A Mathematical Model of Cell Cycle Progression Applied to the MCF-7 Breast Cancer Cell Line

In this paper, we present a model of cell cycle progression and apply it to cells of the MCF-7 breast cancer cell line. We consider cells existing in the three typical cell cycle phases determined using flow cytometry: the G1, S, and G2/M phases. We further break each phase up into model phases in order to capture certain features such as cells remaining in phases for a minimum amount of time. The model is also able to capture the environmentally responsive part of the G1 phase, allowing for quantification of the number of environmentally responsive cells at each point in time. The model parameters are carefully chosen using data from various sources in the biological literature. The model is then validated against a variety of experiments, and the excellent fit with experimental results allows for insight into the mechanisms that influence observed biological phenomena. In particular, the model is used to question the common assumption that a ‘slow cycling population’ is necessary to explain some results. Finally, an extension is proposed, where cell death is included in order to accurately model the effects of tamoxifen, a common first line anticancer drug in breast cancer patients. We conclude that the model has strong potential to be used as an aid in future experiments to gain further insight into cell cycle progression and cell death.     

An ATP-Sensitive K+ Current that Regulates Progression Through Early G1 Phase of the Cell Cycle in MCF-7 Human Breast Cancer Cells

Whole-cell recordings were used to identify in MCF-7 human breast cancer cells the ion current(s) required for progression through G1 phase of the cell cycle. Macroscopic current-voltage curves were fitted by the sum of three currents, including linear hyperpolarized, linear depolarized and outwardly rectifying currents. Both linear currents, but not the outwardly rectifying current, were increased by 1 μm intracellular Ca²⁺ and blocked by 2 mm intracellular ATP. When tested at concentrations previously shown to inhibit proliferation by 50%, linogliride, glibenclamide and quinidine inhibited the linear hyperpolarized current, and quinidine and linogliride inhibited the linear depolarized current; none of these agents affected the outwardly rectifying current. In contrast, tetraethylammonium completely inhibited the outwardly rectifying current, but did not inhibit either linear current. Changing the bath solution to symmetric K⁺ shifted the reversal potential of the linear hyperpolarized current from near the K⁺ equilibrium potential (−84 mV) to −4 mV. Arrest of the cell cycle in early G1 by quinidine was associated with significantly smaller linear hyperpolarized currents, without a change in the linear depolarized or outwardly rectifying currents, but this reduction was not observed with arrest by lovastatin at a site ≈6 hr later in G1. The linear hyperpolarized current was significantly larger in ras-transformed than in untransformed cells. We conclude that the linear hyperpolarized current is an ATP-sensitive K⁺ current required for progression of MCF-7 cells through G1 phase. Received: 22 January 1999/Revised: 11 May 1999     

p16基因导入致人乳腺癌MCF-7细胞端区缩短及细胞周期阻滞

为进一步探讨 p1 6基因在抗肿瘤及细胞衰老中的作用 ,以脂质体介导的方法 ,将重组的含全长 p1 6c DNA的逆转录病毒载体导入人乳腺癌 MCF- 7细胞 ,获得稳定整合有效表达 .检测其对MCF- 7细胞的端区长度、细胞形态、增殖特性及细胞周期的影响 .结果显示 :导入 p1 6c DNA后的MCF- 7细胞端区长度明显缩短、增殖减慢 ,细胞周期阻滞于 G1期 .由此推测 ,野生型 p1 6基因可能通过诱导端区缩短效应及抑制细胞增殖从而抑制肿瘤和启动细胞衰老 .     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

人乳腺癌MCF-7细胞在放射线处理的SCID小鼠中转移的实验研究

目的建立人乳腺癌MCF-7细胞SCID(Severe combined immunodeficiency,SCID)小鼠转移动物模型。方法采用人乳腺癌细胞株MCF-7细胞悬液,分别接种于5只经放射线处理的SCID小鼠腋背部皮下。记录肿瘤生长情况,处死荷瘤鼠并做病理切片,观察各脏器转移情况。结果接种SCID小鼠后6～10d成瘤,成瘤率为5/5只,潜伏期平均(7.4±1.3)d。接种后5只鼠分别于第60～68天拉颈处死,检测荷瘤,平均直径为(26.6±2.2)mm,平均重量为5.28g。病理学检查,转移脏器有3个部位,出现肺转移的为4/5只、骨转移的为3/5只和淋巴结转移的为1/5只。结论建立了人乳腺癌SCID小鼠转移动物模型,该模型可为肿瘤转移研究提供重要的实验工具。     

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.     

