抑癌基因PTEN对乳腺癌ZR-75-1细胞增殖和转移的抑制作用

背景与目的:研究表明,抑癌基因PTEN不仅能抑制肿瘤细胞的增殖,还能抑制其转移,但其机理还不甚明了.本文研究抑癌基因PTEN对人乳腺癌ZR-75-1细胞增殖和转移的作用.方法:以脂质体介导法分别将野生型PTEN质粒和磷酸酶缺陷的PTEN质粒转染人乳腺癌ZR-75-1细胞,用MTT法测定细胞增殖抑制率:转染后用嘌呤霉素筛选阳性克隆.用Western blot法检测细胞中PTEN蛋白的表达.通过细胞-基质粘附实验和人工重组基底膜侵袭实验,检测细胞粘附抑制率与侵袭抑制率.结果:野生型PTEN质粒转染的ZR-75-1细胞增殖明显被抑制,并伴有部分细胞凋亡;该细胞与未经转染的和磷酸酶缺陷的PTEN质粒转染的ZR-75-1细胞比较.细胞增殖抑制率差异均有统计学意义(42.7% vs.0%及2.7%,P＜0.01),细胞增殖抑制效应随细胞培养时间与质粒浓度的增加而增强.而磷酸酶缺陷的PTEN质粒转染的与未经质粒转染的ZR-75-1细胞比较,细胞增殖抑制率差异无统计学意义(2.7%vs.0%,P＞0.05).在两种PTEN质粒转染的ZR-75-1细胞中PTEN蛋白均明显表达,其中转染野生型PTEN质粒的细胞的粘附抑制率与侵袭抑制率分别达65.7%和70.4%,而转染磷酸酶缺陷的PTEN质粒的ZR-75-1细胞的粘附抑制率与侵袭抑制率分别只有8.8%和6.9%(P＜0.05).结论:具有双特异磷酸酶活性的野生型PTEN基因对ZR-75-1细胞的增殖和转移有一定的抑制作用.     

乳腺癌细胞转染野生型PTEN质粒后转移能力的变化

 目的 研究抑癌基因PTEN对乳腺癌细胞转移的影响。方法 脂质体介导法将外源野生型抑癌基因PTEN转染入该基因缺陷的ZR-75-1乳腺癌细胞中，用嘌呤霉素筛选阳性克隆,Western- blot法检测PTEN蛋白表达；重组人工基底膜上检测黏附与侵袭能力。结果 转染后ZR-75-1乳腺癌细胞PTEN蛋白有明显表达；转染后ZR-75-1乳腺癌细胞侵袭抑制率与黏附抑制率分别达70.4 %和60.0 %。结论 PTEN基因对乳腺癌细胞转移具有一定的抑制作用；PTEN基因的缺失与否在一定程度上可以评价乳腺癌患者发生转移的危险程度。     

PTEN磷酸酶活性对乳腺癌细胞ZR-75-1迁移能力的影响

目的:探求PTEN蛋白的磷酸酶活性对乳腺癌细胞ZR-75-1转移能力的影响。方法:采用脂质体介导法分别将野生型PTEN质粒(wt-PTEN)、磷酸酶失活的PTEN质粒(G129R-PTEN)和只具有蛋白磷酸酶活性的PTEN质粒(G129E-PTEN)转染PTEN基因缺失的人乳腺癌细胞株ZR-75-1,Western印迹法检测PTEN蛋白及P397-FAK的表达水平,体外细胞划痕实验观察PTEN磷酸酶活性对ZR-75-1细胞迁移能力的影响,细胞基质黏附试验和人工重组基底膜侵袭试验测定PTEN质粒转染和未转染的ZR-75-1细胞的黏附抑制率和侵袭抑制率,免疫组化法检测MMP-2的水平。结果:wt-PTEN、G129R-PTEN及G129E-PTEN3种质粒均成功转染ZR-75-1细胞并有PTEN蛋白的表达,其中wt-PTEN、G129E-PTEN均能抑制ZR-75-1细胞迁移;wt-PTEN和G129E-PTEN转染细胞之间的黏附抑制率和侵袭抑制率或侵袭细胞相对数均无显著性差异,但与G129R-PTEN转染的和未经转染的ZR-75-1细胞相比有显著性差异(P<0.01)。wt-PTEN和G129E-PTEN质粒转染的ZR-75-1细胞其P397-FAK水平均显著低于G129R-PTEN质粒转染的ZR-75-1细胞;wt-PTEN与G129E-PTEN质粒转染的ZR-75-1细胞MMP-2水平对比于G129R-PTEN质粒转染的和未经质粒转染的ZR-75-1细胞有显著性差异(P<0.01)。结论:具有双特异磷酸酶活性的野生型PTEN基因和只具蛋白磷酸酶活性的PTEN基因均能抑制乳腺癌细胞ZR-75-1的迁移,而磷酸酶失活的PTEN基因则无此作用。     

