TY - JOUR
T1 - Structural and functional analysis of the control region of the human DNA topoisomerase IIα gene in drug-resistant cells
AU - Takano, Hiroshi
AU - Tomoko, Ise
AU - Nomoto, Minoru
AU - Kato, Ken
AU - Murakami, Tadashi
AU - Ohmori, Haruki
AU - Imamura, Toshihiro
AU - Nagatani, Gunji
AU - Okamoto, Tatsuro
AU - Ohta, Ryo
AU - Furukawa, Manabu
AU - Shibao, Kazunori
AU - Izumi, Hiroto
AU - Kuwano, Michihiko
AU - Kohno, Kimitoshi
PY - 1999/4
Y1 - 1999/4
N2 - We have previously shown that the DNA topoisomerase IIα (topo IIα) gene is down-regulated in VP16/VM26-resistant cells at the transcriptional level. To determine the DNA elements responsible for down-regulation, the transcriptional activities of luciferase reporter constructs containing various lengths of the promoter sequences were investigated by transient transfection of two resistant cell lines, KB/VP2 and KB/VM4. The transcriptional activities of the full-length promoter (-295 to +85) and of three deletion constructs (-197, -154 and -74 to +85) were significantly down-regulated in resistant cells. In contrast, the transcriptional activity of the minimal promoter (-20 to +85) in resistant cells was similar to that in parental KB cells. Furthermore, introduction of a mutation in ICE1 abolished the down-regulation of the topo IIα promoter activity in drug-resistant cells. In vivo footprinting analysis of topo IIα gene promoter revealed several specific protein-binding sites, a GC box, ICE1, ICE2 and ICE3. In vivo footprinting analysis also identified a cluster of hypersensitive sites. However, there was no marked difference in protein-binding sites between parental and resistant cells. To confirm our previous results, we have established the VP16-resistant cell lines T12-VP1 and T12-VP2 from T12 cells derived from human bladder cancer T24 cells stably transfected with the chloramphenicol acetyltransferase reporter gene driven by the topo IIα gene promoter. The expression of topo IIα was down-regulated in both cell lines. We also found that CAT gene expression was significantly decreased to one-fifth of that in T12 parental cells. These results suggest that the expression of the topo IIα gene requires the binding of multiple factors to the core promoter and is down-regulated at the transcriptional level, probably through binding of a negative factor to ICE1 in drug-resistant cells.
AB - We have previously shown that the DNA topoisomerase IIα (topo IIα) gene is down-regulated in VP16/VM26-resistant cells at the transcriptional level. To determine the DNA elements responsible for down-regulation, the transcriptional activities of luciferase reporter constructs containing various lengths of the promoter sequences were investigated by transient transfection of two resistant cell lines, KB/VP2 and KB/VM4. The transcriptional activities of the full-length promoter (-295 to +85) and of three deletion constructs (-197, -154 and -74 to +85) were significantly down-regulated in resistant cells. In contrast, the transcriptional activity of the minimal promoter (-20 to +85) in resistant cells was similar to that in parental KB cells. Furthermore, introduction of a mutation in ICE1 abolished the down-regulation of the topo IIα promoter activity in drug-resistant cells. In vivo footprinting analysis of topo IIα gene promoter revealed several specific protein-binding sites, a GC box, ICE1, ICE2 and ICE3. In vivo footprinting analysis also identified a cluster of hypersensitive sites. However, there was no marked difference in protein-binding sites between parental and resistant cells. To confirm our previous results, we have established the VP16-resistant cell lines T12-VP1 and T12-VP2 from T12 cells derived from human bladder cancer T24 cells stably transfected with the chloramphenicol acetyltransferase reporter gene driven by the topo IIα gene promoter. The expression of topo IIα was down-regulated in both cell lines. We also found that CAT gene expression was significantly decreased to one-fifth of that in T12 parental cells. These results suggest that the expression of the topo IIα gene requires the binding of multiple factors to the core promoter and is down-regulated at the transcriptional level, probably through binding of a negative factor to ICE1 in drug-resistant cells.
KW - DNA topoisomerase IIα
KW - Inverted CCAAT element (ICE)
KW - Resistant cells
KW - Transcription
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M3 - Article
C2 - 10405635
AN - SCOPUS:0344258383
SN - 0266-9536
VL - 14
SP - 87
EP - 92
JO - Anti-Cancer Drug Design
JF - Anti-Cancer Drug Design
IS - 2
ER -