RESUMEN
A new cancer gene, HIC-1 (Hypermethylated in Cancer) telomeric to p53 on chromosome 17p may be of clinical importance in sporadic breast cancer. Regional DNA hypermethylation of 17p13.3 resulting in suppression of gene expression has been shown to precede 17p structural changes in human carcinogenesis. In addition, loss of heterozygosity studies have suggested clinically significant involvement of a gene on 17p13.3 associated with poor prognosis in breast cancer. Using RT-PCR analysis, we demonstrate that the MCF7 (wild type p53) cell line expressed HIC-1 transcripts but the MDAMB231 (mutant p53) cell line did not, suggesting loss of HIC-1 expression and p53 malfunction may be synergistic events in sporadic breast cancer. HIC-1 expression was examined using RT-PCR on RNA extracted from 50 primary untreated, human breast cancers and was detected in only 7/50 (14%) cancers. All seven patients with HIC-1 expression were alive without disease recurrence after 8 years follow-up and 5/7 had detectable p53 wild type mRNA expression. This suggests that retained HIC-1 expression may offer a survival advantage. However the seven cancers had 17p13.3 loss of heterozygosity (LOH; four patients), a feature previously associated with poor prognosis, or were homozygous (three patients) suggesting there may be two genes at 17p13.3 involved in breast carcinogenesis. Using a demethylating drug 5-aza-2'-deoxycytidine (DeoxyC), HIC-1 expression was restored in the MDAMB231 cells, also suggesting restoration of HIC-1 function by reversing HIC-1 hypermethylation may offer a therapeutic avenue in breast cancer.
Asunto(s)
Azacitidina/análogos & derivados , Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas de Neoplasias/fisiología , Factores de Transcripción/fisiología , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/mortalidad , Carcinoma/patología , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Estudios de Cohortes , Metilación de ADN/efectos de los fármacos , Decitabina , Inhibidores Enzimáticos/farmacología , Femenino , Estudios de Seguimiento , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen/efectos de los fármacos , Genes p53 , Humanos , Factores de Transcripción de Tipo Kruppel , Pérdida de Heterocigocidad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Pronóstico , Estudios Prospectivos , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Células Tumorales Cultivadas/metabolismoRESUMEN
Molecular and immunohistochemical studies of genetic events on chromosome 17p were prospectively compared with conventional clinical and pathological parameters and disease behaviour at a minimum of 72 months follow-up. In a series of 91 patients with primary operable breast cancer, 37 out of 91 (41%) patients had disease relapse and 23 out of 91 (25%) had died during the follow-up period. Allelic imbalance at the YNZ22 locus (17p13.3), demonstrated in 33 out of 63 (52%) informative patients, was significantly associated with disease recurrence (P < 0.01, 2 d.f. Cox analysis) and showed a trend towards impaired survival (P = 0.08, 2 d.f. Cox analysis) after a mean follow-up of 84 months for survivors. By contrast, p53 mutation (in 10 out of 60, 17% of cancers), p53 allelic imbalance (in 23 out of 56, 41% informative patients), p53 mRNA expression (in 47 out of 87, 54% patients), p53 mRNA overexpression (in 24 out of 87, 28%) or p53 protein expression (detected in 25/76, 32%) were not associated with disease behaviour. There was no significant association between allelic imbalance at YNZ22 and any abnormality of p53 DNA, RNA or protein. Allelic imbalance at 17p13.3 (YNZ22) serves as a marker of poor prognosis in breast cancer. As yet unidentified genes on 17p13.3, distinct from and telomeric to p53, are therefore likely to be of clinical importance in breast cancer.
