Serum regulation of the estrogen responsiveness of the human breast cancer cell line MCF-7

Initially after receiving MCF-7 cells, we were able to confirm their estrogen responsiveness. We observed significant increases in thymidine incorporation, in thymidine kinase activity, and in cell numbers in response to 10(-8) M estradiol. Subsequently, however, the cells failed to show a response to estradiol. A growth response to estradiol could be restored by increasing the serum concentration in the medium. Cells grown in 15% serum (calf or human) responded to estradiol with increased rates of growth and thymidine incorporation and increased activities of thymidine kinase and DNA polymerase. We suggest that there is present in serum a "factor" which can influence the expression of a growth response to estradiol.     

Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture

MCF-7 human breast cancer cells, grown in long-term tissue culture, were found to be highly responsive to prolactin in terms of growth even in the presence of serum. Human prolactin, placental lactogen, and growth hormone (50-250 ng/ml) stimulated MCF-7 cells to grow when added to culture medium of cells in the presence of charcoal-stripped serum. Within 3 days of the hormone addition, a 4.4-fold increase in cell number was achieved with human prolactin at 100 ng/ml in the presence of 10% charcoal-stripped serum. Under these same conditions, estradiol-17 beta at 10(-8) M achieved only a 2-fold increase. After 6 days of culture, both estradiol-17 beta and prolactin gave a total 5-fold increase in cell number. No prolactin effect was achieved in the presence of 10% fetal bovine serum. Stripping fetal bovine serum with dextran-coated charcoal removes as much as 85% of the endogenous lactogens. Removal of these hormones is essential for demonstration of subsequent prolactin-induced growth response in MCF-7 cells, since bovine prolactin binds effectively to lactogen receptors on the surface of the cells but does not transmit a growth signal. When added simultaneously with human prolactin, bovine prolactin blocks the growth response to the former hormone. These results clearly demonstrate that, under the proper conditions of culture, the human breast cancer cell line MCF-7 is highly responsive to growth stimulation by homologous lactogenic hormones. This then affords us an excellent model for further studies on the possible role of prolactin in growth and maintenance of human breast cancer.     

Regulation of human estrogen receptor gene, epidermal growth factor receptor gene, and oncogenes by estrogen and antiestrogen in MCF-7 breast cancer cells

It is generally believed that estrogen may act either as an initiator or as a promoter in carcinogenesis of human breast cancer. This estrogenic action is generally dependent on the estrogen receptor. In the human estrogen receptor, cDNA has a homology to V-erb-A oncogene. Experiments using MCF-7 human breast cancer cells were carried out to study the regulatory effect of estrogen and antiestrogen on RNA activities of oncogenes, estrogen receptor gene, and epidermal growth factor (EGF) receptor gene. The effect of estradiol on activation of estrogen and EGF receptor genes and myc, ras, and fos oncogenes was positive in relation to the concentrations of supplemented estradiol. In addition, the effects of antiestrogen (tamoxifen) were investigated. Tamoxifen suppressed MCF-7 cell growth, and spot hybridization of the RNA of MCF-7 cells revealed that RNA activities of estrogen and EGF receptor genes and myc, ras, and fos oncogenes were suppressed by tamoxifen. These results suggest that the three oncogenes and two receptor genes are partly regulated by estrogen and antiestrogen (tamoxifen) in MCF-7 human breast cancer cells. This regulatory system may have a role in carcinogenesis and in the treatment of human breast cancer.     

Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.     

Effect of prolactin on growth and the estrogen receptor level of human breast cancer cells (MCF-7).

The intracellular specific 17beta-estradiol binding in the human breast cancer cell line, MCF-7, was shown to be modified by prolactin. Both ovine and human prolactin doubled the estradiol receptor (E2R) level, but the latter was at least 10 times more stimulatory on a concentration basis. Most of the E2R complex (approximately 80%) was transported to the nucleus, and the prolactin stimulation was reflected in an elevated nuclear uptake of the tritiated 17beta-estradiol. Neither ovine nor human prolactin altered the growth rate of the cells when E2R stimulation was maximal. Insulin (10 mug/ml) stimulated tritiated thymidine incorporation and total DNA content but had no apparent effect on E2R concentration. At 10(-4) M, N6,O2'-dibutyrylcyclicadenosine 3':5'-monophosphate increased insulin stimulation of tritiated thymidine incorporation and brought about a prolactin stimulation of apparent DNA synthesis. Theophylline (10(-3) M) blocked both of these effects of N6,O2'-dibutyrylcyclicadenosine 3':5'-monophosphate. The possible mechanism implicating prolactin as an effector of differentiation and growth of MCF-7 cells is discussed.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds on the occupied nuclear estrogen receptor in MCF-7 human breast cancer cells

