Shahnaz Hashmi Dairkee

Immunolocalization of a human basal epithelium specific keratin in benign and malignant breast disease

SummaryThis report describes the immunocytochemical localization of a human basal- or myoepithelial-specific antikeratin antibody in benign and malignant breast disease. Reactivity patterns with this antibody have demonstrated the lack of myoepithelial or basal epithelial participation in most benign breast specimens examined including those displaying cystic disease, fibrosis, or hyperplasia. However, in specimens of sclerosing adenosis, strong reactivity with the majority of cells in most ducts suggests a major participation of the myoepithelial cell type. Analysis of 118 breast carcinoma specimens has demonstrated strong, homogeneous reactivity in 4% of the specimens, suggesting a role for the basal epithelial cell in malignancy of the human mammary gland and implications for the prognosis of such tumors. Antigenic characterization of the malignant and benign mammary specimens which are uniformly reactive with the antibody has demonstrated the presence of a 51 kd keratin polypeptide not found in the non-reactive specimens.

Differential retention of rhodamine 123 by breast carcinoma and normal human mammary tissue.

SummaryWe have qualitatively evaluated the retention of the fluorescent dye rhodamine 123 by malignant or non-malignant breast epithelial cells in passively-infused fresh surgical specimens. Our findings demonstrate a microscopically-visible increase in the ability of primary and metastatic tumor cells to retain the dye, as compared to non-malignant epithelium. Some variability in fluorescence intensity was seen within and between tumor specimens. The optimal length of incubation in the presence of the dye was critical in achieving differential fluorescence intensity between normal and malignant cells. This method of examining rhodamine 123 uptake and retention in tissue explants provides a reliable means for direct, comparative visualizationin situ of any tissue and its associated disorders. The results of this study also demonstrate the validity of extending the use of liophilic, cationic compounds such as rhodamine 123 as antitumor agents, from model systems to the treatment of malignant disease.

Early expression of vimenti in human mammary cultures

SummaryThe intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin

Accessibility to intracellular antigens within nutritionally deprived human mammary epithelial cells

We have previously demonstrated immunolocalization of antikeratin antibodies in apparently random subpopulations of malignant cells in fresh surgical specimens of breast carcinoma (S. H. Dairkee and A. J. Hackett, 1988, J. Natl. Cancer Inst. 80, 1216-1220). The goal of the present study was to determine whether deficiencies in essential nutrients contribute toward cellular alterations in membrane integrity, consequently allowing antikeratin to bind to the cytoskeleton within live, unfixed cells. We have demonstrated here that in an in vitro model in which human mammary epithelial cells are subjected to an oxygen-glucose gradient, immunolocalization of antikeratin within the cells is observed in a dose-dependent manner in the depleted regions of the gradient, even though the cells appear to be morphologically unaltered. The potential use of antibodies to intracellular antigens for immunotargeting solid tumors and the use of this method in antibody-loading studies toward understanding functional aspects of specific cellular antigens, as well as determining differential response of various cell types under these culture conditions, are discussed.

Response of primary human mammary tumor cell cultures to a monoclonal antibody-recombinant ricin A chain immunotoxin.

SummaryMalignant epithelial tumor cells were isolated and cultured from ten human mammary specimens of cancerous origin. The 260F9 monoclonal antibody (MAB) bound to frozen sections of all of the human breast tumors tested and to primary cultured cells from the tumors. Cultured cells from all ten breast tumors were sensitive to the clonal inhibitory effects of immunotoxin 260F9 MAB-recombinant ricin A chain. At an immunotoxin concentration of 200 ng/ml (about 1 nM), inhibition of colony formation was >99% for all ten tumors.

Cellular Manifestations of Human Breast Cancer

We hypothesize that the earliest events in carcinogenesis involve a field effect causing subtle changes in a region of an organ such as the human breast. The changes may best be described as a deterioration of the

Colony morphology on agar is a sensitive indicator for growth effectors

Colonies of Chinese hamster ovary (CHO) cells growing on agar exhibit changes in colony morphology and size in response to extremely low doses of hormones and growth factors. Computer-aided densitometric scanning of photographs of these colonies allows the quantitative measurement of these morphological changes. Correlation of these changes with dosage for a variety of growth effectors is a sensitive assay for the effects of these factors, both alone and in combination, and should be useful in comparing effects of agents with similar biological activities and in the search for variant cell types with altered responsiveness. Cells growing attached to solid substrates are often responsive to these low dosages and do not seem to mimic in vivo growth effector phenomena as well as colonies growing in three dimensions on agar.

Human Myoepithelium-specific Monoclonal Antibodya

We have developed a monoclonal antibody, which upon immunohistochemical localization in frozen sections, displays specificity for myoepithelial cells in the resting mammary gland. We have found this monoclonal antibody, 312C8-3, to be specifically reactive with the myoepithelium of 9 of 9 normal mammary specimens. FIGURE 1 illustrates mature normal mammary ducts from three different reduction mammoplasty specimens. The immunoperoxidase staining with the monoclonal antibody, 3 12C8-1, reveals the myoepithelial layer at the base of the epithelium. The antibody is non-reactive with epithelium, stroma, muscle, fat, and vasculature. In order to define the myoepithelial component in malignant breast disease, which has not been very frutiful by conventional techniques, we have studied, using the myoepithelium-specific antibody, 3 12C81, the reactivity of a total of 14 infiltrating ductal breast carcinomas. The distribution of 31268-1 reactivity over the tumor population was either homogeneous or heterogeneous. Only 3 of 14 displayed a strong, homogeneous reactivity over the entire tumor. The most reactive tumor is illustrated in FIGURE 2(a). Among the heterogeneous tumors, in 5 of 14, the antibody circled only a few tumor nests (FIGURE 2, b). In one specimen, the tumor cells were predominantly negative with scattered individual cells positive (FIGURE 2, c). The remaining five specimens were very weakly reactive or negative. In all the heterogeneously reactive tumors, it was difficult to ascertain whether some tumor cells in these specimens were exhibiting the antigen or whether the reactivity represented residual myoepithelium of the ducts. In our biochemical characterization of the antigen@) recognized by the monoclonal antibody, 3 12C8-1, using the nitrocellulose-enzyme immunoassay, we have found it to be extremely reactive with a preparation of human epidermal keratin and nonreactive with preparations of myosin, actin, alpha-lactalbumin, or fibronectin. Our present interpretation of these results is that 312C8-1 is directed towards an epitope of cytokeratin, which is not a major component of secretory mammary epithelium but is abundant in the myoepithelial cells of the human mammary gland.