Metastasis from human breast cancer cell lines

Summary Immunodeficient animals, principally nude mice, when used in appropriately designed studies have been shown to be useful for the experimental analysis of human breast cancer metastasis. As with many other human tumors, the implantation of breast cancer cells into an anatomically appropriate tissue (the mammary fatpad) results in increased tumor take and incidence of metastasis for certain cell lines compared with subcutaneous injection. Testing a number of widely available human breast cancer cell lines identified the MDA-MB-435 cell line as the most metastatic, producing lung and lymph node metastases in a high proportion of nude and severe combined immunodeficient (SCID) mice after injection in the mammary fatpad. Mixing human breast cancer cells with normal fibroblasts or with Matrigel also increases the tumor incidence and growth rates in nude mice. Different routes of injection can be used to assess the ability of human breast cancer cells to form metastatic lesions in the lungs (i.v. injection), the liver (injection in the spleen), the brain (direct or intracarotid artery injection) and the bone marrow and bone (injection into the left ventricle of the heart). These different approaches demonstrate the potential of experimental studies of human breast cancer growth and metastasis using immunodeficient mice; this model is valuable for experiments that test the role of metastasis-associated genes and the efficacy of novel forms of therapy.     

New cell lines of human breast cancer origin

Summary Established human mammary tumor cell lines constitute an important tool in the study of breast cancer. The aim of this work was to isolate and characterize two new mammary tumor cell lines, JCK and GCS, which were obtained from the pleural effusion and ascitic fluid, respectively, from two breast cancer patients. Both cell lines had some properties of transformed cells, namely immortalization and growth in soft agar. The carcinoma cells presented epithelial morphology shown by light and electron microscopy, and antigenic properties shown by different tumor markers such as a cytokeratin cocktail, carcinoembryonic antigen, epithelial membrane antigen, and human milk fat globule membrane antigen. A significant increase was also found (P>0.05) in cell growth and³H-thymidine incorporation into DNA in the JCK and GCS cell lines in the presence of 17 estradiol at concentrations of 10^–9 and 10^–7 M, respectively, after 5 days in culture. These cells presented estradiol receptor levels which were similar in the biopsies and the resulting cell lines. The aromatase activity was also similar in the JCK cell line and the original patient biopsy. However, there was a considerably higher aromatase activity in the GCS cell line than in the biopsy specimen. Southern hybridizations with theneu oncogene showed an additional 12 kb fragment in both cell lines, as also seen in patients with breast cancer. We conclude from these studies that thisin vitro system may provide us with a way to study metastatic cells and improve clinical management of breast cancer patients.     

Transcription profiles of non-immortalized breast cancer cell lines

Background  

Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed.     

Molecular cytogenetic analysis of breast cancer cell lines

The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/ approximately pawefish.     

Hormonal control of human breast cancer cell lines

The most widely studied steroid hormone responsive cancers are those of the prostate, endometrium and mammary endothelium and the leukaemias. Steroids may in some cases be involved in tumour initiation, and they induce critical events in the malignant 'progression' of these cancers once they develop (after exposure to carcinogens, for example). This paper focuses on the role of the oestrogen receptor as a trigger of cellular growth and invasiveness and as modulator of growth factor secretion in human breast cancer. Growth factor secretion may be a common final pathway of diverse tumorigenic stimuli in a variety of cancers. The finding of external growth control mechanisms suggests possible new ways of therapeutic intervention in breast cancer.     

黑色素瘤抗原基因A1在乳腺癌组织和四株乳腺癌细胞株中的表达

目的研究乳腺癌组织和细胞株中Mage A1基因的表达情况，为Mage A1基因编码蛋白用于乳腺癌免疫治疗提供依据。方法采用RT—PCR检删33例乳腺癌组织和4株常用乳腺癌细胞株MCF-7、Sk-Br-3，MDA-MB-435s和TM40D中Mage A1基因的表达。结果33例乳腺癌组织中12％(4／33)Mage—A1阳性，细胞株MCF-7和Sk-Br-3中Mage-A1阳性表达，MDA-MB-435s和TM40D中Mage A1基因的表达阴性。结论不同的乳腺癌细胞表达Mage A1基因有差别，Mage A1蛋白可望成为乳腺癌免疫治疗的靶抗原。     

