Establishment and characterization of a cell line (T201) derived from a human larynx squamous cell carcinoma.

The purpose of this report was the initiation and further maintenance of tumor cells from a primary larynx squamous cell carcinoma. A tumor fragment was mechanically dissociated, the cells were grown in RPMI medium, being the primary culture dependent on the presence of epidermal growth factor and insulin; during subsequent passages the adaptation to conventional growth conditions was obtained. Cells grew in monolayer with an epitheliod shape, showing a pavement-like arrangement; at confluence, cells piled up without contact inhibition maintaining the same morphology. Population doubling time was about 48 h with a colony-forming efficiency of 10%. Immunocytochemical characterization was performed with a panel of monoclonal antibodies reactive against tumor associated antigens, including mucin glycoproteins and related carbohydrate antigens, carcinoembryonic antigen (CEA), p53 as well as cytokeratins, vimentin and desmin. T201 expressed CEA, sialyl Lewis x, Lewis x, Lewis y, MUC1 mucin, Tn hapten, p53, vimentin and cytokeratins. On the other hand, a modal chromosome diploid number of 46 occurring in 74% of cells was detected. Present data confirmed that the methodology employed was adequate for the establishment and characterization of a new cell line which can provide a useful model to study biological and immunological aspects of larynx squamous cell carcinoma.

Expression of tumour associated antigens in normal, benign and malignant human mammary epithelial tissue: a comparative immunohistochemical study.

Breast carcinoma cells may express a variety of clinically relevant epitopes, some of which are associated with aberrant glycosylation of MUC1 mucin molecules, as well as determinants which are commonly expressed on their normal molecular counterparts. The present investigation is primarily an immunochemical analysis of MUC1 epitopes and other tumour associated antigenic determinants, as defined by their reaction with monoclonal antibodies and expressed in normal, benign and malignant epithelia. It was determined that malignant tissues of the breast expressed MUC1 mucin, as well as the Le(y) hapten and CEA, at different intensities, cellular distribution and patterns and percentages of positively stained cells. Conversely, benign tissues expressed a low intensity of MUC1 which was restricted to apical cell surface membranes and lumen debris; a similar pattern was found in some normal breast sections. It was concluded that MUC1 mucin exhibits heterogeneous antigenicity (as defined by its reactivity with a panel of related anti-MUC1 monoclonal antibodies) which is predominantly related to the progression of malignant disease. Le(y) is a marker of breast neoplasia, while CEA was found on only a small proportion of tumours. These immunohistochemical findings are considered in the context of improving breast cancer diagnosis and therapy.

Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549).

The morphology, cell growth, antigenic expression and tumorigenicity of cell subpopulations from the A549 lung adenocarcinoma isolated by Percoll gradient separation have been analysed. Four subpopulations were obtained (subpopulations A, B, C and D). Immunocytochemical analysis of several antigens was performed with monoclonal antibodies (MAbs): MUC1 mucin (C595, HMFG1 and HMFG2), MUC5B (PANH2); gp230 (PANH4); carbohydrate antigens including sialyl Lewis x (KM93), Tn antigen (83D4), Lewis y (C14); 5, 6, 8, 17 and 19 cytokeratins and p53. The cell population D tended to form cell aggregates that piled up on the monolayer similar to overgrowth cultures of the A549 parental cell line, whereas A, B and C cell subpopulations formed well spread monolayers. Both parental A549 and subpopulation D secreted abundant mucus. The topographic distribution and secretion production were correlated with tumorigenic assays since only subpopulation D grew in nude mice exhibiting reduced latency period; these characteristics correlated with the fast growth of the subpopulation D in vitro. Immunocytochemical analysis demonstrated that subpopulation D showed greater expression of MUC1 mucin and carbohydrate antigens such as Tn antigen, sialyl Lewis x and Lewis y and less expression of cytokeratins, p53, MUC5B and gp230; conversely, subpopulations A, B and C showed the opposite antigenic profile. Our results illustrate heterogeneity in the A549 cell line; subpopulations A, B and C retained characteristics of more differentiated adenocarcinoma while subpopulation D displayed features of a less differentiated tumor line.

Immunohistopathological characterizatin of spontaneous metastases in a human lung mucoepidermoid adenocarcinoma (HLMC) xenograft.

The most common clinical form of lung cancer is a disseminated disease with distant metastases; several years of cancer progression precede presentation, and this ultimately limits the efficacy of curative therapy. In this immunohistochemical study, we examined a mucinous adenocarcinoma cell line, maintained by xenogeneic transplantation, and a spontaneous metastatic variant which produces distant tumors (in liver, spleen and kidney). The aim was to investigate possible parameters which characterize the metastatic process. Histopathological comparison between the two subcutaneous transplanted tumor lines showed that both lines presented a similar cellular morphology, a different pattern of cellular growth and an increased vascularization in the metastatic line with respect to its parent. All the tumor sections expressed differential immune reactivity with monoclonal antibodies against Lewis y (MAb C14), sialyl-Lewis x (MAb SNH3) and Lewis x (MAb FH2) determinants. Neither expressed MUC 1 mucins detectable with monoclonal antibodies reactive with the mucin protein core (MAbs C595 and SM3) nor was carcinoembryonic antigen (MAb C365) expressed. Neoplastic cells were reactive with an anti-pan cytokeratin monoclonal antibody confirming their epithelial histogenesis. Our findings have been evaluated with respect to defining metastatic phenotypes in lung cancer by examination of distinct histopathological and immunological parameters.

