Characterization of insulin-like growth factor 1 receptors (IGF1-R) in human breast cancer cell lines

The binding characteristics of IGF1 on membranes prepared from 5 human breast cancer cell lines were investigated in detail. The presence of one class of high affinity IGF1 binding sites was demonstrated (BT-20: n = 230 fmol/mg protein, Ka = 0.7 nM-1; MCF-7: n = 124 fmol/mg protein, Ka = 1 nM-1; T-47D: n = 61 fmol/mg protein, Ka = 1.1 nM-1; HBL-100: n = 18 fmol/mg protein, Ka = 3.2 nM-1; MDA-MB-231: n = 7 fmol/mg protein, Ka = 2.8 nM-1). Chemical cross-linking of 125I IGF1 to breast cancer cell membranes then sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed one major band of relative molecular weight 130000. The specificity of these receptors was studied: native or recombinant IGF1 had the same potency to inhibit 125I IGF1 binding; IGF2 was able to compete for binding, whereas insulin competed with a potency lower than 1/100 that of IGF1. These characteristics of IGF1 binding sites in breast cancer cell membranes correspond to the previously described binding unit of type I IGF receptors (IGF1-R). Finally we determined that for a routinely used standard assay, it was necessary to incubate for 5 h at 4 degrees C a high amount of membrane protein (400 micrograms) and 200,000 cpm of tracer. Considering the known effect of IGF1 on breast cancer cell multiplication, it is tempting to suggest that this factor might play a major role in the growth of breast cancer: the measurement of IGF1-R, using this standardized method, will give an assessment of these tumors IGF1 sensitivity; it can be performed on the membrane fraction obtained when preparing cytosol for steroid receptor assay.     

多药耐药相关蛋白1在三种乳腺癌细胞系中表达差异的研究

 目的　比较多药耐药相关蛋白1（MRP1）在人类乳腺癌敏感细胞（MCF-7／S）和耐药细胞（MCF-7／TAM、MCF-7／ADR）中的表达差异。初步探索乳腺癌细胞对三苯氧胺（TAM）和多柔比星（ADR）的耐药性的发生机制。方法　分别采用实时荧光定量PCR、Western blot技术检测MRP1基因和MRP1蛋白在3种乳腺癌细胞中的表达差异。结果　MRP1 mRNA在MCF-7／TAM和MCF-7／ADR细胞中表达量分别是MCF-7／S细胞的（2.63±0.18）倍和（8.38±0.76）倍,差异均有统计学意义（P＝0.004,P＝0.003）。MRP1蛋白在MCF-7／TAM细胞和MCF-7／ADR细胞中表达均高于MCF-7／S细胞,与MRP1 mRNA的趋势相一致。结论　MRP1的高表达可能是乳腺癌细胞对TAM和ADR产生耐药的共同发生机制。     

Caspase expression profile and functional activity in a panel of breast cancer cell lines

Caspases play an essential role in the initiation/regulation of apoptosis. Aberrant apoptotic regulation has been associated with carcinogenesis and therapeutic resistance. To explore the possible involvement of altered caspase expression in breast cancer, we have systematically examined the expression of both protein and mRNA levels of 7 caspases in a panel of 18 breast cancer cell lines. We found that variation of caspase expression can occur at both protein and RNA levels. Down-regulation of these caspases, especially caspase-8 and -10, was frequently observed. Functional screening of these selected cell lines using TNF-alpha, doxorubicin and radiation induced cell injury showed that a lack of functional caspase-8 resulted in resistance to TNF-alpha-induced apoptosis. Array style examination of caspase expression profiles in breast cancer cell lines yields massive information that is valuable in establishing cell line models to study the role of caspase down-regulation/deficiency in breast cancer development and therapeutic resistance.     

Lack of functional erythropoietin receptors of cancer cell lines

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.     