表达MT1-MMP的乳腺癌细胞株对蒽环类药物敏感性研究

目的观察表达MT1-MMP的乳腺癌细胞株对蒽环类化疗药物敏感性的影响。方法用脂质体Lipofectamine 2000将pcDNA3.1-MT1-MMP真核表达载体转染乳腺癌细胞株MDA-MB-453,然后用含G418的培养基筛选获得过表达MT1-MMP的乳腺癌细胞株。蛋白印迹（Western blotting）技术检测MDA-MB-453细胞内MT1-MMP的表达情况。四唑蓝（MTT）法检测各组细胞对化疗药物的敏感性。结果 Western blotting实验证实,重组载体pcDNA3.1-MT1-MMP真核表达载体成功转入乳腺癌细胞内,并稳定过表达MT1-MMP。MTT法检测发现,阿霉素（ADH）、表阿霉素（EPI）、吡喃阿霉素（THP）转染组细胞的抑制率分别为41.38±1.34%、37.69±1.93%和57.96±3.19%,均明显低于未转染组（70.22±2.55%、70.35±1.21%、76.97±2.70%）和空白质粒组（68.94±1.89%、69.43±1.27%、73.06±1.65%,P〈0.05）。结论表达MT1-MMP的乳腺癌细胞株MDA-MB-453对蒽环类化疗药物具有耐药性,MT1-MMP有可能成为一个新的蒽环类化疗药物敏感性预测因子。     

THE EFFECTS OF ESTRADIOL ON BREAST CANCER CELL LINES

The estradiol effects on breast cancer cell lines including estrogen receptors (ER) positive and negative were studied with flow cytometry analysis, scanning and transmission electron microscopy (SEM and TEM), immunogold and immunofluorescence staining techniques. The results showed that estradiol markedly stimulated the division and proliferation of the ER( ) MCF-7 cells at 10 nM, but had no marked effect on the cell cycle of the ER(-) H466B cells at the same concentration, and that tamo-xifen inhibited the stimulation of estradiol on MCF-7 cells. Estradiol obviously influenced the ultrastruc-ture of MCF-7 cells. Immunocytochemical localization of epidermal growth factor receptor (EGFR) on the MCF-7 cell membrane surface indicated that one of the mechanisms involving the growth of MCF-7 breast cancer cell and the stimulating effect on MCF-7 cells growth by estradiol is autocrine secretion.     

Differential effects of bisphosphonates on breast cancer cell lines

Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.     

Sensitivity of human hematopoietic cell lines to an alkyl-lysophospholipid-derivative

A panel of 16 human hematopoietic cell lines was tested for sensitivity to racemic 1-0-octadecyl-2-0-methyl-glycero-3-phosphocholine (ALP). Effects on proliferation rate, cell cycle distribution, viability and morphology were examined. Cell growth was heavily retarded for all lines at an ALP concentration of 10 micrograms ml-1, which induced profound cell death. 1 microgram ml-1 of ALP was a suitable concentration to make comparisons between the different cell lines. In sensitive lines cell growth was retarded due to induction of multinucleated cells and cytotoxicity. The multinucleated cells could proceed through the cell cycle but cell division was inhibited. The most ALP sensitive cell lines (MN-60, P3Hr-1, Um-42 and RPMI 8226) were B-cell lines, whereas the most resistant lines (U-61M, K562-4 and CCRF-CEM) were of different cell origin. In conclusion, a variable response to ALP was found in 16 human hematopoietic cell lines. As a rule sensitivity to ALP was demonstrated as increased cell death and induction of cells with less than or equal to 2 nuclei, leading to a decreased cell number as compared to control cells.     

Platinum resistance in breast and ovarian cancer cell lines

Background

MDR1 gene encoding P-glycoprotein is an ATP-dependent drug efflux transporter and related to drug resistance of yolk sac carcinoma. Ultrasound microbubble-mediated delivery has been used as a novel and effective gene delivery method. We hypothesize that small interfering RNA (siRNA) targeting MDR1 gene (siMDR1) delivery with microbubble and ultrasound can down-regulate MDR1 expression and improve responsiveness to chemotherapeutic drugs for yolk sac carcinoma in vitro.

Methods

Retroviral knockdown vector pSEB-siMDR1s containing specific siRNA sites targeting rat MDR1 coding region were constructed and sequence verified. The resultant pSEB-siMDR1 plasmids DNA were encapsulated with lipid microbubble and the DNA release were triggered by ultrasound when added to culture cells. GFP positive cells were counted by flow cytometry to determine transfection efficiency. Quantitative real-time PCR and western blot were performed to determine the mRNA and protein expression of MDR1. P-glycoprotein function and drug sensitivity were analyzed by Daunorubicin accumulation and MTT assays.

