雌激素调节人乳腺癌细胞CD147表达的研究

目的：探究雌激素对人乳腺癌细胞系中CD147表达的调节作用。方法：用雌激素处理人乳腺癌细胞系,RT-PCR及Real-time PCR定量检测用药前后细胞中CD147 mRNA表达水平变化,Western blot检测用药前后细胞中CD147蛋白表达水平变化。结果：经雌激素处理后的ER阳性人乳腺癌细胞系中CD147 mRNA及蛋白的表达均呈上升趋势,而ER阴性人乳腺癌细胞系则无明显变化。结论：雌激素通过ER激活CD147的表达,在乳腺癌的发生发展过程中发挥了重要的作用。     

P-glycoprotein expression in human breast cancer cells

Multidrug resistance in Chinese hamster ovary cells is associated with the Mr 170,000 surface glycoprotein. Using our monoclonal antibody to this protein, we have isolated a complementary DNA clone from an expression vector library. This complementary DNA recognizes a 4.5-kilobase mRNA in drug-resistant but not-sensitive Chinese hamster ovary cells; it also recognizes a 5.0-kilobase mRNA in our Adriamycin-resistant subline of the MDA-231 human breast cancer cell line which is not expressed in the drug-sensitive parent line. Southern blot analysis shows that the P-glycoprotein sequences are greatly amplified in resistant Chinese hamster ovary cells but not in the resistant human breast cancer cells, indicating that amplification and expression of the Mr 170,000 P-glycoprotein gene are not necessarily coordinate events. Amplification of this gene may not be required for multidrug resistance in human cells.     

TIMP1 overexpression mediates resistance of MCF-7 human breast cancer cells to fulvestrant and down-regulates progesterone receptor expression

High levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP1) are associated with poor prognosis, reduced response to chemotherapy, and, potentially, also poor response to endocrine therapy in breast cancer patients. Our objective was to further investigate the hypothesis that TIMP1 is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while TIMP1 levels did not influence the sensitivity to 4-hydroxytamoxifen. An inverse correlation between expression of the progesterone receptor and TIMP1 was found, but TIMP1 levels did not correlate with estrogen receptor levels or growth-promoting effects of estrogen (estradiol, E2). Additionally, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss of progesterone receptor expression combined with fulvestrant resistance. Our findings in vitro may have clinical implications as the data suggest that high tumor levels of TIMP1 may be a predictive biomarker for reduced response to fulvestrant.     

Tumor suppressor p53 down-regulates expression of human leukocyte marker CD43 in non-hematopoietic tumor cells

CD43 (leukosialin, sialophorin), a cell surface protein on most hematopoietic cells, is an important regulator of immune cell function and is involved in regulation of cell adhesion and proliferation. Aberrant expression of CD43 is a common event observed in human tumors of non-hematopoietic origin suggesting a role in tumor development. We have previously shown that overexpression of CD43 causes activation of the ARF-p53 tumor-suppressor pathway and results in cell death. In a non-functional ARF-p53 background, the cells overexpressing CD43 display an increased cell growth rate due to higher survival. Here we show that p53 specifically downregulates the expression of CD43 at the protein and mRNA level. Transactivating properties of p53 are necessary to affect the expression of exogenous CD43. The downregulation of CD43 mRNA is caused by p53-dependent transrepression, at least in part, via a histone deacetylation mechanism. These studies establish that under certain conditions there exists a negative feedback loop between p53 and CD43: CD43-dependent signaling activates p53, which in turn downregulates the expression of CD43.     

Flavopiridol down-regulates antiapoptotic proteins and sensitizes human breast cancer cells to epothilone B-induced apoptosis

The molecular mechanisms underlying the cell cycle growth-inhibitory and apoptotic effects of flavopiridol (FP) were determined in human breast cancer cells. Treatment with FP caused accumulation in the G(1) phase of the cell cycle and induced apoptosis of SKBR-3 and MB-468 cells. This was associated with down-regulation of the levels of cyclins D1 and B1, as well as with inhibition of cyclin-dependent kinase (cdk) 1, cdk2, and cdk4. FP-induced apoptosis was accompanied by a conformational change and mitochondrial localization of Bax. This resulted in the accumulations of cytochrome c, Smac, and Omi/HtrA2 in the cytosol and induced the poly(ADP-ribose) polymerase cleavage activity of caspase-3. Treatment with FP also attenuated the mRNA and protein levels of XIAP, cIAP-2, Mcl-1, Bcl-x(L), and survivin. In MB-468 cells with overexpression of Bcl-2 (468/Bcl-2), FP-induced Bax conformational change and apoptosis were inhibited, whereas the FP-mediated decline in the levels of IAP proteins, Mcl-11 and Bcl-x(L) remained unaltered. The effects of cotreatment with FP and the nontaxane tubulin-polymerizing agent epothilone (Epo) B were also determined in MB-468 cells. Sequential treatment with Epo B followed by FP induced significantly more apoptosis of MB-468 cells than treatment with the reverse sequence of FP followed by Epo B or treatment with either agent alone (P < 0.05). Treatment with Epo B followed by FP induced more Bax conformational change and was associated with a greater decline in the levels of XIAP, cIAP-2, Mcl-1, and Bcl-x(L). However, MB-468/Bcl-2 cells remained relatively resistant to Epo B followed by FP. Taken together, these findings suggest that the superior sequence-dependent anti-breast cancer activity of Epo B followed by FP may be due to FP-induced Bax conformational change and down-regulation of the antiapoptotic IAP, Bcl-x(L), and Mcl-1 proteins, but this treatment may not overcome the resistance to apoptosis of breast cancer cells conferred by overexpression of Bcl-2.     

