Molecular Mechanisms of Loss of β₂-Microglobulin Expression in Drug-Resistant Breast Cancer Sublines and Its Involvement in Drug Resistance

Abstract

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.

β2-microglobulin induces apoptosis in HL-60 human leukemia cell line and its multidrug resistant variants overexpressing MRP1 but lacking Bax or overexpressing P-glycoprotein

“…These results suggest that b 2 m may utilize an apoptosis pathway distinct from Class I MHC antigen complexinduced apoptosis (Genestier et al, 1997a,b;WallenOhman et al, 1997). The decreased expression levels of b 2 m in HL-60 drug resistant variants con®rms our previous report demonstrating that the loss of b 2 m expression in breast cancer cells is associated with resistance to DOX, and that inhibition of its expression in MCF-7 cells by an antisense b 2 m expression vector induced the drug resistance phenotype (Ogretmen et al, 1998). Partial or complete loss of b 2 m in several aggressive metastatic cancers or cell lines derived from such cancers has been observed (Kakamanis et al, 1995;Vitale et al, 1998;Restifo et al, 1993;Benitez et al, 1998).…”

“…We previously reported that the loss or decreased expression of b 2 m results in drug resistance in sublines of MCF-7 cells selected for resistance to the chemotherapeutic agent doxorubicin (DOX), and in a DOX-resistant variant of the T-47D human breast cancer cell line (Ogretmen et al, 1998). These data suggested that the loss of b 2 m in cancer cells may cause both resistance to chemotherapy as well as an escape from immune surveillance.…”

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101

“…Semiquantitative reverse transcriptase-PCR (RT-PCR) protocols that compare relative transcript levels of target genes with those of housekeeping genes (including 16S or 18S rRNA) also use the internal control to titrate the exact amount of total RNA added to each reaction (Nicoletti and SassyPrigent, 1996;Ogretmen et al, 1998;Wang et al, 2002). The expression of N-regulated genes, ntcA, napA and nifH, was studied using this semiquantitative RT-PCR method with minor modifications.…”

Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant

“…Nuclear run-on analysis was performed as previously described (Ogretmen et al, 1998). Briefly, nuclei from HL-60, HL-60/ADR cells were isolated and stored in 100 ml of freezing buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl 2 and 0.1 mM EDTA).…”

“…Equal counts of TCA-precipitated labeled transcripts were used for hybridizations of slot blots containing 0.4 mg 360 bp human PR3 and 1.8 kb human b-actin (CloneTech, Palo Alto, CA, USA) cDNA fragments. Hybridizations were performed at 458C for 72 -96 h and then filters were washed at 578C with 26SSC -1% SDS and 0.16SSC -1% SDS for 1 -2 h as we previously described (Ogretmen et al, 1998). Bands were visualized by autoradiography at -808C for 7 -10 days, and quantitated by densitometry using Photoshop version 4 software.…”