Results

Transfection efficiency of pSEB-siMDR1 DNA was significantly increased by ultrasound microbubble-mediated delivery in rat yolk sac carcinoma L2 (L2-RYC) cells. Ultrasound microbubble-mediated siMDR1s delivery effectively inhibited MDR1 expression at both mRNA and protein levels and decreased P-glycoprotein function. Silencing MDR1 led to decreased cell viability and IC₅₀ of Vincristine and Dactinomycin.

Conclusions

Our results demonstrated that ultrasound microbubble-mediated delivery of MDR1 siRNA was safe and effective in L2-RYC cells. MDR1 silencing led to decreased P-glycoprotein activity and drug resistance of L2-RYC cells, which may be explored as a novel approach of combined gene and chemotherapy for yolk sac carcinoma.     

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.     

HMG-I/Y in human breast cancer cell lines

The HMG-I/Y gene encodes the HMG-I and -Y architectural, chromatin binding proteins originally identified based on their association with chromosomal DNA. HMG-I/Y proteins bind to AT-rich regions in chromosomal DNA and alter gene expression. Increased HMG-I/Y protein expression also correlates with neoplastic transformation. Previous work from our laboratory has shown that HMG-I/Y is a direct c-Myc target gene involved in neoplastic transformation in Burkitt's lymphoma. We also observed that HMG-I/Y proteins have several oncogenic properties. In this report, we show that HMG-I/Y proteins are increased in several human breast cancer cell lines compared to a human breast cell line derived from normal breast cells. Decreasing HMG-I/Y proteins using an antisense ribozyme approach inhibits transformation in human breast cancer cells, suggesting that HMG-I/Y is important for the transformed phenotype observed in these cells. In addition, increased expression of the HMG-I isoform in normal human breast cells leads to transformation. These results suggest that HMG-I/Y is an oncogene important in the pathogenesis of human breast cancer. Although additional studies with animal models are needed, the antisense experiments, which result in blocking transformation suggest that this approach may have therapeutic potential in patients with breast cancer characterized by increased HMG-I/Y expression.     

Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein nigrin b

The cytotoxicity of the type 2 ribosome-inactivating proteins (RIPs) ricin and nigrin b was determined in a variety of cancer cells. Nigrin b, considered to be a novel non-toxic type 2 RIP as compared with ricin, was approximately 10(4)-10(5) times less toxic than ricin in all cancer cells studied, with the exception of melanoma cells. Cancer cells displayed considerable heterogeneity in their sensitivity to ricin, melanoma cells being the least sensitive. Rabbit polyclonal anti-nigrin b antibodies did not cross-react with ricin as analyzed by enzyme-linked immunosorbent assays. The low non-specific toxicity of nigrin b as compared with that of ricin and the lack of immunological cross-reaction between anti-nigrin b antibodies and ricin supports the use of nigrin b in the construction of cytotoxic conjugates as an alternative to ricin when anti-ricin antibodies are produced during cancer therapy.     

Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment

The pure steroidal anti-estrogens ICI 164,384 and ICI 182,780 are very potent growth inhibitors of the estrogen receptor-positive human breast cancer cell line MCF-7. However, long-term treatment of MCF-7 cells with 10^?7 M concentrations of these compounds results in selection of proliferating colonies of resistant cells. Our report describes 4 ICI 164,384- and 3 ICI 182,780-resistant MCF-7 sublines established after long-term treatment. Resistant sublines are estrogen receptor-positive, and all sublines have lost expression and estrogen inducibility of the progesterone receptor protein. Based on IC₅₀ concentrations, all tested resistant sublines had a reduced sensitivity to pure anti-estrogens on the order of 100- to 1000-fold compared with parent MCF-7 cells. All resistant cell lines have survived propagation for more than 15 subcultivations in the presence of 10^?7 M pure anti-estrogen. The MCF-7/182^R-6 subline has been tested for stability of resistance and appeared to be stably resistant after 13 weeks of propagation without the selective pressure of ICI 182,780. Cell lines resistant to the ICI 182,780 compound are cross-resistant to the ICI 164,384 compound and vice versa. However, the sublines resistant to pure anti-estrogens are sensitive to tamoxifen. Our results show that although the pure steroidal anti-estrogens are very potent growth inhibitors, they do not circumvent development of resistance. © 1995 Wiley-Liss, Inc.     

