Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells.
The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (-123 to -108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.
Daschner
P J
PJ
Intramural Research Support Program, SAIC, NCI-FCRDC, Frederick, MD 21702, USA.
Ciolino
H P
HP
Plouzek
C A
CA
Yeh
G C
GC
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D017239:Paclitaxel / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014747:Vinblastine; D004317:Doxorubicin; D004247:DNA; D017239:Paclitaxel