人类增殖性乳腺疾病细胞谱系模型中对细胞凋亡的渐进性抗性

Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease.

作者信息

Starcevic S L, Elferink C, Novak R F

机构信息

Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.

出版信息

J Natl Cancer Inst. 2001 May 16;93(10):776-82. doi: 10.1093/jnci/93.10.776.

DOI:10.1093/jnci/93.10.776

PMID:11353788

Abstract

BACKGROUND

Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice.

METHODS

The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided.

RESULTS

Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series.

CONCLUSION

Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.

摘要

背景

增生性乳腺疾病（PBD）可能会增加女性患乳腺癌的风险，这或许是通过降低细胞对凋亡的敏感性来实现的。为了确定在PBD过程中是否会产生凋亡抗性，我们在一系列能重现PBD在裸/米色小鼠体内形态学变化的细胞系中，研究了通过Fas途径引发的凋亡。

方法

所使用的细胞系包括MCF-10A细胞（人乳腺上皮前肿瘤亲本细胞）、MCF-10AT细胞（用T(24) Ha-ras转化）以及MCF-10ATG3B细胞（进展为癌的衍生细胞）。当Fas单克隆抗体结合并激活这些细胞上的Fas受体时所诱导的Fas介导的凋亡，通过形态学和流式细胞术进行评估。通过免疫印迹法测定Fas介导的凋亡相关蛋白水平以及聚（腺苷二磷酸-核糖）聚合酶（PARP）的裂解情况，PARP是半胱天冬酶激活的终产物。通过共免疫沉淀法检测Bcl-2和Bax异源二聚化。所有统计检验均为双侧检验。

结果

细胞对Fas介导的凋亡的敏感性随细胞的致瘤潜能降低：MCF-10A细胞极易感，MCF-10AT细胞较不易感，而MCF-10ATG3B细胞具有抗性。凋亡细胞的百分比分别从24%降至8%再降至6%。所有细胞系均产生Fas配体（FasL），且Fas受体、FasL、Fas相关死亡结构域蛋白以及半胱天冬酶3和6的水平相当。MCF-10A和MCF-10AT细胞中半胱天冬酶8的水平相似，但MCF-10ATG3B细胞中的水平约低30%（P>.01但<.05）。MCF-10AT细胞中半胱天冬酶10的水平约低20%（P>.005但<.01），MCF-10ATG3B细胞中的水平比MCF-10A细胞低约59%（P>.01但<.05）。在MCF-10A和MCF-10AT细胞中检测到PARP裂解，但在MCF-10ATG3B细胞中未检测到。所有细胞系中Bax、Bid和Bak蛋白的水平相似，但MCF-10AT和MCF-10ATG3B细胞中Bcl-2的水平低于MCF-A细胞，并且该细胞系中Bcl-2 - Bax异源二聚化逐渐下降。