Autologous and homologous immunofluorescent antibody to established breast cancer cell lines

Summary Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients. This work was supported in part by Grant CA 16789 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-term human breast carcinoma cell lines of metastatic origin: Preliminary characterization

Summary Nineteen human breast carcinoma cell lines have been established as continous cultures during the past 6 years in our laboratory. This preliminary report is designed to list the lines by their designated code numbers (MDA-MB) and present a brief summary of their morphological, cytogenetic and biochemical characteristics. Sixteen of our lines were obtained from pleural effusions, two from brain metastases, and one from pericardial fluid. All lines have been shown to be distinct entities and are uncontaminated by HeLa cells or each other. A lq marker chromosome is present in all but one of the lines examined. This research was sponsored by the National Cancer Institute Contract NO1-CB-23869; Institutional Grant 5S 07 RR 5511-15 awarded by the Division of Research Resources, and a Kelsey-Leary Grant NO 974.     

Cellular localization of tissue factor in human breast cancer cell lines

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and differentiation of breast cancer cells in culture.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Metastases from metastases: comparative metastatic potential of human cancer cell lines originated from primary tumors or metastases in various tissues

Although metastases from original (primary) tumors are highly studied, metastases from metastatic sites (secondary tumors) are far less studied. Here, using data from metastasis map (MetMap) project reported in a recent study (Jin et al. in Nature 588(7837): 331–336. 10.1038/s41586-020-2969-2, 2020), we found that human cancer cell lines isolated from metastatic sites have higher potential to metastasize to another site in mice, compared to human cancer cell lines isolated from primary sites, for certain types of cancer including liver, lung and pancreas cancer. In contrast, for cancer types such as ovarian and skin cancer, human cancer cell lines originated from primary tumors have increased metastatic potential in mice, compared to human cancer cell lines originated from metastatic sites. This preliminary analysis points that the potential of metastases to further metastasize compared to that of primary tumors might be cancer type-dependent, and further research is needed to understand why certain cancer cell lines isolated from metastatic sites are more likely to spread to other organs.     

Influence of fetal bovine serum and hormones on primary vs. long-term cultures of human breast cancers

Summary We investigated the influence of fetal bovine serum, complemented or otherwise with estradiol or insulin or both, on the proliferation of mammary cancer cells from long-term and primary cultures. The long-term culture corresponded to mouse MXT and MCF-7 cell lines whereas the primary culture corresponded to primitive breast cancers squashed onto histologic slides and maintained in cultures for between 12 and 48 h. Cell proliferation was evaluated by means of digital cell image analysis of Feulgen-stained nuclei. Our results show that the addition of estradiol and insulin slightly but nevertheless significantly increases the proportion of cells still living at Hour 48 of culture. Fetal bovine serum, necessary for the growth of MXT and MCF-7 mammary cells, was highly cytotoxic with respect to the primary cultures of the 20 breast cancers under study. We are now conducting new experiments using chemically defined media to study the influence of new antineoplastic compounds on primary cultures of breast cancers.     

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.     

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy

Summary To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu (nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients’ tumors, allowing assessment of the effect of therapy when confronted with different breast tumors’ genotype and phenotype.     

Fish cell lines: Establishment and characterization of nine cell lines from salmonids

Summary Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24°C. All survived storage for at least 1 yr at −65°C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology. This work is a result of research sponsored in part by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant NA79AA-D-0016 and by the Oregon Department of Fish and Wildlife under PL-89304 Anadromous Fish Act and is Oregon Agricultural Experiment Station Technical Paper 6857.     

Characterization of invertebrate cell lines

Summary Cell extracts of five mosquito cell lines and a tick cell line were examined for four cellular isozymes using a cellulose-acetate electrophoretic technique. This method distinguished the cell lines that were derived from the different species. Intraspecies distinctions were not made using the cell lines tested; the significance of this finding is discussed. The usefulness of this technique in identifying a potentially mislabeled cell line was demonstrated. This research was supported by contracts, DADA 17-72C-2170 of the U.S. Army and N00014-78C-0104 of the U.S. Office of Naval Research and grants from the World Health Organization and the Rockefeller Foundation.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