外源性p53基因导入对乳腺癌细胞系MCF-7中MDM2表达的影响

目的:探讨野生型p53导入对乳腺癌细胞系MCF-7生存的影响,及对MDM2表达的影响。方法:人腺病毒p53注射液处理人乳腺癌细胞系MCF-7,将野生型p53基因导入细胞中,用MTT和流式细胞仪研究两组MCF-7细胞的增殖凋亡情况,Western blot检测MDM2蛋白表达情况。结果:外源性野生型p53基因导入组增殖能力低于对照组,凋亡率明显高于对照组,且外源性野生型p53基因导入组MDM2表达明显高于对照组。结论:野生型p53导入可以诱导人乳腺癌细胞系MCF-7凋亡增加,且可以上调MDM2的表达,这可能与外源性p53增加后其负反馈泛素化降解酶也增加有关。     

抑癌基因PTEN抑制乳腺癌ZR-75-1细胞转移作用机制的探讨

目的 探讨抑癌基因PTEN抑制乳腺癌细胞ZR-75-1转移的作用机制.方法 用脂质体介导法分别将野生型PTEN质粒(wt)、磷酸酶活性缺失的PTEN质粒(G129R)和只具蛋白磷酸酶活性的PTEN质粒(G129E)转染PTEN基因缺失的乳腺癌细胞ZR-75-1.转染细胞以嘌呤霉素筛选后,用PCR和Western-blot分别检测PTEN基因及其蛋白;通过黏附、侵袭实验,比较3种质粒转染细胞和未转染细胞之间的黏附、侵袭能力的差异;用Western-blot法检测各组转染细胞总的黏着斑激酶(FAK)和磷酸化黏着斑激酶(P397-FAK)的表达水平;以免疫组化法检测基质金属蛋白酶-2(MMP-2)和E-钙连素(E-Cd)的表达,并用RT-PCR检测各组转染细胞的p53mRNA水平.结果 3种质粒均成功转染ZR-75-1细胞,并证实3种转染细胞内均有PTEN基因存在及PTEN蛋白表达;wt、G129R、G129E等 3种质粒转染的细胞黏附抑制率分别为65.7%、8.8%和43.5%;侵袭抑制率分别为70.4%、6.9% 和63.5%.将wt或G129E转染细胞的黏附抑制率及侵袭抑制率与G129R转染细胞的比较,均有显著性差异(P＜0.05);但wt与 G129E转染细胞比较,均无显著性差异(P＞0.05 ).3种转染细胞总FAK水平虽无显著性差异(P＞0.05 ),但wt和G129E转染细胞其P397-FAK和 MMP-2水平都显著低于G129R转染细胞(P＜0.05).3种转染细胞间的E-Cd水平未见显著差异.RT-PCR分析显示,wt、G129E和G129R 3种转染细胞p53 mRNA水平无显著性差异,但均显著高于未转染细胞.结论 野生型PTEN基因所表达的蛋白具脂质和蛋白双特异磷酸酶活性,对乳腺癌细胞ZR-75-1的转移具有抑制作用,其机制与磷酸酶活性有关,其中蛋白磷酸酶活性可能起主要作用.     