磺化壳聚糖对MCF-7的体外抑制作用研究

目的:研究磺化壳聚糖(SCTS)对体外培养的人乳腺癌细胞的增殖抑制和凋亡的作用.方法:用不同浓度磺化壳聚糖对体外培养人乳腺癌细胞MCF-7进行干预,MTT法检测SCTS对MCF-7细胞增殖的抑制作用;显微荧光法、流式细胞术检测细胞凋亡.结果:磺化壳聚糖抑制MCF-7细胞增殖,且呈时间、剂量依赖性;镜下可见凋亡细胞的形态学改变、FCM显示G0/G1期细胞增加,而S期细胞减少.结论:磺化壳聚糖可有效抑制人乳腺癌细胞MCF-7增殖,促进细胞凋亡.     

人体乳腺癌细胞系Bcap－37和MCF－7的细胞遗传学研究

应用低温同步法与秋水酰胺处理,对人体乳腺癌细胞系Ｂｃａｐ－３７和ＭＣＦ－７的中期及早中期细胞进行Ｇ－显带分析。研究表明,Ｂｃａｐ－３７细胞染色体众数为６３,可识别其结构的标记染色体１７条;ＭＣＦ－７细胞染色体众数为５６,可识别其结构的标记染色体１３条。结合文献报道以及本研究结果显示,乳腺癌中最常涉及到第１、３、５、７、１１、１３和１７号染色体结构及数目的异常,染色体断裂点１ｐ１１（１ｑ１１）、１ｐ１３、３ｐ２１、３ｑ１１、５ｑ１１、６ｑ１３、６ｑ２３、７ｑ２２、１１ｐ１３和１１ｐ１５也经常涉及;它们可能与癌相关基因的激活和抗癌基因的丢失有关,从而在乳腺癌发生发展中起一定作用。     

二烯丙基二硫诱导人乳腺癌MCF-7细胞凋亡及机制的研究

目的:研究二烯丙基二硫(DADS)诱导人乳腺癌MCF-7细胞凋亡及其分子机制。方法:AO／EB荧光染色法、流式细胞仪检测细胞凋亡率;Western blot法检测DADS对caspase-3剪切片断的影响,及对MAPKs通路相关蛋白,包括p-JNK、JNK表达的影响。结果:DADS对乳腺癌细胞株MCF-7生长具有明显的抑制作用,经AO／EB形态变化分析,可见明显的细胞凋亡特征;DADS处理MCF-7细胞6、12、24、48 h,流式细胞仪检测细胞的凋亡率分别为3．74％、9．22％、20.2％、42％,而对照组细胞的凋亡率仅为3．03％(P<0．05);不同浓度的DADS作用于MCF-7细胞24 h后,Western blot法检测发现caspase-3出现断裂片断,并随着浓度的增加断裂更明显。进一步研究发现,DADS处理MCF-7细胞后,JNK磷酸化水平明显升高。结论:DADS能诱导乳腺癌细胞株MCF-7细胞凋亡,JNK信号通路抑制可能是DADS诱导其调亡的分子机制之一。     

GALNT14对人乳腺癌MCF-7细胞迁移的影响

构建真核表达载体pcDNA 3.1-Flag-T14,重组质粒经酶切分析及测序鉴定后,利用脂质体将重组质粒转染人乳腺癌细胞系MCF-7细胞,经G418筛选并建立稳定转染GALNT14细胞株.应用半定量RT-PCR、Western blot检测稳定细胞株GALNT14 mRNA及蛋白表达水平,细胞划痕修复及穿膜试验检测GALNT14基因对MCF-7迁移能力的影响,同时RT-PCR检测GALNT14对MMP-2,MMP-9,TGF-β1及VEGF等肿瘤浸润转移相关因子表达的影响.结果显示成功构建了真核重组表达载体pcDNA 3.1-Flag-T14,经RT-PCR和Western blot检测显示成功获得了稳定表达GALNT14的MCF-7细胞株;GALNT14能够提高MCF-7细胞株的迁移能力,且能增加侵袭转移相关因子MMP-2,MMP-9,TGF-β1及VEGF的表达.结论:GALNT14可明显促进MCF-7细胞的迁移,可能在肿瘤侵袭转移中起重要作用.     

Human Adipocytes Stimulate Invasion of Breast Cancer MCF-7 Cells by Secreting IGFBP-2

Background and Aims

A better understanding of the effects of human adipocytes on breast cancer cells may lead to the development of new treatment strategies. We explored the effects of adipocytes on the migration and invasion of breast cancer cells both in vitro and in vivo.

Methods

To study the reciprocal effects of adipocytes and cancer cells, we co-cultured human mature adipocytes and breast cancer cells in a system devoid of heterogeneous cell-cell contact. To analyze the factors that were secreted from adipocytes and that affected the invasive abilities of breast cancer cells, we detected different cytokines in various co-culture media. To study the communication of mature adipocytes and breast cancer cells in vivo, we chose 10 metastatic pathologic samples and 10 non-metastatic pathologic samples to do immunostaining.

Results

The co-culture media of human MCF-7 breast cancer cells and human mature adipocytes increased motility of MCF-7 cells. In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells. Based on our co-culture medium chip results, we chose four candidate cytokines and tested their influence on metastasis individually. We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors. In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.