Treatment of MCF-7 human breast cancer cells with 17 beta-[3H]estradiol resulted in a rapid accumulation of occupied nuclear estrogen receptor complex in which levels were maximized within 1 h and decreased after 3 h. Pretreatment of the cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 6 or 12 h prior to the addition of the radiolabeled hormone resulted in a 38 and 63% reduction in the levels of the occupied nuclear estrogen receptor, respectively, whereas addition of TCDD 1 h prior to the radioligand did not cause any significant change in the levels of the occupied nuclear receptor using velocity sedimentation analysis. Moreover, it was also shown with estrogen receptor antibodies that the TCDD-mediated dose-dependent decrease in occupied nuclear receptor levels was paralleled by a comparable decrease in immunoreactive protein at concentrations of 10(-8) to 10(-11) M TCDD. The reduction in levels of the occupied nuclear estrogen receptor was not due to increased estradiol metabolism since a significant reduction was observed at TCDD concentrations (10(-11) to 10(-13) M) which do not induce cytochrome P-450-dependent monooxygenase enzyme activities in MCF-7 cells. Treatment of the MCF-7 cells with actinomycin D or cycloheximide resulted in a greater than 2-fold increase in levels of the occupied nuclear estrogen receptor, and cotreatment of the cells with both TCDD and these inhibitors significantly decreased levels of the nuclear estrogen receptor complex. The structure-activity relationships for TCDD and several congeners were similar for both the reduction of occupied nuclear estrogen receptor levels and several aryl hydrocarbon (Ah) receptor agonist activities, and these results support a role of the Ah receptor in these processes.     

Regulation of estrogen sulfotransferase by estrogen in MCF-7 human mammary cancer cells

Summary The importance of the steroid hormone microenvironment within cells is now recognised in studies on endocrine-related neoplasms such as breast cancer. This focuses attention on ezymes which control the intracellular levels of estradiol-17 (E₂). One such enzyme, estrogen sulfotransferase, which converts E₂ to inactive E₂-3 sulfate, has now been shown to be regulated by estrogen in MCF-7 human mammary cancer cells. Hydroxysteroid sulfotransferase, which sulfurylates the adrenal-derived estrogen 5-androstene-3,17-diol, is also under estrogen control. Evidence is provided which shows that one function of these enzymes may involve elimination of estrogen from the cell following processing of the ligand-charged estrogen receptor (ER).     

Interleukin 1 alpha blocks estradiol-stimulated growth and down-regulates the estrogen receptor in MCF-7 breast cancer cells in vitro

We studied the effect of interleukin 1 alpha (IL-1 alpha) on estradiol stimulation of cell growth and estrogen receptor (ER) content in MCF-7 human breast cancer cells in vitro to determine if IL-1 alpha altered cellular estradiol responsiveness. We found that IL-1 alpha blocked estradiol-stimulated growth of these cells in a dose-dependent manner (complete antagonism at 1000 units/ml: day 7 mean growth = vehicle, 47.7 micrograms DNA; estradiol 10(-10) M, 95.1; IL-1 alpha/estradiol, 44.6) and at all concentrations of estradiol from 10(-8) to 10(-11) M. IL-1 alpha in combination with trans-hydroxytamoxifen further inhibited estradiol-stimulated growth (vehicle = 44.8 micrograms DNA, estradiol = 108.3, estradiol/trans-hydroxytamoxifen = 47.8, IL-1 alpha/estradiol/trans-hydroxytamoxifen = 3.0, P less than 0.01). Inhibition with trans-hydroxytamoxifen was IL-1 alpha dose dependent (maximum = 97% at 1000 units/ml, P less than 0.01) and estradiol dose dependent (reversible with 10(-8) M estradiol, maximum inhibition at 10(-10) M estradiol). Concomitantly, IL-1 alpha down-regulated ER concentration by 38.0-43.7% (P less than 0.01) as measured by immunoreactivity or Scatchard analysis, respectively. This occurred as early as 3 h without a change in the Kd (vehicle = 0.23 nM, IL-1 alpha = 0.24 nM), persisted for at least 48 h, was dose dependent (maximum, 43.7% at 1000 units/ml, P less than 0.01), and was blocked by cycloheximide. IL-1 alpha, however, did not block estradiol stimulation of progesterone receptor content (vehicle = 221.9, IL-1 alpha = 238.9 fmol/mg protein) and did not block estradiol down-regulation of ER content. Furthermore, IL-1 alpha alone did not alter levels of ER mRNA and did not alter estradiol down-regulation of ER mRNA. These findings indicate that while IL-1 alpha antagonizes estradiol stimulation of growth and reduces ER content, its mechanism may involve other non-estrogen-regulated pathways.     