Gynaecological cancers and their cell lines

Cell lines are widely used for various research purposes including cancer and drug research. Recently, there have been studies that pointed to discrepancies in the literature and usage of cell lines. That is why we have prepared a comprehensive overview of the most common gynaecological cancer cell lines, their literature, a list of currently available cell lines, and new findings compared with the original studies. A literature review was conducted via MEDLINE, PubMed and ScienceDirect for reviews in the last 5 years to identify research and other studies related to gynaecological cancer cell lines. We present an overview of the current literature with reference to the original studies and pointed to certain inconsistencies in the literature. The adherence to culturing rulesets and the international guidelines helps in minimizing replication failure between institutions. Evidence from the latest research suggests that despite certain drawbacks, variations of cancer cell lines can also be useful in regard to a more diverse genomic landscape.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.     

Viral validation strategy for recombinant products derived from established animal cell lines

For products derived from continuous cell lines, regulatory agencies worldwide require that the purification process be validated for its ability to remove or inactivate potential contaminants such as viruses and virus-like particles. New guidance suggests a requirement for statistical evaluation of these studies but the industry has yet to develop such standards. The task of estimating excess capacity is also complicated by variable assays, accumulation of variability in clearance estimates over unit operations, dependence of clearance capacity on operating parameters, and expense of experiments. We propose an experimental strategy to determine the excess clearance capacity of a biopharmaceutical process and to provide statistical estimation of excess capacity in an efficient way. Clearance estimates and their variances are calculated for each orthogonal unit operation and estimates are combined to form an interval estimate of overall process clearance capacity. Poisson regression is suggested as an efficient technique for data analysis of clearance studies. We believe that this approach should meet regulatory guidelines in a cost effective way, while clarifying the roles of qualitative and quantitative components in setting requirements.     

三苯氧胺对乳腺癌和宫颈癌细胞增殖的影响

目的：研究三苯氧胺(tamoxifen,TAM)对人乳腺癌Bcap-37和宫颈癌HeLa细胞增殖的影响并探讨其可能的机制。方法：采用细胞培养、细胞计数、MTT、流式细胞术和激光共聚焦显微镜技术。结果：TAM(10^-6mol／L)使Bcap-37细胞的生长曲线下移,使HeLa细胞的生长曲线上移。TAM(10^-8～10^-6mol／L)剂量依赖性的抑制Beap-37细胞的增殖,促进HeLa细胞的增殖作用。TAM(10^-6mol／L)使Bcap-37细胞发生凋亡,凋亡率达到97．5％,而使HeLa细胞周期由G1期加速向S期转化,G1期的DNA含量由对照组的55．5％下降到加药组的32．8％,S期的DNA含量由对照组的29．0％上升到加药组的49．4％。激光共聚焦检测到TAM(10^-6mol／L)可使Bcap-37细胞和HeLa细胞内的Ca^2 浓度显著升高。结论：TAM可以通过调节细胞周期各阶段DNA含量和胞内Ca^2 浓度水平,从而调节Bcap-37细胞和HeLa细胞的增殖活动,提示使用TAM治疗乳腺癌时可能会对子宫颈产生副作用。     

Production of human-human hybridomas secreting monoclonal antibodies reactive to breast cancer cell lines

Summary We produced human-human hybridomas by fusing lymph node lymphocytes of breast cancer patients with a fusion partner, HO-323 cells, in the presence of 50% polyethylene glycol, and screened hybridomas producing monoclonal antibodiy (MoAb) reactive to a breast cancer cell line, MCF-7. Among 11 hybridomas secreting IgM reactive to MCF-7, four hybridomas (H15A3, H15F2, K15B4, and K15C3) produced IgM reacting specifically to breast cancer cell lines (MCF-7 and HBC-5). Among them, H15A3 proliferated in a serum-free (RDF) medium supplemented with insulin, transferrin, ethanolamine, selenium, and endothelial cell growth supplement, as fast as in 10% FCS-RDF medium. According to transfer blotting analysis, the H15F2 MoAb reacted with a 40 000 dalton antigen in MCF-7 cells. Editor's Statement Human monoclonal antibodies obviously hold great potential in diagnosis and treatment of disease. Although the antibodies described in this report are not entirely specific for breast cancer cells, the reactivity profile and ease of production as described here may make them a highly useful tool in both basic and applied biomedical research.     

Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels

Summary Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial-related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast. This work was supported by the North West Cancer Research Fund and the Cancer and Polio Research Fund.