Sigma-1 receptors bind cholesterol and remodel lipid rafts in breast cancer cell lines

Lipid rafts are membrane platforms that spatially organize molecules for specific signaling pathways that regulate various cellular functions. Cholesterol is critical for liquid-ordered raft formation by serving as a spacer between the hydrocarbon chains of sphingolipids, and alterations in the cholesterol contents of the plasma membrane causes disruption of rafts. The role that sigma receptors play in cancer is not clear, although it is frequently up-regulated in human cancer cells and tissues and sigma receptors inhibit proliferation in carcinoma and melanoma cell lines, induce apoptosis in colon and mammary carcinoma cell lines, and reduce cellular adhesion in mammary carcinoma cell lines. In this study, we provide molecular and functional evidence for the involvement of the enigmatic sigma 1 receptors in lipid raft modeling by sigma 1 receptor-mediated cholesterol alteration of lipid rafts in breast cancer cell lines. Cholesterol binds to cholesterol recognition domains in the COOH terminus of the sigma 1 receptor. This binding is blocked by sigma receptor drugs because the cholesterol-binding domains form part of the sigma receptor drug-binding site, mutations of which abolish cholesterol binding. Furthermore, we outline a hypothetical functional model to explain the myriad of biological processes, including cancer, in which these mysterious receptors are involved. The findings of this study provide a biological basis for the potential therapeutic applications of lipid raft cholesterol regulation in cancer therapy using sigma receptor drugs.     

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。     

Antiestrogens modulate MT1 melatonin receptor expression in breast and ovarian cancer cell lines

An interaction between cellular estrogen response and melatonin signaling mediated by G-protein coupled receptors is present in breast cancer cells. In this study, the effect of antiestrogens on basal and melatonin-modulated expression of MT1 melatonin receptor in breast and ovarian cancer cells was examined. For this purpose, the effects of the selective estrogen receptor modulator tamoxifen and pure antiestrogen ICI 182,780 on MT1 expression in estrogen receptor (ER) alpha-positive and -negative breast and ovarian cancer cell lines cultured in medium supplemented with 1 nM 17-beta estradiol were assessed by Western blot analysis. We were able to detect expression of the MT1 receptor in SK-OV-3 and OVCAR-3 cells and report its up-regulation by melatonin in both ovarian cancer cell lines. MT1 expression was observed to be significantly weaker in ERalpha-positive MCF-7 and OVCAR-3 cells than in ERalpha-negative MDA-MB-231 and SK-OV-3 cells. Treatment with the pure antiestrogen ICI 182,780 increased MT1 receptor expression in OVCAR-3 ovarian cancer cells, but decreased MT1 expression in MCF-7 breast cancer cells. No effect of ICI 182,780 on MT1 expression was observed in the ERalpha-negative cell lines SK-OV-3 and MDA-MB-231. After treatment with 4-OH tamoxifen, down-regulation of basal MT1 receptor expression in ERalpha-positive MCF-7 cells and inhibition of melatonin-induced up-regulation of MT1 in OVCAR-3 ovarian cancer cells were observed. In contrast, treatment with 4-OH tamoxifen increased the MT1 receptor level in ERalpha-negative SK-OV-3 ovarian cancer cells. Our findings support the existence of close interaction between estrogen and melatonin signaling. Moreover, our data suggest that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells.     

Regulation of ICAM-1 (CD54) expression in human breast cancer cell lines by interleukin 6 and fibroblast-derived factors

Four human breast cancer cell lines (T47D, ZR-75-1, MCF7D and HS578T) were examined for the effects of cytokines on expression of cell surface antigens. Interferon (IFN)γ up-regulated the expression of 1CAM-1 (CD54) in all the cell lines and coordinately up-regulated both CD54 and CD40 expression in T47D. Tumour necrosis factor (TNF)α, interieukin (IL)Iα ILIβ and IL6 also up-regulated the expression of CD54 in all the cell lines but CD40 was unaffected. Levels of expression of CD IIa, CDI8, CD49b, CD58 and CD71 were unaltered by these cytokines. Conditioned medium (CM) generated from human fibroblasts, and Fn particular from foetal cells, was highly effective in up-regulating expression of ICAM-I but not of CD40 in the breast cancer cell lines. 1CAM-1 induction correlated with IL6 bioactivity in these CMs. Combinations of IL6 with other cytokines, such as ILI, resulted in further increases in ICAM-1 expression. Our observations suggest that IL6 is involved in intercellular signalling between mesenchyme and breast cancer epithelium. © 1994 Wiley-Liss, Inc.     