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% +/- 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% +/- 6.53% of the infected T47D cells and 41.52% +/- 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100-500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.     

The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells

Introduction  

Loss of the cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in breast cancer. The decrease in p27 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S phase kinase protein 2 (Skp2). The mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphoinositol 3' kinase (PI3K)/Akt pathway that down-regulates p27 levels in breast cancer. Rapamycin was found to stabilize p27 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression is unknown.     

VEGF EXPRESSION IS INHIBITED BY APIGENIN IN HUMAN BREAST CANCER CELLS

Objective： To study the effects of apigenin on vascular endothelial growth factor （VEGF） in human breast cancer cells （MDA-MB-231. Methods： MTT assay was used to detect the cell proliferation inhibitory effect of apigenin on MDA-MB-231 cell. ELISA was used to determine the protein level of VEGF secreted by MDA-MB-231 cells. RT-PCR was used to detect mRNA levels of VEGF in MDA-MB-231 cells. The protein levels of HIF-1α, p-AKT, p-ERK1/2, and p53 were detected by Western Blotting. Results： Apigenin did not inhibit the cell viability of MDA-MB-231 cell. Apigenin reduced the secretion and mRNA levels of VEGF in MDA-MB-231 cells. Additionally, apigenin decreased the expressions of HIF-1α, p-AKT and p-ERK1/2, but induced the expression of p53. Conclusion： Apigenin can inhibit VEGF expression in human breast cancer cells, and this may be achieved through decreasing HIF-1α.     

重组人GM—CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定…

建立逆转录病毒介导的ＧＭ－ＣＳＦ基因转移系统,为ＧＭ－ＣＳＦ基因悠我肿瘤细胞疫苗研究奠定实验基础。应用基因重组技术将ＧＭ－ＣＳＦｃＤＮＡ克隆于逆转录病毒载体ｐＤＯＲｎｅｏ,获得重组载体ｐＤＯＲＧＭ。经脂质体介针其转染于病毒包装细胞系ＰＡ３１７细胞,经ＮＩＨ３Ｔ３细胞检测病毒滴度,ＮＩＨ３Ｔ３放大实验检测辅助病毒,并用同熵度病毒感染人乳腺癌细胞系ＭＣＦ－７。     

重组人GM-CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定表达

目的　建立逆转录病毒介导的 GM- CSF基因转移系统 ,为 GM- CSF基因修饰的肿瘤细胞疫苗研究奠定实验基础。方法　应用基因重组技术将 GM- CSF c DNA克隆于逆转录病毒载体p DORneo,获得重组载体 p DORGM。经脂质体介导将其转染于病毒包装细胞系 PA317细胞 ,经NIH3T3细胞检测病毒滴度 ,NIH3T3放大实验检测辅助病毒 ,并用高滴度病毒感染人乳腺癌细胞系MCF- 7。结果　GM- CSF基因克隆入逆转录病毒载体 ,经 PA317细胞包装产生病毒 ,最高的病毒滴度为 1× 10 6CFU/ ml,且没有发现辅助病毒存在。重组病毒感染的人乳腺癌 MCF- 7细胞在体外长期培养中保持稳定分泌 GM- CSF,活性在 50 0～ 80 0 U/ 10 6细胞 / 2 4小时。结论　建立的逆转录病毒介导的GM- CSF基因转移系统安全、有效 ,为 GM- CSF应用于肿瘤基因治疗提供了实验基础。     

Estradiol enhances osteolytic lesions in mice inoculated with human estrogen receptor-negative MDA-231 breast cancer cells in vivo

