Cell surface expression of urokinase receptor in normal mammary epithelial cells and breast cancer cell lines

Background: The urokinase receptor (uPAR) is important in the process of extracellular matrix degradation occurring during cancer cell invasion and metastasis. We wished to quantify uPAR on the surfaces of normal mammary epithelial cells (HMEC) and 6 well-known breast cancer cell lines using flow cytometry. Materials and Methods: Cell surface uPAR was labelled with a monoclonal antibody, and this was detected with a florescent-labelled second antibody and accurately measured using flow cytometry. The measured fluorescent signals of the stained cells were interpolated with those of Quantum Simply Cellular bead standards to determine the number of uPAR sites per cell. Results: The breast cancer cell lines ranged from 13,700 to 50,800 uPAR sites per cell, whilst HMEC cells had only 2,500 sites. Conclusions: This simple and reliable method showed that the expression of cell surface uPAR is higher in the breast cancer cell lines than in the normal mammary cells.

Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell-cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.

Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.