, each with two or more living members with prostate cancer, provides some additional support for the existence of a prostate cancer susceptibility gene at Xq27-28.
Asunto(s)
Ligamiento Genético , Predisposición Genética a la Enfermedad , Neoplasias de la Próstata/genética , Cromosoma X , Anciano , Mapeo Cromosómico , Femenino , Marcadores Genéticos , Genotipo , Humanos , Masculino , Linaje , Factores Sexuales , Estadísticas no ParamétricasRESUMEN
We report the isolation of the murine ortholog of AIM1, a human gene whose expression is associated with the reversal of tumorigenicity in an experimental model of melanoma. Mouse and human AIM1 are more than 90% identical in amino acid sequence in the betagamma-crystallin repeats and the C-terminal domain, and more than 75% identical in the extended N-terminal domain. Consistent with the isolated cDNA representing the authentic AIM1 ortholog, linkage analysis localized mouse Aim1 to proximal mouse Chromosome (Chr) 10 in a conserved linkage group with genes localized to human Chr band 6q21. Searches of EST databases identified a second AIM1-like gene in both mouse and human, suggesting the existence of a gene family. Northern analysis demonstrates Aim1 is expressed most abundantly in adult skin, lung, heart, liver, and kidney and is temporally regulated during embryogenesis. Aim1 is expressed highly in the shaft region of the hair follicles and the presumptive ectoderm, but not at detectable levels in melanocytes or melanocyte precursor cells.
Asunto(s)
Cristalinas/genética , Proteínas de la Membrana , Familia de Multigenes/genética , Proteínas/genética , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Mapeo Cromosómico , Cromosomas Humanos Par 6/genética , Clonación Molecular , Embrión de Mamíferos/química , Embrión de Mamíferos/embriología , Regulación del Desarrollo de la Expresión Génica , Humanos , Melanocitos/química , Melanocitos/citología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Análisis de Secuencia de ADN , Piel/química , Piel/citología , Simportadores , gamma-CristalinasRESUMEN
Chromosome 6-mediated suppression of tumorigenicity in malignant melanoma cell lines provides a model system to identify genes associated with the reversion of the tumorigenic phenotype. Using subtractive cDNA selection, we recently identified a series of novel genes which are differentially expressed in association with chromosome 6-mediated suppression. We now report the molecular characterization of a novel gene termed AIM2 for (Absent In Melanoma), which represents a 1485 bp cDNA. An open reading frame of 1032 base pairs, corresponding to 344 amino acid residues, is predicted. The predicted protein shares a conserved sequence domain of approximately 200 amino acids with known interferon-inducible genes of both human and mouse. We demonstrate that the AIM2 gene encodes a transcript of approximately 2 kb which is expressed in spleen, small intestine, and peripheral blood leukocytes. In addition, we have localized AIM2 to the long arm of human chromosome 1 (band q22) in a highly conserved region which also contains the known interferon-inducible genes IFI16 and MNDA. We have also demonstrated that, like IFI16 and MNDA, AIM2 is induced in HL60 cells by interferon gamma. Our findings support the existence of a family of genes in this region similar to the well-characterized mouse Ifi200 gene family.
Asunto(s)
ADN Complementario/análisis , Interferón gamma/farmacología , Melanoma/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Clonación Molecular , Células HL-60 , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
AIM1 is a novel gene whose expression is associated with the experimental reversal of tumorigenicity of human malignant melanoma. The predicted protein product of the major 4.1-kb transcript shows striking similarity to the betagamma-crystallin superfamily. All known members of this superfamily contain two or four characteristic motifs arranged as one or two symmetrical domains. AIM1, in contrast, contains 12 betagamma motifs, suggesting a 6-domain structure resembling a trimer of beta- or gamma-crystallin subunits. The structure of the AIM1 gene shows remarkable similarity to beta-crystallin genes, with homologous introns delineating equivalent protein structural units. AIM1 is the first mammalian member of the betagamma superfamily with a primarily non-lens role. Other parts of the predicted AIM1 protein sequence have weak similarity with filament or actin-binding proteins. AIM1 is a good candidate for the putative suppressor of malignant melanoma on chromosome 6, possibly exerting its effects through interactions with the cytoskeleton.
