Direct Involvement of the Y-box Binding Protein YB-1 in Genotoxic Stress-induced Activation of the Human Multidrug Resistance 1 Gene

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.The overexpression of P-glycoprotein (P-gp) 1 appears to be closely associated with multidrug resistance in human malignancies, suggesting that P-gp is a useful prognostic marker for assessing therapeutic efficacy (1, 2). The human multidrug resistance 1 (MDR1) gene encoding P-gp is highly susceptible to transcriptional activation and gene amplification during the selection of drug-resistant cell lines (3-5). 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 also in some clinical malignancies in vivo after cancer chemotherapy (16,17). Expression of MDR1 gene is enhanced in cultured human cancer cells and in vivo after transient exposure to both P-gp-and non-P-gp-targeted cytotoxic anticancer agents (7-10) and also in some clinical tumors after cancer chemotherapy (16,17).We previously identified the DNA binding protein, which recognized the cis-regulatory element, Y-box, on the MDR1 promoter (6,18,19). The Y-box binding family proteins are widely distributed from bacteria to mammals, and Y-boxes are located on the promoter of many genes such as the major histocompatibility complex class II gene, epidermal growth factor receptor, proliferating cell nuclear antigen, DNA polymerase ␣, thymidine kinase, and topoisomerase II␣ (20, 21). Furthermore, we found that the human Y-box binding protein (YB-1) gene spans ...

Hammerhead ribozyme against γ-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins

“…Involvement of SP1 in the regulation of the expression has been reported for MDR1 48 and MRP1. 49 There are other transcriptional factors for the expression of ABC transporter genes, such as YB -1 for MDR1 50,51 or AP -1 for MDR1. 52 In the electrophoretic gel mobility shift assay ( Fig 9 ), down -regulation of the SP1 -DNA binding activity was observed in the anti --GCSh ribozyme -transfected cells.…”

Some p53-binding proteins that can function as arbiters of life and death

“…Nuclear translocation of YB1 occurs in response to a variety of stresses that include UV exposure, DNA-damaging agents, and hypothermia. [37][38][39] Recent evidence suggests that YB1 must first be cleaved, and that only the N-terminal portion translocates to the nucleus. 40 This issue is discussed further below.…”

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells

“…In the cytoplasm, YB-1 regulates mRNA stability and translational regulation (Evdokimova et al, 2001;Ashizuka et al, 2002;Fukuda et al, 2004), while in the nucleus, it plays a pivotal role in transcriptional regulation through specific recognition of the Y-box promoter element (Ladomery and Sommerville, 1995;Kohno et al, 2003). Interaction of YB-1 with its cognate Y-box-binding site (inverted CCAAT box) is promoted by cytotoxic stimuli, including actinomycin D, cisplatin, etoposide, ultraviolet (UV) and heat shock, leading to the activation of a representative ABC transporter MDR1/ABCB1 and DNA topoisomerase IIa genes (Asakuno et al, 1994;Furukawa et al, 1998;Ohga et al, 1998). YB-1 also selectively interacts with damaged DNA or RNA, and protects from cytotoxic effects following cellular exposure to cisplatin, mitomycin C, UV and oxygen radicals (Ohga et al, 1996;Ise et al, 1999).…”