Abstract
The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.
Highlights
The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy
The MDR1 gene is up-regulated in response to ultraviolet light (UV), anticancer agents, serum starvation, heat shock, phosphatase inhibitors, and phorbol ester in cultured human cancer cells (6 –15) and in some clinical malignancies in vivo after cancer chemotherapy [16, 17]
We recently demonstrated that YB-1 protein is localized mainly in the cytoplasm but is translocated into the nucleus when cells are treated with either UV irradiation or anticancer agents [26]
Summary
Plasmids and Construction of CAT Expression Vectors—Clone pYB-29 and plasmid pRc/CMV (Invitrogen, San Diego, CA) were used to construct two YB-1 antisense expression plasmids as described previously [18]. Two cDNA fragments, EcoRI-EcoRI 1060-bp and EcoRI-SalI 735-bp fragments, were isolated from pYB-29, which contains the full-length YB-1 cDNA. The 5Ј overhangs of linearized DNA were filled in with the Klenow fragment of DNA polymerase I and inserted into the HindIII site of pRc/CMV after HindIII linker ligation. These two YB-1 antisense expression plasmids were designated AS 1.1 YB-1 and AS 0.7 YB-1, respectively. Cell Culture—Four stable transfectants derived from KB cells, Kst-6, Kac-7, Kxh-28, and Khp-2, were grown in modified Eagle’s medium
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