Reduction of estrogen receptor concentration in MCF-7 human breast carcinoma cells following exposure to chemotherapeutic drugs

To study the effects of commonly used chemotherapeutic drugs on estrogen receptor (ER) of human breast cancer, we investigated the specific binding of [3H]estradiol within intact MCF-7 human breast cancer cells after 1 to 4 hr of exposure to methotrexate (0.5 to 50 micrograms/ml), 5-fluorouracil, and vincristine in serum- and hormone-free medium. Intracellular [3H]estradiol binding was either slightly increased (methotrexate and 5-fluorouracil) or not changed (vincristine) during the first 2 hr of drug exposures at 37 degrees, but slightly decreased at the third hr. After 4 hr of drug treatments, [3H]estradiol binding within MCF-7 cells was reduced by 30 to 70%; the response was dose dependent. Most (80 to 83%) of the intracellularly bound [3H]estradiol was found within the nuclei, and the drug-induced reduction of ER was reflected by a depleted nuclear uptake of [3H]estradiol. The Scatchard plot showed a large decrease of receptor number per cell with no apparent alteration in the binding affinity. The reduction of ER was reversible; regeneration of receptors to the control level occurred at either 4 hr (methotrexate and 5-fluorouracil) or 8 hr (vincristine) after removal of these drugs. The restoration was followed by an increase of ER beyond the control level. The dose-dependent depletion of ER by these cytotoxic drugs was also detectable in a second ER-positive cell line, MDA-MB-134. These data indicate that the cytotoxic drugs may cause a dose-dependent, reversible depletion of ER in human breast cancer, and the effect seems to be due to inhibition of receptor synthesis rather than inhibition of the binding of estradiol to its receptors.     

乳腺癌耐药细胞ER表达状态影响细胞对屈洛昔芬和阿霉素的敏感性

背景与目的：雌激素受体（estrogen receptor,ER)阳性乳腺癌细胞株来源的耐药细胞株中,ER表达缺失或下降,且细胞生长速度减慢,本文的目的是研究MCF－7／Adr乳腺癌耐药细胞株中ER表达状态与细胞对出洛昔芬（droloxifene,Dro)和阿霉素（Adriamycin,Adr)敏感性之间的关系。方法：Western blot法检测MCF－7／Adr及其亲本MCF－7细胞中ER蛋白的表达,构建ER的真核细胞表达质粒（pCER),利用LipofectAMINE^TM将ER基因导入MCF－7／Adr细胞,经G418抗性筛选获得阳性克隆（MTER／Adr),PCR,Western blot法鉴定并检测ER基因的整合和蛋白表达,流式细胞仪检测细胞周期变化,MTT法检测Dro及Adr对细胞增殖的影响。结果：在MCF－7细胞中可检测到ER蛋白 的表达,而MCF－7／Adr细胞中使用Westernblot检测不到ER蛋白的表达,成功构建真核细胞表达质粒pCER并转染MCF－7／Adr细胞,阳性克隆MTER／Adr整合了ER基因并获得表达。经MTT分析,10μmol/LDro对MCF－7细胞的生长有明显的抑制作用。而到20μmol/L时对MCF－7／Adr细胞的生长有抑制作用。ER转染MCF－7／Adr细胞后,15μmol/L的Dro对其生长出现抑制作用,使细胞多分布于G0／G1期。同时细胞对Adro的敏感性下降,结论：MCF－7／MCF－7／Adr细胞部分恢复对Dro和Adr的敏感性。     