Asunto(s)
Alelos , Neoplasias de la Mama/genética , Cromosomas Humanos Par 17/genética , Genes p53 , Proteínas de Neoplasias/genética , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Anciano , Neoplasias de la Mama/mortalidad , Femenino , Estudios de Seguimiento , Marcadores Genéticos , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Análisis de SupervivenciaRESUMEN
The pattern of loss of heterozygosity (LOH) on chromosome 17 in human breast cancer is complicated and shows many different regions of loss. In an attempt to narrow down the relevant regions of LOH on chromosome 17, we have studied the deletion pattern and its association with clinical parameters in 1280 breast carcinoma-venous blood lymphocyte pairs. In total, 42 different chromosome 17 loci were investigated, and between 25 and 625 cases were analyzed at each locus. The frequency of LOH observed on the p arm was much higher than that observed on the q arm. The opposite effect was observed in 52 ovarian cancer cases investigated, with less LOH on 17p than on 17q. Patterns of loss consistent with interstitial and terminal deletions, as well as loss of either the p or q arm or monosomy 17 were observed. To determine whether loss at particular loci may be associated with biological features of breast tumors, clinical data including age of onset, family history of breast cancer, tumor histopathology, tumor size, estrogen receptor (ER) status, and occurrence of lymph node or distant metastases were collected for each case. Overall, large-sized, ER-negative, lymph node-positive ductal tumors showed the highest frequencies of LOH, with ER-negative and ductal tumors showing LOH for markers along the majority of the chromosome. Eight regions of chromosome 17 appear to be associated with human breast cancer, two on 17p and six on 17q. These regions were not necessarily in the areas exhibiting the highest frequencies of LOH but were defined by interstitial and terminal deletions in multiple independent cases. Seven of these regions showed statistically significant differences in LOH associated with clinical parameters. These data strongly suggest that loci on chromosome 17 may determine aspects of tumor presentation and disease behavior in human breast cancer and pinpoint candidate tumor suppressor gene loci.
Asunto(s)
Alelos , Neoplasias de la Mama/genética , Cromosomas Humanos Par 17 , Pérdida de Heterocigocidad , Adulto , Neoplasias de la Mama/patología , Femenino , Genes Supresores de Tumor , Marcadores Genéticos , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia/genéticaRESUMEN
Dinucleotide repeat sequences ('microsatellites') have been used as polymorphic genetic markers following amplification in the polymerase chain reaction (PCR). We have compared several methods of analysing the PCR products. The most reliable and unambiguous results were obtained when the PCR products were probed with a specific dinucleotide repeat oligonucleotide, so that only the microsatellite-containing products were detectable.
Asunto(s)
Marcadores Genéticos , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Neoplasias de la Mama/química , ADN/sangre , ADN/genética , ADN de Neoplasias/genética , Etidio , Humanos , Leucocitos Mononucleares/química , Coloración y EtiquetadoAsunto(s)
Autoanticuerpos , Antígeno H-Y/inmunología , Animales , Femenino , Masculino , Ratones , RatasRESUMEN
We describe a method of immunofluorescence which is a lateral application of the principles of the APAAP immunohistochemical technique. Immune complexes of R-phycoerythrin and monoclonal anti-R-phycoerythrin (PEAPE complexes) were used in an indirect immunofluorescence technique to detect the binding to cells of monoclonal antibodies directed to IgM, HLA-DR and B cell activation and differentiation antigens. PEAPE complexes were linked to cell surface bound mAbs by unlabelled anti-mouse Ig antibodies to produce high levels of fluorescent staining. The sensitivity of this method of indirect immunofluorescence was enhanced by the sequential application of several cycles of anti-mouse Ig and PEAPE complexes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Técnica del Anticuerpo Fluorescente , Ficoeritrina/inmunología , Pigmentos Biológicos/inmunología , Animales , Complejo Antígeno-Anticuerpo , Antígenos de Superficie/análisis , Humanos , RatonesRESUMEN
We have examined the MHC class II beta chains in lymphoblastoid cell lines from over 200 individuals and describe one line which possesses, in addition to normal beta chains, a species of beta chain of unusually high Mr and abnormal pI which appears to be a product of the DR locus. This abnormality in Mr, detected by SDS-gel electrophoresis, was apparent only in the presence of mercaptoethanol and was shown to be due to difference in polypeptide chain length rather than to extra glycosylation.
Asunto(s)
Linfocitos B/inmunología , Antígenos HLA-D/aislamiento & purificación , Línea Celular , Glicósido Hidrolasas , Antígenos HLA-D/genética , Humanos , Inmunoelectroforesis , Mercaptoetanol , Peso Molecular , Conformación ProteicaRESUMEN
When mice were immunized with a mixture of human MHC class II alpha and beta glycoprotein chains, the predominant antibody response was anti-alpha, and from a subsequent fusion experiment over 60 hybridomas showing anti-alpha activity were generated, compared with 11 anti-beta secretors. These findings contrast with the relative paucity of anti-alpha monoclonals described previously. Use of a miniaturized Western blot screening protocol was a critical factor in the present study since the anti-alpha monoclonals do not bind to the surface of living B cells and would therefore be missed in conventional screening assays. After glutaraldehyde fixation of target B lymphocytes or B-cell lines, the majority of anti-alpha monoclonals do react in a radio-immunobinding assay, although none binds as strongly as pan-reactive anti-beta chain antibodies. This suggests that the immunogenic epitopes of alpha chains are normally concealed by the three-dimensional folding of the alpha beta dimer. The anti-alpha monoclonals were all monomorphic but varied in the extent of their reactivity with alpha chains separated on one-dimensional and two-dimensional IEF gels. The most reactive antibodies identified up to seven distinct components among mature class II antigens from solubilized cell membranes.
