Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, M18, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody M18 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Abstract. Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, Ml8, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody Ml8 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

HL-A antigens in North American black families

The polymorphic HL-A histocompatibility system has been studied in North American black families. The family studies show that haplotype frequencies differ between black and white populations. Seven haplotypes (W28,W5; W28,W17; W28, undefined four; W23,W5; W19,W5; undefined LA,W5; and undefined LA, undefined four) were significantly more frequent in blacks than whites, while haplotypes 1,8 and 3,7 were significantly less frequent. Some of these differences may be accounted for by differences in gene frequencies between the two groups; other differences may be explained by linkage disequilibrium in the white population. No significant linkage disequilibrium between the LA and FOUR loci was found in the black population.     

Irradiation of synchronized cell cultures

Summary When HeLa cells are synchronized by the double thymidine block method, it is shown that the mitotic delay produced by 450 R X-rays is longer when cells are irradiated in G₂ than in G₁ but longer still when they are irradiated in the early part of S. DNA synthesis, measured by³H-Thymidine incorporation, is not reduced considerably (± 25%) and only for a few hours when 600 R are given at this time. A more pronounced inhibition occurs when irradiation is given later in S. There is also no decrease in incorporation of³H-orotic acid in RNA when the cells are irradiated in S or in G₂. These results are discussed and it is concluded that the long mitotic delays cannot he due to a modification of nucleic acids metabolism.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

Expression of proliferation associated antigens in the cell cycle of synchronized mammalian cells.

Flow cytometric bivariate analysis was used to investigate the expression of PCNA, p120 and p145 during the cell cycle of a mammalian cell line (CHO-K1). Initially, aliquots of cells in exponential and plateau (G0) phase were analyzed for proliferation associated antigen expression. Expression of PCNA and p145 during G0 was markedly depressed (less than 12% positive) while 54% of the G0 cells stained positive for p120. The fluorescent intensity (mean channel fluorescence) of these G0 positive p120 cells, however, was only slightly above the mean channel fluorescence (MCF) of cells stained with a negative isotype control. In asynchronous cultures, all three antigens were expressed in greater than 70% of the cells, with PCNA staining being greater than 95%. Cells were then synchronized using mitotic selection (mitotic index of 97%) and antigen levels were measured as cells progressed synchronously through the cell cycle. From DNA analysis histograms, it appeared that the degree of synchrony was approximately 90% throughout the remainder of the cell cycle. The bivariate DNA/PCNA, DNA/p120, and DNA/p145 histograms for mitotic cells indicated that both p120 and p145 expression were elevated (percent positive and MCF) while PCNA levels were near controls (MCF). In early G1, all three markers were depressed (less than 12% positive); however PCNA levels rose precipitously in mid-G1 (greater than 50% positive). In late G1 to early S, p145 levels increased concomitantly with increases in p120. All three antigens were elevated throughout S phase and began to decline as cells moved from G2/M to G1 of the next cell cycle with p145 expression decreasing first. This report indicates that all three proliferation associated antigens studied are differentially expressed in the cell cycle and therefore may be useful in detecting and assessing the proliferation state.     

The identification of HL-A antigens on fresh and cultured human endothelial cells.

Human endothelial cells were obtained from the umbilical cord veins of 16 newborns by methods previously described and tested for HL-A antigens by a microcytotoxicity method. HL-A antigens were present on all endothelial cell lines tested. When the HL-A phenotypes of fresh endothelial cells and autologous fetal lymphocytes were compared, a concordance of 70% was observed. When the HL-A phenotypes of maternal lymphocytes and fresh endothelial cells were compared, a maternal contribution to the endothelial cell phenotype was evident in 72% of the possible commmon antigens. Some HL-A antigens were deleted from 11 of 16 endothelial cell lines that were re-typed after 2 weeks in tissue culture. The majority (90%) of deleted antigens were from the second HL-A locus. When three lines of endothelials cells were again re-typed after 6 weeks in culture, no further changes in antigenicity were noted. These findings: a) demonstrate that HL-A antigens are present on human endothelium and suggest that endothelial cells are actively involved in establishing the immunogenicity of a graft, and b) demonstrate that the HL-A antigens on human endothelial cells may be modulated by in vitro culture.     

Pattern of chromosomal replication in synchronized lymphocytes

Summary The BrdUrd-Hoechst 33258-Giemsa technique was employed to study patterns of chromosomal replication in human lymphocytes synchronized by Methotrexate (MTX). It is proposed that in the presence of MTX, a major portion of the cell population is blocked in an advanced stage of the S-phase and not in the G₁/S border of the cell cycle. At this point, the replication of the chromosomal segments corresponding to the R-bands is terminated, and the replication of the G-bands and the inactive X-chromosomes is being initiated. The use of this method in the study of higher resolution patterns of chromosomal replications is proposed.