Calbindin-D28k and calcium sensing receptor cooperate in MCF-7 human breast cancer cells

The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+]o, and neomycin sulfate, and spatial interactions of CaR with calbindin-D28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2 or neomycin sulfate and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+]i. The normalized dose response curves fitting yielded Hill co-efficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly co-operative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 micro M for CaCl2 and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D28k were co-localized when cells were exposed to 200 micro M neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.     

瘦素对乳腺癌MCF-7细胞增殖的影响及调控机制的研究

目的：研究瘦素对人乳腺癌MCF-7细胞增殖的影响及对雌、孕激素受体和VEGF的mRNA表达水平的影响，进而揭示瘦素对乳腺癌MCF-7细胞系作用的分子生物学机制。方法：体外培养人乳腺癌MCF-7细胞系，分为对照组、瘦素组和雌激素组。给药后用MTT法测570nm分光光度值。实时荧光定量PCR法观察各组雌、孕激素受体及VEGF的mRNA表达水平。结果：与对照组相比，瘦素可以呈时间和剂量依赖的方式促进MCF-7细胞增殖，并上调细胞中雌、孕激素受体及VEGF的mRNA表达水平，P〈0．05。结论：瘦素有促进乳腺癌细胞增殖的作用，该作用的发挥可能与其上调雌、孕激素受体与VEGF的表达有关。为乳腺癌的内分泌治疗提供了新的理论依据。     

瘦素对乳腺癌MCF-7细胞增殖的影响及调控机制的研究

目的:研究瘦素对人乳腺癌MCF-7细胞增殖的影响及对雌、孕激素受体和VEGF的mRNA表达水平的影响,进而揭示瘦素对乳腺癌MCF-7细胞系作用的分子生物学机制。方法:体外培养人乳腺癌MCF-7细胞系,分为对照组、瘦素组和雌激素组。给药后用MTT法测570nm分光光度值。实时荧光定量PCR法观察各组雌、孕激素受体及VEGF的mRNA表达水平。结果:与对照组相比,瘦素可以呈时间和剂量依赖的方式促进MCF-7细胞增殖,并上调细胞中雌、孕激素受体及VEGF的mRNA表达水平,P<0.05。结论:瘦素有促进乳腺癌细胞增殖的作用,该作用的发挥可能与其上调雌、孕激素受体与VEGF的表达有关。为乳腺癌的内分泌治疗提供了新的理论依据。     

乳腺癌MCF-7细胞体外干细胞培养条件下雌激素受体表达与治疗敏感性初探

目的通过比较观察激素敏感的MCF-7细胞在体外干细胞培养和常规培养条件下雌激素受体（ER）变化及对内分泌治疗药物的敏感性变化,初步探讨肿瘤干细胞与内分泌耐药的关系。方法分别于常规培养及干细胞培养条件下（悬浮球培养）培养激素敏感的MCF-7细胞株,流式细胞仪检测分子表型CD44^＋CD24^-/low与CD44＋CD24＋亚群细胞比例变化,免疫细胞化学法测定ERa和ERl3的表达变化,MTT法检测细胞对他莫西芬的敏感程度。分别以t检验、卡方检验、方差分析进行统计分析。结果干细胞培养条件下CD44^＋CD24^-/low亚群细胞的比例为（1.60±0．08）％,比常规培养条件下的（0．27±0．08）％显著增加（t=-12．10,P=0．00）,而CD44＋CD24＋亚群细胞比例由（5．59±0．88）％增至（30．63±4．40）％（t=-5．58,P=0．00）。干细胞培养条件培养下ERα和ERβ表达率较常规培养下调,分别由85．27％和90．53％降至69．43％和73．20％,差异有统计学意义（χ^2=214．64,P=0．00;χ^2=303．58,P=0．00）,且对他莫西芬的敏感性降低,IC50值由（9．82±0．31）μmol／L升至（16．46±0．50）μmol／L,再次诱导分化后ER并未出现上调,对他莫西芬的敏感性仍旧降低（F=113．63,P=0．00）。结论与常规培养相比较,体外干细胞培养条件下可以培养出含高比例具有干细胞特性的CD44＋CD24^-/low与CD44＋CD24＋亚群细胞的微球囊,其ER仍为阳性表达,但对他莫昔芬治疗敏感性减低,推测其可能是乳腺癌内分泌耐药的原因。     

