Ogretmen B, McCauley M D, Safa A R
Medical University of South Carolina, Hollings Cancer Center, Department of Experimental Oncology, Charleston 29425, USA.
Biochemistry. 1998 Aug 18;37(33):11679-91. doi: 10.1021/bi980573c.
In this study, we investigated the mechanism of the loss or decreased expression of beta 2-microglobulin (beta 2m) in several drug-resistant sublines of MCF-7 and in a doxorubicin (DOX)-resistant variant of the T-47D breast cancer cell line. beta 2m protein and RNA are not expressed in highly metastatic, multidrug-resistant MCF-7/Adr cells with high resistance to DOX. Nuclear run-on transcription and RNA stability assays demonstrate that while beta 2m in MCF-7/Adr cells is transcribed, its mRNA is rapidly degraded after synthesis in these cells, indicating that it is controlled by post-transcriptional mechanisms. We also show that an MCF-7 subline (MCF-7/Adr-5) expressing a very low level of resistance to DOX has a decreased level of beta 2m expression. Treatment with actinomycin D revealed that the half-life of beta 2m mRNA in MCF-7 and MCF-7/Adr-5 cell lines was comparable. Nuclear run-on transcription analysis revealed a decreased rate of beta2m transcription in MCF-7/Adr-5 cells compared to that in MCF-7 cells. Moreover, beta 2m mRNA remained undetectable in MCF-7/Adr cells following cycloheximide treatment. However, in MCF-7 cells, increased beta 2m mRNA was observed after 12 h, and a similar level of increased mRNA expression was observed after 36 h of cycloheximide treatment in MCF-7/Adr-5 cells; these results suggest that one of the mechanisms controlling beta 2m mRNA expression might be a negative regulatory protein in MCF-7/Adr-5 cells. Analysis of the beta 2m status of other drug-resistant MCF-7 sublines revealed that deregulation of beta 2m is not limited to DOX resistance, but can also be detected in cells selected for resistance to mAMSA and DOX-verapamil. In addition, our data show that reduced beta 2m expression correlates with the decreased levels of estrogen receptor (ER) expression in the DOX-resistant MCF-7/Adr and T-47D/Adr-4 human breast cell lines. Furthermore, we provide evidence that the partial inhibition of beta 2m by antisense RNA results in 2-3-fold decreased sensitivity of MCF-7 cells to DOX and mAMSA. Moreover, the addition of exogenous beta 2m protein near its physiological human serum concentration can modulate the DOX sensitivity of the MCF-7 antisense beta 2m and control transfectants. Therefore, these results indicate that lost or decreased beta 2m expression is involved in the development of the drug-resistant phenotype and correlates with the loss of ER in human breast cancer cell lines.
在本研究中，我们调查了β2-微球蛋白（β2m）在MCF-7的几个耐药亚系以及T-47D乳腺癌细胞系的阿霉素（DOX）耐药变体中表达缺失或降低的机制。β2m蛋白和RNA在对DOX具有高抗性的高转移性、多药耐药的MCF-7/Adr细胞中不表达。核延伸转录和RNA稳定性分析表明，虽然MCF-7/Adr细胞中的β2m被转录，但其mRNA在这些细胞中合成后迅速降解，这表明它受转录后机制控制。我们还表明，对DOX耐药水平非常低的MCF-7亚系（MCF-7/Adr-5）中β2m的表达水平降低。用放线菌素D处理表明，MCF-7和MCF-7/Adr-5细胞系中β2m mRNA的半衰期相当。核延伸转录分析显示，与MCF-7细胞相比，MCF-7/Adr-5细胞中β2m的转录速率降低。此外，用环己酰亚胺处理后，MCF-7/Adr细胞中仍未检测到β2m mRNA。然而，在MCF-7细胞中，12小时后观察到β2m mRNA增加，在MCF-7/Adr-5细胞中用环己酰亚胺处理36小时后观察到类似水平的mRNA表达增加；这些结果表明，控制β2m mRNA表达的机制之一可能是MCF-7/Adr-5细胞中的一种负调控蛋白。对其他耐药MCF-7亚系的β2m状态分析表明，β2m的失调不仅限于对DOX的耐药性，在对mAMSA和DOX-维拉帕米耐药的细胞中也能检测到。此外，我们的数据表明，在DOX耐药的MCF-7/Adr和T-47D/Adr-4人乳腺癌细胞系中，β2m表达降低与雌激素受体（ER）表达水平降低相关。此外，我们提供的证据表明，反义RNA对β2m的部分抑制导致MCF-7细胞对DOX和mAMSA的敏感性降低2-3倍。此外，在接近其生理人血清浓度的情况下添加外源性β2m蛋白可以调节MCF-7反义β2m转染细胞和对照转染细胞对DOX的敏感性。因此，这些结果表明，β2m表达缺失或降低与耐药表型的发展有关，并且与人乳腺癌细胞系中ER的缺失相关。