Comparative analysis of xanafide cytotoxicity in breast cancer cell lines

Xanafide, a DNA-intercalating agent and topoisomerase II inhibitor, has previously demonstrated comparable cytotoxicity to the parent drug amonafide (NSC 308847). The current study was conducted to investigate further the anti-proliferative effects of xanafide in human breast cancer cell lines, in vitro and in vivo. The in vitro activity of xanafide against MCF-7, MDA-MB-231, SKBR-3 and T47D cell lines was compared to that of paclitaxel, docetaxel, gemcitabine, vinorelbine and doxorubicin. In MCF-7, xanafide demonstrated comparable total growth inhibition (TGI) concentrations to the taxanes and lower TGI values than gemcitabine, vinorelbine and doxorubicin. MCF-7 (oestrogen receptor (ER)+/p53 wild-type) was the most sensitive cell line to xanafide. MDA-MB-231 and SKBR-3 exhibited similar sensitivity to xanafide. T47 D (ER+/p53 mutated), showed no response to this agent. The in vivo activity of xanafide was further compared to that of docetaxel in MCF-7 and MDA-MB-231 cell lines using the hollow fibre assay. Xanafide was slightly more potent than docetaxel, at its highest dose in MCF-7 cell line, whereas docetaxel was more effective than xanafide in MDA-MB-231 cell line. Our results show that there is no relationship between sensitivity of these cell lines to xanafide and cellular levels of both isoforms of topoisomerase II and suggest that ER and p53 status and their crosstalk may predict the responsiveness or resistance of breast cancer patients to xanafide.     

Molecular cytogenetic analysis of 11 new breast cancer cell lines

We describe a survey of genetic changes by comparative genomic hybridization (CGH) in 11 human breast cancer cell lines recently established in our laboratory. The most common gains took place at 8q (73%), 1 q (64%), 7q (64%), 3q (45%) and 7p (45%), whereas losses were most frequent at Xp (54%), 8p (45%), 18q (45%) and Xq (45%). Many of the cell lines displayed prominent, localized DNA amplifications by CGH. One-third of these loci affected breast cancer oncogenes, whose amplifications were validated with specific probes: 17q12 (two cell lines with ERBB2 amplifications), 11q13 (two with cyclin-D1), 8p11-p12 (two with FGFR1) and 10q25 (one with FGFR2). Gains and amplifications affecting 8q were the most common genetic alterations in these cell lines with the minimal, common region of involvement at 8q22-q23. No high-level MYC (at 8q24) amplifications were found in any of the cell lines. Two-thirds of the amplification sites took place at loci not associated with established oncogenes, such as 1q41-q43, 7q21-q22, 7q31, 8q23, 9p21-p23, 11p12-p14, 15q12-q14, 16q13-q21, 17q23, 20p11-p12 and 20q13. Several of these locations have not been previously reported and may harbour important genes whose amplification is selected for during cancer development. In summary, this set of breast cancer cell lines displaying prominent DNA amplifications should facilitate discovery and functional analysis of genes and signal transduction pathways contributing to breast cancer development.     

Production of PDGF-like growth factor by breast cancer cell lines

The breast cancer cell line T47D produces factors which show mitogenic activity for 3T3 cells. Here we describe the partial purification of one of these factors and show that it has properties similar to platelet-derived growth factor (PDGF). The T47D factor is a heat-stable hydrophobic protein with a molecular weight of around 30,000, which inhibits the binding of 125I-EGF in a temperature-dependent manner. This 30 kd protein does not act synergistically with PDGF or fibroblast-derived growth factor (FDGF; also PDGF-like) in stimulating DNA synthesis; moreover, like these two factors, its mitogenic activity can be inhibited by an antiserum raised against human PDGF. A PDGF-like growth factor was also found in the serum-free medium of the breast cancer cell line MDA-MB-157. Since PDGF acts on mesenchymal cells, the production of PDGF-like growth factors by breast cancer cells suggests a basis for the intense stromal reaction seen in human breast cancers.     

Differentiation state and invasiveness of human breast cancer cell lines

Summary Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO-1, vimentin, keratin and ₁ and ₄ integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in anin vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in thein vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best within vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47D_CO, the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.