不同人乳腺肿瘤细胞系中Jagged1基因和蛋白的表达水平

目的:研究Jagged1蛋白和mRNA在人乳腺癌细胞系中的表达。方法:分别使用蛋白印迹法及实时定量聚合酶链式反应检测Jagged1蛋白和基因在人乳腺癌细胞系中的表达情况。结果:Jagged1在不同的人乳腺肿瘤细胞系中蛋白和基因的表达水平不同。结论:Jagged1蛋白在人乳腺癌细胞系ZR-75-30和MDA-MB-231中高表达,Jagged1基因在人乳腺癌细胞系MDA-MB-231,HCC 1937中高表达,Jagged1可能是在人乳腺癌的发展进程中发挥着重要的作用。     

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.     

雌激素对人乳腺癌细胞系基质细胞衍化因子-1影响的观察

目的:探讨雌激素对基质细胞衍化因子-1(SDF-1)的影响.方法:选取乳腺癌细胞系MCF-7和MDA-MB-231为研究对象,分成对照组、雌激素组和雌激素+雌激素受体阻断组,分别加入不同生理浓度的17-β雌二醇作用一定时间以及同一浓度17-β雌二醇作用不同时间点,用酶联免疫吸附实验(ELISA)方法测定培养液中SDF-1的浓度,半定量逆转录聚合酶链反应(RT-PCR)法检测细胞SDF-1 mRNA的表达.结果:MDA-MB-231细胞系加与未加雌激素,均未检测到SDF-1的分泌.而MCF-7细胞基础培养液中可检测到SDF-1分泌.不同生理浓度的17-β雌二醇均可增加MCF-7细胞SDF-1的分泌水平.当加入1×10-7 mol/L的生理高浓度17-β雌二醇时,细胞分泌SDF-1水平在2 h达到高峰,是对照组的6倍[(1 823.17±325.18)ρg/mL vs (307.23±5.42)ρg/mL,F=201.02,P<0.01],该作用可被雌激素受体拮抗剂(ICI182,780)消除.此外,SDF-1mRNA的表达水平与测得的SDF-1蛋白水平相一致.结论:在某些乳腺癌细胞系,生理浓度的雌激素可促进SDF-1的分泌,而这种作用主要是通过雌激素受体来实现.雌激素可通过SDF-1来影响乳腺癌的生物学特性.     

RGS5在乳腺癌组织和细胞系中的表达和定位

目的:探讨G蛋白信号转导调节蛋白5（RGS5）在人乳腺癌细胞系中的表达。方法:提取人正常乳腺上皮细胞HBL100,人乳腺癌细胞系MCF-7,ZR-75-1,ZR-75-30,MDA-MB-231,HCC1937的总蛋白和总RNA,提取细胞的总蛋白和总RNA进行蛋白免疫印迹和实时定量PCR检测RGS5的表达。共聚焦激光扫描显微镜观察RGS5胞内定位;免疫组化检测RGS5在乳腺肿瘤组织中表达情况。结果:MDA-MB-231细胞系中RGS5基因表达上调,MCF7和ZR-75-1细胞系中RGS5蛋白表达提高。结论:RGS5在乳腺癌细胞的高表达可能参与其生长和转移,揭示RGS5可能是人乳腺肿瘤的治疗靶点。     

p57kip2蛋白在乳腺癌组织和细胞系中的表达和定位

目的：研究p57kip2在人乳腺癌组织和细胞系中的表达和定位.方法：免疫印迹法检测人乳腺肿瘤细胞系中p57kip2的表达.人乳腺肿瘤组织进行免疫组化染色,检测p57kip2在组织中的表达.激光共聚焦扫描显微镜检测内源p57kip2在MCF-7细胞中的定位.结果：p57kip2在乳腺癌细胞系内表达,免疫组化染色分析和激光共聚焦扫描显微镜证实p57kip2主要定位于细胞核内.结论：p57kip2在乳腺癌细胞内表达,p57kip2主要定位于乳腺癌组织细胞核内,可能是参与肿瘤细胞基因转录调控和蛋白表达的重要蛋白之一.     

Insulin receptor expression and function in human breast cancer cell lines.