The effect of 17--estradiol (E₂) on the induction of osteolytic lesions by estrogen receptor (ER)-negative breast cancer cells was investigated in 4-week-old female nude mice. Exposure to exogenous E₂ was found to increase osteolytic areas on radiographs up to 5.3 times in mice inoculated intracardially with MDA-231 human breast cancer cells. The MDA-231 cells were ER-negative, both before inoculation, and after isolation from osteolytic lesions, and the corresponding cell cultures were insensitive to E₂. The induction of skeletal lesions by E₂ in this mouse model was mainly effectuated at the early development of bone metastases, since exposure to E₂ for 8 days around MDA-231 inoculation increased osteolysis to the same level, as did E₂ given throughout the entire 31-day experimental period, and because E₂-exposure for just the final 14 days had no effect. Independently of exposure to E₂, histology revealed cancer cells in hind limp long bones of approximately 80% of the mice, and tumors were absent in non-skeletal organs. In vitro studies showed that the number and activity of osteoclasts generated from mouse bone marrow cells were increased 5–6 times when co-cultured with MDA-231 cells. Addition of 0.1–10 nM E₂ further dose-dependently increased the osteoclastogenesis and associated bone resorption in these co-cultures. In conclusion, E₂ was found to increase the morbidity in mice inoculated with ER-negative MDA-231 cells, and to stimulate osteoclast formation and bone resorption in co-cultures of bone marrow cells and MDA-231, suggesting that the progression of osteolytic metastases by ER-negative breast cancer cells can be induced by E₂ due to stimulation of osteoclastogenesis.     

Tetraspanin CD9 determines invasiveness and tumorigenicity of human breast cancer cells

Interaction of breast cancer cells (BCCs) with stromal components is critical for tumor growth and metastasis. Here, we assessed the role of CD9 in adhesion, migration and invasiveness of BCCs. We used co-cultures of BCCs and bone marrow-derived multipotent mesenchymal stromal cells (MSCs), and analyzed their behavior and morphology by dynamic total internal reflection fluorescence, confocal and scanning electron microscopy. 83, 16 and 10% of contacts between MDA-MB-231 (MDA), MA-11 or MCF-7 cells and MSCs, respectively, resulted in MSC invasion. MDA cells developed long magnupodia, lamellipodia and dorsal microvilli, whereas long microvilli emerged from MA-11 cells. MCF-7 cells displayed large dorsal ruffles. CD9 knockdown and antibody blockage in MDA cells inhibited MSC invasion by 95 and 70%, respectively, suggesting that CD9 is required for this process. Remarkably, CD9-deficient MDA cells displayed significant alteration of their plasma membrane, harboring numerous peripheral and dorsal membrane ruffles instead of intact magnupodium/lamellipodium and microvillus, respectively. Such modification might explain the delayed adhesion, and hence MSC invasion. In agreement with this hypothesis, CD9-knockdown suppressed the metastatic capacity of MDA cells in mouse xenografts. Our data indicate that CD9 is implicated in BCC invasiveness and metastases by cellular mechanisms that involve specific CD9⁺ plasma membrane protrusions of BCCs.     

肿瘤细胞内层粘蛋白表达与乳腺癌预后的关系

目的 探讨肿瘤细胞内层粘蛋白表达与乳腺癌预后的关系。方法 用免疫组化和微波抗原修复技术,检测７８例原发乳腺癌细胞内层粘蛋白的表达。结果 本组乳腺癌患者的术后５年生存率为６１．５％,术后平均生存时间５９．６月。死亡组和生存组的层粘蛋白表达率分别为６６．７％和５４．２％。腋淋巴结状况、肿瘤大小、组织学分组和ＴＮＭ分期均是影响乳腺癌预后的重要因素。层粘蛋白阴性表达组术后平均生存时间明显较阳性组长（Ｐ〈０     

Prognostic significance of CD55 expression in breast cancer

PURPOSE: Our recent study revealed that CD55-high population in breast cancer cell line was resistant to apoptosis and formed colonies in vitro more efficiently than CD55-low population. The present study was conducted to examine whether CD55-high population in breast cancer cell line possesses higher tumorigenic potential in vivo and presence of CD55-high cells in breast cancer affects clinicopathologic behavior of patients. EXPERIMENTAL DESIGN: CD55-high and CD55-low population was sorted from breast cancer cell line, injected into immunodeficient mice, and the resultant tumor volume was measured. CD55 expression was immunohistochemically examined in clinical samples from 74 cases with breast cancers, and cases with >1% of tumor cells showing high level of CD55 expression were categorized as CD55 high. RESULTS: The xenotransplanted tumor volume derived from CD55-high population was significantly larger than that from CD55-low population. Fifty (67.6%) of 74 cases of breast cancer were CD55-high. A significant correlation was observed between CD55-high character and relapse rate (P < 0.001). Univariate analysis showed that tumor size (P = 0.005) and CD55 expression (P = 0.005) were unfavorable prognostic factors. Multivariate analysis revealed that the tumor size (P = 0.013) and CD55 expression (P = 0.011) were independent prognostic factors. CONCLUSIONS: CD55 play an important role in tumorigenesis of breast cancer, and presence of small population of cells with strong CD55 expression would be sufficient to predict poor prognosis of patients.