Assessment of methods for primary tissue culture of human breast epithelia.

A serie of 29 human normal, benign and malignant breast tissues were cultivated in an attempt to isolate and propagate primary breast epithelial cells in vitro. Explants methodology as well as disaggregation techniques (enzymatic and mechanical) were employed to obtain better culture conditions. Cells derived from breast malignant tissue were propagated in an appropriate and survived in culture for at least 6 months, exhibiting a marked preference to grow in suspension (independent anchorage). Tumorigenicity assays in nude mice were performed with malignant cells obtained from primary culture cells as well as with cells achieved from successive passages. The rate of long time cell survival from malignant and non-malignant tissues demonstrated the accuracy of the methodology but it also emphasised the need for improving technology to obtain cell lines with long survival.

MUC1 oncogene amplification correlates with protein overexpression in invasive breast carcinoma cells.

The MUC1 gene is aberrantly overexpressed in approximately 90% of human breast cancers. Several studies have shown that MUC1 overexpression is due to transcriptional regulatory events. However, the importance of gene amplification as a mechanism leading to the increase of MUC1 expression in breast cancer has been poorly characterized. The aim of this study was to evaluate the role of MUC1 gene amplification and protein expression in human breast cancer development. By means of real-time quantitative polymerase chain reaction and immunohistochemical methods, 83 breast tissue samples were analyzed for MUC1 gene amplification and protein expression. This analysis showed MUC1 genomic amplification and a positive association with the histopathological group in 12% (1 out of 8) of benign lesions and 38% (23 out of 60) of primary invasive breast carcinoma samples (P = 0.004). Array-comparative genomic hybridization meta-analysis of 886 primary invasive breast carcinomas obtained from 22 studies showed MUC1 genomic gain in 43.7% (387 out of 886) of the samples. Moreover, we identified a highly statistical significant association between MUC1 gene amplification and MUC1 protein expression assessed by immunohistochemistry and Western blot test (P < 0.0001). In conclusion, this study demonstrated that MUC1 copy number increases from normal breast tissue to primary invasive breast carcinomas in correlation with MUC1 protein expression.

MUC1 cytoplasmic tail detection using CT33 polyclonal and CT2 monoclonal antibodies in breast and colorectal tissue.

UNLABELLED: The immunohistochemical detection (IHC) of MUC1-CT employing a polyclonal antibody (CT33) in relation to CT2 monoclonal antibody (MAb) was analyzed. Western blot (WB) was used to determine the molecular mass of CT. MATERIALS AND METHODS: We studied 163 breast and 89 colorectal cancer specimens, 10 breast and 14 colorectal benign conditions, and 12 breast and 20 colorectal normal samples. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and WB. A nonparametric statistical analysis was employed; data were standardized and a Kendall-Tau correlation was applied. RESULTS: By IHC, 146/163 (90%) and 151/163 (93%) of breast cancer were positive with CT33 and CT2, respectively; a statistically significant correlation was obtained (t=0.5199). Seven out of ten (70%) benign breast specimens were positive with CT33 while all samples stained with CT2; in normal breast sample tissues, all were positive with both Abs. In colorectal cancer samples, both antibodies stained 47/89 (53%) samples; CT2 reacted in 13/14 (93%) of benign samples while CT33 showed a positive reaction in 9/14 (64%) of benign specimens. In normal samples, CT2 showed staining in 17/20 (85%) of samples and CT33 was reactive in 12/20 (60%). By WB, in breast and colorectal cancer samples, similar results were obtained with both antibodies: a main band at about 30kDa which represents the smaller subunit. CONCLUSION: CT33 polyclonal antibody has demonstrated its efficacy to detect MUC1 in breast and colorectal cancer tissues with similar reactivity to CT2. It is worthwhile to affirm that CT33 is a good indicator of MUC1 expression.

Delitos sexuales: relevamiento de las solicitudes de cotejo durante el 2001 / Sexual offenses: relevance of comparison requests for 2001

El Laboratorio de Inmunogenética es un organismo dependiente de la Suprema Corte de Justicia de la provincia de Buenos Aires. Durante el año 2001 ingresaron 295 pedidos de delitos sexuales, abarcando violaciones (41,4 por ciento), violaciones agravadas (18,7 por ciento), violaciones seguidas de muerte (2,2 por ciento), abuso sexual (28,4 por ciento), abuso deshonesto (7,2 por ciento) y estupro (2,1 por ciento). En un 22 por ciento las víctimas fueron menores. En un 34 por ciento el autor permanece desconocido. En el 45 por ciento del total de los casos analizados, se observó correspondencia entre el genotipo detectado en las evidencias y el imputado, mientras que un 20 por ciento resultaron exclusiones. Como conclusión del relevamiento surge la necesidad de implementar una base de datos (AU)