Ricca A, Biroccio A, Del Bufalo D, Mackay A R, Santoni A, Cippitelli M
Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
Int J Cancer. 2000 Apr 15;86(2):188-96. doi: 10.1002/(sici)1097-0215(20000415)86:2<188::aid-ijc7>3.0.co;2-w.
bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF-kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF-kappaB binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells.
在多种肿瘤类型中，bcl-2表达常与预后不良相关；然而，该分子在乳腺癌中的作用仍存在争议。我们之前发现，在人乳腺癌细胞系（MCF7(ADR)）中bcl-2的过表达通过诱导转移相关特性，如基质金属蛋白酶-9（mmp-9）分泌增加，增强了其致瘤性和转移潜能。在本研究中，我们调查了bcl-2过表达对转录因子NF-κB活性的影响，NF-κB是参与肿瘤进展和侵袭的基因的重要调节因子。瞬时转染实验表明，在MCF7(ADR)细胞系中bcl-2的过表达增强了NF-κB依赖性转录活性。凝胶迁移分析显示，在bcl-2过表达克隆中NF-κB与DNA的结合增加，这与NF-κB细胞质抑制剂IkappaBalpha水平降低相关。此外，BH1和BH2结构域内2个高度保守残基的点突变消除了bcl-2与bax的相互作用，或N端BH4结构域的缺失，完全消除了该分子上调NF-κB依赖性反式激活的能力。由于mmp-9是一个受NF-κB调节的基因，我们还研究了bcl-2过表达是否上调mmp-9转录。我们发现在瞬时转染实验中，mmp-9 mRNA的诱导与mmp-9启动子报告基因构建体的激活相关，并且(-600)mmp-9-NF-κB结合元件的突变消除了这种效应。总体数据表明，bcl-2介导的对NF-κB转录因子活性的调节可能是促进MCF-7(ADR)细胞恶性行为的重要机制。