MDR1及MDR3短发夹RNA逆转乳腺癌细胞阿霉素耐药的实验研究

目的 研究靶向多药耐药(MDR)1及MDR3基因的短发夹RNA(shRNA)在逆转人乳腺癌阿霉素耐药细胞株MCF-7/Adr耐药中的作用.方法 真核质粒介导的针对MDR1及MDR3基因的shRNA转染细胞,空载体转染作为对照.Annexin-Ⅴ和PI双标法、流式细胞术、四甲基偶氮唑蓝(MTT)、逆转录聚合酶链反应(RT-PCR)、免疫组化分别检测细胞凋亡、细胞内阿霉素蓄积、细胞增殖活性及对阿霉素的IC50、MDR1及MDR3 mRNA及P-糖蛋白(P-gp)表达.结果 转染后,MDR1组及MDR3组MCF-7/Adr细胞凋亡率分别为30.21%±1.65%和22.07%±2.17%,与未转染组和空载体转染组比较差异有统计学意义(P＜0.01);MCF-7/Adr细胞内的阿霉素积聚浓度显著增加;MCF-7/Adr细胞存活率显著下降,MCF-7/Adr细胞对阿霉素IC50显著降低;相对于空载体转染组,MCF-7/Adr细胞中MDR1和MDR3 mRNA最高分别下降89.5%±0.8%和85.1%±1.2%,mRNA下降水平与孵育时间有关;P-gp表达明显降低,与未转染组和空载体转染组比较差异有统计学意义(P＜0.05).结论 shRNA可特异性地沉默MDR1及MDR3基因的表达,逆转P-gp介导的乳腺癌细胞阿霉素耐药,而MDR1的这种作用更为显著.     

重组腺病毒介导的ING-4用于人MCF-7乳腺癌细胞的体外基因治疗

目的研究重组腺病毒介导的ING-4感染人MCF-7乳腺癌细胞后对其的生长抑制作用。方法将搭载有ING-4基因的重组腺病毒载体Ad-ING-4感染人MCF-7乳腺癌细胞,用荧光显微镜、RTPCR和Western-Blot法检测ING-4在MCF-7细胞中的转录和表达;CCK法与流式细胞技术检测ING-4基因对MCF-7细胞的生长抑制和促凋亡作用;半定量RT-PCR法检测ING-4基因表达对MCF-7细胞中相关凋亡基因表达的影响。结果在MCF-7细胞中ING-4基因的表达对其细胞增殖有明显抑制作用,并显著促进其凋亡,ING-4表达使MCF-7细胞中Bax表达上调,Bcl-2、Survivin表达下调。结论 MCF-7细胞在转染ING-4基因后其增殖受到了明显抑制,且更易凋亡,可能是通过改变Bax,Bcl-2及Survivin表达水平来实现。     

siRNA-TRF2对人乳腺癌MCF-7细胞增殖的影响

目的 构建表达端粒重复序列结合因子2基因小干扰RNA的腺病毒表达载体（rAd-shRNA-TRF2）,探讨其对人乳腺癌MCF-7细胞增殖的影响。方法 根据预实验筛选出的针对人TRF2 mRNA的特异性siRNA靶序列,构建表达siRNA-TRF2的重组腺病毒载体rAd-shRNA-TRF2,转染人乳腺癌MCF-7细胞后,用MTT比色法检测MCF-7细胞增殖情况,流式细胞仪检测细胞周期分布情况。结果 rAd-shRNA-TRF2转染MCF-7细胞后,MCF-7细胞增殖抑制、细胞周期阻滞于G₀/G₁期、增殖指数显著下降。结论 通过RNAi抑制TRF2基因表达进而抑制人乳腺癌MCF-7细胞增殖的方法有望成为肿瘤基因治疗的一个新策略。     