We have previously reported that insulin receptor expression is increased in human breast cancer specimens (V. Papa et al., J. Clin. Invest., 85:1503-1510, 1990). In the present study, in order to further understand the role of the insulin receptor in breast cancer, insulin receptor expression and function were characterized in three human breast cancer cell lines, MCF-7, ZR-75-1, and T-47D, and compared to a nonmalignant human breast epithelial cell line, 184B5. Insulin receptor content, measured by radioimmunoassay, was elevated 5- and 3-fold in MCF-7 and ZR-75-1 breast cancer cell lines, respectively, when compared to the nonmalignant cell line 184B5. In contrast, the insulin receptor content of T-47D cells was not increased. The increase in insulin receptor content in MCF-7 and ZR-75-1 cells was not due to amplification of the insulin receptor gene. Also, total insulin receptor mRNA content was not increased in breast cancer cells in respect to nonmalignantly transformed 184B5 breast epithelial cells. However, significant differences in the content of receptor mRNA species were observed. The insulin receptors in the breast cancer cell lines were functional: (a) In all 4 cell lines, high-affinity insulin-binding sites were detected, and, in concert with the insulin receptor radioimmunoassay data, binding capacity was highest in MCF-7 and then in ZR-75-1 cells. (b) In all cell lines, insulin stimulated insulin receptor tyrosine kinase activity. However, the effect of insulin was greater in breast cancer cell lines than in nonmalignant breast cells. (c) In all cell lines, insulin at concentrations of 1 nM or less stimulated [3H]thymidine incorporation. This effect of insulin was inhibited by 50% in MCF-7 cells and by 60% in 184B5 cells when alpha-IR3, a monoclonal antibody to the insulin-like growth factor I receptor, was present. In these cells, therefore, insulin was active via both its own receptor and the IGF-I receptor. In contrast, alpha-IR3 antibody was without effect in T-47D and ZR-75-1 cells, suggesting that in these cell lines insulin acted only via its receptor. In the breast cancer cells, MA-5, an agonist monoclonal antibody to the insulin receptor, stimulated [3H]thymidine incorporation. This present study indicates therefore that in breast cancer cell lines there are functional insulin receptors that regulate breast cancer cell growth.     

Molecular characterization of breast cancer cell lines by a low-density microarray

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors.     

Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in breast cancer cell lines

The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a tumor suppressor gene that is altered mainly by deletion in a large fraction of human tumors, including breast cancers. To evaluate the potential of FHIT gene therapy in this type of cancer, we have studied the biological effects of adenoviral FHIT transduction (Ad-FHIT) in breast cancer cell lines. The results showed that, after FHIT restoration in BT-549, MDA-MB-436, and HCC1806 cells, they underwent apoptosis by activation of the intrinsic pathway. In all three cell lines infected with Ad-FHIT, we have found activation of caspase-2, which is required for permeabilization of mitochondria, release of cytochrome c, and apoptosis. Furthermore, Fhit overexpression produces alteration in cell cycling properties, as well as reduction of the tumorigenic potential in nude mice.     

Metabolism of resveratrol in breast cancer cell lines: impact of sulfotransferase 1A1 expression on cell growth inhibition

Resveratrol is a polyphenolic compound present in grapes and wine with anticancer activities that undergoes pronounced metabolism in humans. In order to determine whether metabolism of resveratrol also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MB-MDA-231 human breast cancer cells. Along with resveratrol, it was possible to identify one metabolite, namely, resveratrol-3-O-sulfate in both cell lines. Its concentration in the cytoplasm and culture medium was 5.4- to 9-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 3.1-fold higher IC(50) value in the ZR-75-1 cell line (74 microM compared to 38 microM). By using RT-PCR, expression of sulfotransferase (SULT)1A1 mRNA, but not of other SULTs investigated, showed a close correlation with resveratrol 3-O-sulfate formation which was particularly high in ZR-75-1 and very low in MDA-MD-231 cells. In conclusion, we demonstrate that SULT1A1-based biotransformation reduces the anticancer activity of resveratrol in breast cancer cells, which must be considered in humans following oral uptake of dietary resveratrol as a chemopreventive agent.