Asunto(s)
Cristalinas/genética , Melanoma/patología , Proteínas de la Membrana , Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Bovinos , Clonación Molecular , ADN Complementario , Exones , Humanos , Intrones , Melanoma/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas/genética , Homología de Secuencia de AminoácidoRESUMEN
Despite dramatic advances in the identification of human expressed sequence tags (ESTs), techniques that facilitate isolation of chromosome or chromosome band-specific ESTs would be of considerable value. This report demonstrates the feasibility of identifying chromosome-specific ESTs following microdissection of a single-copy chromosome region. For this study, a reduced complexity cDNA library was linkered and hybridized to normal human metaphase chromosomes. After stringency washes, the entire long arm of chromosome 6 (6q) was microdissected. Following PCR amplification using linker-specific primers, captured cDNAs were subcloned and 187 individual clones picked at random. These 187 clones were then sorted by filter cross-hybridization into 34 unique groups. Of these 34 groups, 19 (56%) mapped to chromosome 6 by Southern blot. We identified three previously known genes, human cytovillin (ezrin) mapped previously to 6q25-26, human cardiac gap junction protein (connexin 43) mapped previously to 6q21-23.2 and prolyloligopeptidase, which had not been mapped previously. BLASTN identified three clone groups with homology to known ESTs and 12 representing novel cDNA sequences. Six of the groups were sublocalized to specific band regions of 6q using a chromosome 6 hybrid mapping panel, five representative clones were tested on Northern analysis to verify their expression, and finally, nine clones were mapped against the Gene bridge 4 reduction hybrid panel to confirm their genetic map location on 6q. These results demonstrate that microdissection of single-copy sequences has sufficient specificity for isolation of chromosome-specific cDNAs.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 6/genética , ADN Complementario/genética , Lugares Marcados de Secuencia , Animales , Northern Blotting , Southern Blotting , Células CHO , Células Cultivadas , Clonación Molecular , Conexina 43/genética , Cricetinae , Proteínas del Citoesqueleto , Biblioteca de Genes , Humanos , Hibridación in Situ , Proteínas de la Membrana/genética , Metafase/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Prolil Oligopeptidasas , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/genéticaRESUMEN
We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.
Asunto(s)
Genes Supresores de Tumor , Melanoma/genética , Neoplasias Cutáneas/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , ADN Complementario/genética , ADN de Neoplasias/genética , Humanos , Datos de Secuencia Molecular , Monosomía , Transducción Genética , Células Tumorales CultivadasRESUMEN
Melanocytic transformation is thought to occur by the sequential accumulation of genetic alterations. Evidence implicating human chromosomes as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic reversion of tumorigenicity following the introduction of a normal chromosome 6 by microcell-mediated chromosome transfer (Trent et al., 1990). Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline. A modified PCR-based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells. A total of 32 randomly selected clones from the suppressed sublibrary were isolated and examined, with 24 detecting a transcript by Northern analysis. Of these 24 clones, 21 (88%) demonstrated elevated expressed by Northern analysis in the suppressed subline relative to the tumorigenic parental cell line. In 6/21 differentially expressed clones (29%), expression was exclusive to the suppressed subline. Partial sequence analysis and database searching of these clones indicated that 5/6 were novel with one representing a previously characterized gene. Chromosomal localization of the five novel clones was performed following PCR amplification of a human/rodent somatic cell hybrid mapping panel or fluorescent in situ hybridization. One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21). This novel approach should facilitate the identification of genes whose expression is causally related to the suppressed phenotype.
Asunto(s)
Cromosomas Humanos Par 6 , Genes Supresores de Tumor , Melanoma/genética , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario/biosíntesis , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Humanos , Hibridación Fluorescente in Situ , Melanoma/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.
Asunto(s)
ADN de Neoplasias/genética , Doxorrubicina/farmacología , Resistencia a Medicamentos/genética , Amplificación de Genes/genética , Secuencia de Bases , Southern Blotting , Carcinoma de Células Pequeñas/genética , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 7 , Clonación Molecular , Cartilla de ADN , Fibrosarcoma/genética , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/genética , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia de ADN/métodos , Células Tumorales CultivadasAsunto(s)
Publicidad , Enfermería , Administración de Personal , Selección de Personal , Humanos , Reino Unido , Recursos HumanosRESUMEN
A catheter-tip velocity transducer with two high-fidelity pressure manometers was used to evaluate the left ventricular (LV) hemodynamic effects of intravenous propranolol (10 mg). Nine patients without clinical evidence of heart failure were studied. Pulsatile ascending aortic blood flow velocity and pressure and LV pressure were measured continuously during drug administration. Beat-to-beat changes in stroke volume index, stroke work index, LV end-diastolic pressure, maximum blood flow velocity and acceleration, and maximum LV dP/dt were determined. Propranolol produced a decrease in maximum blood flow velocity (from 58 +/- 4.7 to 42 +/- 5.1 cm/sec, p less than 0.002), and acceleration (from 1181 +/- 130 to 847 +/- 117 cm/sec2, p less than 0.002, max dP/dt (from 1361 +/- 70 to 1146 +/- 63 mm Hg/sec, p less than 0.002), stroke volume index (from 47 +/- 3.0 to 38 +/- 3.2 ml/m2, p less than 0.002) and total stroke work index (from 702 +/- 33 to 603 +/- 44 mJ/m2 p less than 0.04), with little change in mean aortic pressure, peak systolic pressure and LV end-diastolic pressure. Depression in myocardial function was detectable within 1 minute after initiation of propranolol and persisted when negative chronotropic effects were eliminated by atrial pacing. The multisensor catheter technique allows rapid and safe detection of changes in cardiovascular function during propranolol administration in conscious man.