他莫昔芬对转染ERβ1基因的MCF-7细胞生长特性的影响

目的研究在不同处理因子作用下,外源基因ERβ1的表达对MCF-7乳腺癌细胞系生长特性的影响。方法利用脂质体转染方法将ERβ1真核表达载体pcDNA3.1-EGFPERβ1导入MCF-7乳腺癌细胞系。采用Western blot方法检测转染细胞中ERβ1的蛋白表达水平,筛选阳性克隆。以亲本细胞MCF-7为对照,分别在雌激素和雌激素受体拮抗剂他莫昔芬作用下观察细胞的生长特点。结果在转染ERβ1基因的MCF-7细胞系中,Western blot检测证实ERβ1的蛋白表达水平显著增高。在无处理因子的情况下,外源基因ERβ1在MCF-7细胞系中的表达能抑制细胞生长。与亲本细胞MCF-7细胞相比,转染ERβ1的MCF-7细胞对雌激素的敏感性下降,但对他莫昔芬的敏感性无明显变化。结论外源性ERβ1基因在MCF-7乳腺癌细胞中的稳定表达不增加对他莫昔芬的耐药性,但使之对雌激素的敏感性下降。     

紫杉醇耐药基因TXR1 对乳腺癌细胞耐药性的影响

目的:构建人紫杉醇耐药基因1（TXR 1）的表达载体并对MCF-7 人乳腺癌细胞系进行转染且检测其表达。方法:RT-PCR 方法扩增TXR 1 CDS 片段,并将扩增的片段插入pEGFP-C3 真核表达载体,构建成含TXR 1 cDNA 的重组载体pEG ?FP-TXR1,以脂质体介导该质粒转染人乳腺癌细胞系MCF-7,应用RT-PCR、Western Blot和荧光显微镜鉴定转染细胞中TXR 1 的表达。用MTT 法对转染前后的MCF-7 细胞进行紫杉醇耐药性分析。结果:成功构建含TXR 1 cDNA 的重组载体pEGFP-TXR1,转染MCF-7 细胞后成功表达TXR 1 蛋白,在转染的细胞中,证实有TXR 1 mRNA 和蛋白表达上调。MTT 法显示转染了TXR 1 的MCF-7 细胞获得紫杉醇耐药性。结论:成功构建TXR 1 表达载体,并在人乳腺癌细胞系MCF-7 得到表达,获得紫杉醇耐药性。      

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

Reduction of epidermal growth factor binding in human breast cancer cell lines by an alkyl-lysophospholipid

The effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl lysophospholipid derivative, on the binding of epidermal growth factor (EGF) to human breast cancer cell lines (MCF-7, ZR-75-1, and BT-20), the human epidermoid cancer cell line (A431), and the rat fibroblast cell line (NIH3T3) were investigated. The addition of 10 micrograms/ml ET-18-OCH3 to the growth medium reduced the binding of EGF to hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) and A431 but did not change that to the hormone-independent breast cancer cell line (BT-20). ET-18-OCH3 suppressed the EGF-binding prior to the onset of its inhibitory action on cell growth in MCF-7 and ZR-75-1. Scatchard plot analysis demonstrated that ET-18-OCH3 reduced the number of EGF receptor sites without affecting the affinity of EGF receptors in MCF-7 and ZR-75-1. Both EGF-binding and cell growth in NIH3T3 were not changed by treatment with 10 micrograms/ml ET-18-OCH3. These results suggest that ET-18-OCH3 inhibits the growth of hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) by reducing the binding capacity of EGF receptors and consequently by disturbing the transfer of a variety of growth-promoting signals.     

Effect of 17-beta-estradiol on doxorubicin cytotoxicity in human breast cancer cell culture

Pre-treatment with 17-beta-estradiol appeared to improve the cytotoxic efficacy of doxorubicin on MCF-7 but not on ZR-75-1 and EVSA-T human breast cancer cell lines. MCF-7 and ZR-75-1 are both estrogen receptor-positive cell lines: however, only ZR-75-1 showed improved proliferation in the presence of estradiol. On the other hand MCF-7 appeared basically more resistant to doxorubicin compared to the other cell lines. The results indicate that estrogenic pre-treatment is a potential tool for partially overcoming human breast cell resistance to doxorubicin; moreover, they suggest that the mechanism of interaction could be not exclusively related to actual cytokinetics modulation.     