Conclusions

IGFBP-2 secreted by mature adipocytes plays a key role in promoting the metastatic ability of MCF-7 breast cancer cells.     

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G₁/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis.     

Raf-Induced Cell Cycle Progression in Human TF-1 Hematopoietic Cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to b-estradiol-regulated DRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21Cip1, which are associated with G1 progression. Activated DRaf-1:ER and DA-Raf:ER but not DB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16Ink4a, a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16Ink4a suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.

Key Words:

Cell cycle, Raf, p21Cip1, p27Kip1, Cyclins, Cdks, Hematopoietic cells     

Cell Death of MCF-7 Human Breast Cancer Cells Induced by EGFR Activation in the Absence of Other Growth Factors

The Epidermal Growth Factor (EGF) Receptor (EGFR) plays an important role in the growth and progression of breast cancer. Overexpression of EGFR or the high activity of EGFR signal pathway has been related with increases in cell proliferation and a poor prognosis in patients with breast cancer. Several human breast cancer cell lines depend on estrogen for their proliferation. EGF may bypass the requirement of estrogen for the proliferation of breast cancer cells. To evaluate this hypothesis, MCF-7 breast cancer cells were stimulated with EGF and the effects on cell proliferation, signal pathways, and cell cycle progression were determined. The results demonstrate that EGF stimulation in the absence of others growth factors induced a modest effect on cell proliferation and the induction of a cellular arrest in the G1 phase of the cell cycle. Although phosphorylation of AKT and ERK proteins were detected, this phosphorylation was insufficient to support of cell cycle progression. Cellular arrest in G1 phase was accompanied by an increase in p21CIP1 protein, down regulation of the BCL-2 protein, induction of caspase-8, and ARHI/NOEY2 an imprinted tumor suppressor gene. These results indicate that EGFR activation by itself is not sufficient for the proliferation of breast cancer cells and suggest the existence of a mechanism that induces apoptosis upon EGFR activation.     

Inhibition of Human MCF-7 Breast Cancer Cells and HT-29 Colon Cancer Cells by Rice-Produced Recombinant Human Insulin-Like Growth Binding Protein-3 (rhIGFBP-3)

Background

Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells.

Methodology/Principal Findings

We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ±3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice.

Conclusion/Significance

These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.     

Redox Cycling in MCF-7 Human Breast Cancer Cells by Antitumor Agents Based on Mitozantrone

In a series of hydroxyethylaminoalkylaminoanthraquinones (AQ's) based on mitozantrone, 1-AQ (340%) and 1,8-AQ (137%) stimulated basal rate NADPH oxidation (72 + 18pmol min^-lmg S9 protein^-1) whilst 1,4-AQ, 1,5-AQ and mitozantrone had no effect. A similar trend was observed for O_2? generation (measured as nmol acet. cyt c reduction min-¹ mg protein^-1) by these compounds in MCF-7 S9 fraction: 1-AQ (9.5) and 1,8-AQ (7.9), whilst 1,5-AQ, 1,4-AQ and mitozantrone showed no significant effect. All the AQs including mitozantrone were cytotoxic to MCF-7 cells in a dose dependent manner with EC₅₀ values as follows: 1-AQ (0.01 μm) > doxorubicin (0.4μM) > mitozantrone (0.6μM) > 1,8-AQ (2.O μM) > 1,5-AQ (4.0μM) > 1,4-AQ (8,0 μM). Thus the redox active AQs were also the most cytotoxic. Mitozantrone however was not redox active but was more cytotoxic than all but 1-AQ hence it would appear that factors other than free radical generation contribute to the antitumor activity of this group of compounds.     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.     

CDKs和CKIs在胃癌细胞周期调控中的相关性

研究 CDKs和 CKIs在调节胃癌细胞周期进程中的作用表明 ,全反式视黄酸 ( ATRA)通过诱导细胞滞留在 G1/G0 期而抑制胃癌细胞生长 .Western blot分析显示 ,ATRA可上调 p2 1 waf1/ cip1的表达 ,而抑制 p1 6ink4 的表达 .免疫沉淀及活性测定表明 ,CDK2 激酶活性可被 ATRA抑制 ,而CDK4 活性先被诱导上升 ,2 4 h后逐渐下降 .另外 ,ATRA可以调节 Rb蛋白的磷酸化和 c- myc蛋白的表达 .由此证实 ,ATRA诱导胃癌细胞滞留于 G1/G0 期与其上调 p2 1 waf1/ cip1的表达和抑制CDK2 和 CDK4 激酶活性 ,进而抑制 Rb蛋白的磷酸化和 c- myc的表达有关 . Rb蛋白是 ATRA抑制胃癌细胞生长的下游调节因子 .另外 ,p1 6ink4 的功能在胃癌细胞中可能丧失 .     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。