Asunto(s)
Epítopos/análisis , Antígenos HLA-D/análisis , Animales , Anticuerpos Monoclonales , Antígenos de Superficie/análisis , Linfocitos B/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Ratones , Ratones Endogámicos BALB CRESUMEN
The rejection by inbred female mice of skin from syngeneic males is provoked by the male-specific transplantation antigen H- Y. Here D. N. Crichton and C. M. Steel discuss conflicting claims surrounding the detection of H-Y antigen with polyclonal and monoclonal antibodies.
RESUMEN
Binding of 125I-iodohydroxybenzylpindolol to beta-adrenoceptors has been examined in lymphoblastoid cell lines from members of 5 families affected by manic-depressive disorder. Cell lines from 6 manic-depressives, 7 unaffected relatives and 11 non-psychiatric controls were examined. Binding was reduced to less than half of control values in cell lines from 4 out of 6 manic-depressives and only 1 out of 18 unaffected relatives or controls. All the cell lines with reduced beta-adrenoceptor binding came from 3 families; members of the remaining 2 families showed normal binding. These findings suggest that genetic heterogeneity is present in manic-depressive disorder and that a beta-adrenoceptor defect may influence genetic susceptibility to the disorder.
Asunto(s)
Trastorno Bipolar/genética , Receptores Adrenérgicos beta/genética , 5'-Nucleotidasa , Adolescente , Adulto , Linfocitos B/metabolismo , Línea Celular , Femenino , Humanos , Masculino , Nucleotidasas/metabolismo , Ouabaína/metabolismo , Linaje , Pindolol/análogos & derivados , Pindolol/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMEN
Hybridomas secreting monoclonal antibodies reactive with murine spermatozoa were produced by fusion of myeloma cells with spleen cells from C57BL/6J mice immunized with spermatozoa from mice of the same strain. All antisperm antibodies were of the mu (mu) immunoglobulin heavy chain class; only one (MS-1) bound S. aureus protein A. Antibody MS-1 recognized an antigen present on the sperm acrosomal cap, on the surface of cells from liver and kidney and from some cultured cell lines. The subunit molecular weight (69000) of the polypeptide reactive with MS-1 was determined by SDS-PAGE analysis of sperm membrane proteins followed by their electrophoretic transfer to nitrocellulose.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/análisis , Proteínas de la Membrana/análisis , Espermatozoides/inmunología , Animales , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Hibridomas , Masculino , Ratones , Peso Molecular , Espermatozoides/análisisRESUMEN
Putative anti-H-Y antisera have been examined for H-Y-specific antibody in a sensitive antiglobulin radioimmunoassay. All the sera tested were weakly bound by both male and female spleen cells. Absorption of the antisera with cells of either sex was equally effective in reducing the amount of antiglobulin bound revealing that their activity was not specific for H-Y gene products. The failure in this study to demonstrate H-Y-specific antibody is discussed.
Asunto(s)
Antígenos de Histocompatibilidad/inmunología , Cromosomas Sexuales/inmunología , Cromosoma Y/inmunología , Animales , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Femenino , Sueros Inmunes , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Radioinmunoensayo , Bazo/inmunologíaRESUMEN
The expression of H-Y antigen on mouse red blood cells has been investigated by studying their life span in vivo. The survival of 51Cr-labeled cells from males was unaltered when transfused into isogeneic females or female mice which had rejected a male skin graft. These observations are consistent with the view that the H-Y antigen is not present on mouse erythrocytes.
Asunto(s)
Eritrocitos/inmunología , Antígeno H-Y/análisis , Animales , Envejecimiento Eritrocítico , Femenino , Pruebas de Hemaglutinación , Masculino , Ratones , Ratones Endogámicos C57BL , Piel/inmunología , Trasplante de Piel , Bazo/inmunología , Testículo/inmunologíaRESUMEN
Autopsy examination of young adult mice revealed a characteristic pigmentation of the anterior splenic pole occurring in a high proportion (8-34 per cent) of three mouse strains and two sublines. Histological studies identified the pigment as lipofuscin and electron microscopy provided supporting evidence. Preliminary results are consistent with the hypothesis that lipofuscin may represent non metabolisable debris from cellular breakdown associated with lysosomal activity.