RhG-CSF对乳腺癌细胞MCF-7增殖作用的影响

目的：探讨人重组粒细胞集落刺激因子（RhG-CSF）对乳腺癌细胞株MCF-7的增殖作用的影响。方法：体外培养人乳腺癌细胞株MCF-7,将含不同浓度RhG-CSF的培养液与MCF-7细胞共同培养不同时间,采用MTT比色法计算细胞生长增殖率;Anexxin V/PI双染试剂盒检测细胞凋亡率。实验结果用SPSS.v16.0软件分析。结果：不同浓度RhG-CSF处理细胞后,可以显著促进MCF-7细胞株的增殖（P〈0.01）,且当浓度为10μg/L时增殖作用最强。同时各浓度的RhG-CSF亦可抑制MCF-7细胞凋亡（P〈0.01）。结论：RhG-CSF可以显著促进人乳腺癌细胞株MCF-7增殖。当浓度小于10μg/L时,增殖呈浓度依赖性。大于10μg/L时,增殖能力反而逐渐下降。同时RhG-CSF亦可抑制MCF-7细胞凋亡。     

Detection of an elevated HER2 expression in MCF-7 breast cancer cells overexpressing estrogen receptor beta1

The estrogen receptor (ER) expression and HER2 amplification are important factors in determining the prognosis and therapy of breast cancer. Interactions between the two signaling pathways for example resulted in ERalpha-dependent regulation of HER2 expression in breast cancer cells. In this study, we investigated to what extent ERbeta is able to affect the HER2 expression. For this purpose, we analyzed HER2 levels in ERbeta1-overexpressing clones of the breast cancer cell lines MCF-7 and SK-BR-3 and of the ovarian cancer cell lines SK-OV-3 and OVCAR-3 by both RT-PCR and Western blot analysis. Treatment with ligand 17-beta estradiol diminished the HER2 expression in MCF-7 wild-type cells, an effect partially inhibited by treatment with 4-OH tamoxifen. MCF-7 breast cancer cells stably overexpressing ERbeta1 exhibited elevated >5-fold HER2 mRNA levels and elevated >3-fold HER2 protein levels even in the absence of estradiol. In contrast, ERbeta1 overexpression did not affect HER2 protein levels in the ERalpha-positive OVCAR-3 ovarian cancer cells and in the HER2 overexpressing, hormone-independent SK-BR-3 and SK-OV-3 cells. By demonstrating the elevated HER2 expression in a hormone-dependent breast cancer cell line overexpressing ERbeta1, our data suggest the presence of cross-talk between the two receptors. This is one of the molecular mechanisms underlying the significant ERbeta/HER2 co-expression observed in recent clinical studies.     

RhG-CSF对乳腺癌细胞MCF-7增殖作用的影响

目的:探讨人重组粒细胞集落刺激因子(RhG-CSF)对乳腺癌细胞株MCF-7的增殖作用的影响。方法:体外培养人乳腺癌细胞株MCF-7,将含不同浓度RhG-CSF的培养液与MCF-7细胞共同培养不同时间,采用MTT比色法计算细胞生长增殖率;Anexxin V/PI双染试剂盒检测细胞凋亡率。实验结果用SPSS.v16.0软件分析。结果:不同浓度RhG-CSF处理细胞后,可以显著促进MCF-7细胞株的增殖(P<0.01),且当浓度为10μg/L时增殖作用最强。同时各浓度的RhG-CSF亦可抑制MCF-7细胞凋亡(P<0.01)。结论:RhG-CSF可以显著促进人乳腺癌细胞株MCF-7增殖。当浓度小于10μg/L时,增殖呈浓度依赖性。大于10μg/L时,增殖能力反而逐渐下降。同时RhG-CSF亦可抑制MCF-7细胞凋亡。