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.     

Inhibition by an alkyl-lysophospholipid of the uptake of epidermal growth factor in human breast cancer cell lines in relation to epidermal growth factor internalization

The effects of 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3), an alkyl-lysophospholipid derivative, on the binding and uptake of labeled epidermal growth factor (EGF) in hormone-dependent (MCF-7 and ZR-75-1) and hormone-independent (BT-20) breast cancer cell lines were investigated at 4 degrees C and 37 degrees C. The total (bound and intracellular) EGF associated with breast cancer cells tested were largely temperature dependent. By pretreatment of the cells with ET-18-OCH3 (10 micrograms/ml) for 12 h, the EGF uptake at 37 degrees C was greatly reduced in both MCF-7 and ZR-75-1 (ET-18-OCH3-susceptible) but not in BT-20 (ET-18-OCH3-resistant) cell lines. The ET-18-OCH3 pretreatment slightly decreased the EGF uptake at 4 degrees C in MCF-7 and ZR-75-1 and had little effect on that in BT-20. The EGF binding at 37 degrees C was unaffected by ET-18-OCH3 in MCF-7 and BT-20 and slightly decreased in ZR-75-1. The EGF binding at 4 degrees C was not changed by ET-18-OCH3 in all cell lines tested. These results suggest that labeled EGF is taken up by the cells in a temperature-dependent manner and ET-18-OCH3 may inhibit this internalization process only in ET-18-OCH3-sensitive human breast cancer cell lines. It is inferrable that the inhibition of the internalization process for EGF may be one of the modes of antitumoral action of ET-18-OCH3.     

The DNA binding and accumulation of p53 from breast cancer cell lines and the link with serine 15 phosphorylation

Stress treatment generally causes the post-translational modification and accumulation of the p53 protein, although the role of these aspects has not been always understood in relation to this protein's tumor suppressor activity. We analyzed these attributes of p53 in eight different breast cancer cell lines, with either wild-type or mutant p53 protein, in response to oxidative stress. We found that the wild-type p53 protein from MCF-7 and ZR-75-1 cells binds with different affinity to 12 gene sequences covering several pathways regulated by p53. Treatment of MCF-7 cells with H 2O 2 caused an increase in this binding affinity while this same treatment of ZR-75-1 cells caused the p53 protein to lose binding affinity to several genes. The mutant p53 proteins from all cell lines had minimal to weak binding to these sequences even after treatment with H 2O 2. The p53 protein from the ZR-75-1 cells and three cell lines with mutant p53 showed serine 15 phosphorylated protein, but we found no correlation between that modification and the levels or localization of this protein although DNA binding affinity of wild-type protein might be affected by this modification. From this and other work, it appears that the mutation status of the TP53 gene alone cannot predict the activity of this tumor suppressor since cell lines with the same genetic information do not show the same properties of this protein.     

Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells.

All-trans retinoic acid inhibits growth associated with downregulation of cyclin D1 and can cause low level apoptosis in estrogen receptor positive breast cancer cell lines. The cyclin D1 gene is amplified and/or the protein overexpressed in about one-third of breast cancers. Constitutive expression of cyclin D1 in estrogen receptor positive MCF-7 and ZR-75 breast cancer cells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S phase and reduced the G1 accumulation following retinoic acid treatment compared with control cells. However, culture of MCF-7(cycD1) with 1 microM all-trans retinoic acid resulted in about threefold greater growth inhibition compared with vector-transfected cells. Hoechst staining of DNA and in situ DNA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultures contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vector-transfected cells. Retinoic acid treatment of vector-transfected clones resulted in Bax protein activation as assessed by exposure of the NH(2)-terminus of Bax but the proportion of cells containing activated Bax was increased in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translocation of cytochrome c into the cytosol following RA-treatment. Retinoic acid markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingly, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to retinoic acid-induced apoptosis. We conclude that constitutive expression of cyclin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induced mitochondrial death pathway involving Bax activation, cytochrome c release and caspase-9 cleavage.