Cisplatin represses transcriptional activity from the minimal promoter of the O6-methylguanine methyltransferase gene and increases sensitivity of human gallbladder cancer cells to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-2-chloroethyl)-3-nitrosourea.
O(6)-methylguanine methyltransferase (MGMT) repairs O(6)-alkylguanine in cellular DNA introduced by the clinically used alkylating drug 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Thus, cancer cells with MGMT expression are resistant to ACNU treatment. Cisplatin has been reported to suppress MGMT expression; however, the molecular mechanism by which cisplatin reduces MGMT expression remains to be elucidated. Using gallbladder cancer cells (KMG-C) expressing MGMT, we analyzed whether a low dose of cisplatin suppresses MGMT expression, followed by an enhanced drug effect of ACNU in vitro and in vivo. We also investigated the promoter region critical for the transcriptional repression of MGMT gene by cisplatin using 5 deletion mutants in reporter promoter assays. In RT-PCR analysis, the expression of MGMT mRNA in KMG-C cells was dose- and time-dependently repressed. Drug sensitivity to ACNU was increased 2-fold by pretreatment with cisplatin, compared with only ACNU treatment, in MTT assays as well as tumor-bearing nude mice. Although the 5'-flanking region is deleted as far as -69 bp upstream of the transcription start site, cisplatin dose dependently inhibited luciferase activity. However, cisplatin did not cause such repression when 59 bp region from -69 to -10 bp was deleted. We confirmed that cisplatin enhanced sensitivity to ACNU in KMG-C cells expressing MGMT both in vitro and in vivo. Furthermore, a low dose of cisplatin repressed the transcription of the MGMT promoter. The 59 bp region in the MGMT promoter was crucial for repression by cisplatin. These results might form the basis of a chemotherapeutic strategy involving alkylating agents via prior cisplatin-induced biochemical modulation.
Sato
Ken
K
Department of Surgery, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501, Japan.
Kitajima
Yoshihiko
Y
Nakagawachi
Tetsuji
T
Soejima
Hidenobu
H
Miyoshi
Atsushi
A
Koga
Yasuo
Y
Miyazaki
Kohji
K
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001483:Base Sequence; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000627:therapeutic use*; D017931:DNA Primers; D005706:Gallbladder Neoplasms / Q000188:drug therapy*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D015376:Nimustine / Q000627:therapeutic use*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects*
D000970:Antineoplastic Agents; D017931:DNA Primers; D015376:Nimustine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002945:Cisplatin