Evidence for transcriptional control of human mdr1 gene expression by verapamil in multidrug-resistant leukemic cells.
We investigated the mechanism of verapamil (VRP) effects on mdr1 gene expression in two leukemic multidrug-resistant (MDR) cell lines, K562/ADR and CEM VLB100. Exposure to VRP for 24 hr resulted in a decrease in mdr1 mRNA levels that was dose related at concentrations between 15 and 50 microM. The maximal decrease of mdr1 mRNA levels was found to be 6-fold in the K562/ADR cells and 3-fold in the CEM VLB100 cells. The effect of VRP on mdr1 mRNA levels was, however, biphasic. At 100 microM VRP, which strongly inhibited cell proliferation, a 2-fold increase of mdr1 mRNA levels was observed in the K562/ADR cells. To determine whether the decrease of mRNA levels resulted from post-transcriptional mechanisms, mRNA stability was studied after blocking of transcription with actinomycin D in VRP-treated cells and in control cells. This study revealed that mdr1 mRNA was stable in both cell lines and no increase in mdr1 mRNA degradation was observed in the 30 microM VRP-treated cells versus control cells (half-lives of 23 hr versus 14 hr for the K562/ADR cells and 15.5 hr versus 10.0 hr for the CEM VLB100 cells). The suggestion of a transcriptional mechanism was confirmed by nuclear run-on assays. A 4-fold decrease in the mdr1 gene transcription rate was observed in the 30 microM VRP-treated CEM VLB100 cells. The decreased transcription rate could be due to the decrease in mdr1 proximal promoter activity observed in CEM VLB100 cells transiently transfected with the mdr1 promoter fused to the chloramphenicol acetyltransferase gene. Indeed, after exposure to 30 microM VRP, chloramphenicol acetyltransferase activity was decreased by 2-fold. This study reports for the first time a down-regulation of mdr1 gene transcription by a pharmacological agent. These results provide further identification of the regulatory mechanisms involved in the overexpression of mdr1 in MDR cells and may help in the development of new strategies for MDR reversal.
Muller
C
C
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, Toulouse, France.
Goubin
F
F
Ferrandis
E
E
Cornil-Scharwtz
I
I
Bailly
J D
JD
Bordier
C
C
Bénard
J
J
Sikic
B I
BI
Laurent
G
G
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D001483:Base Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects*; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics*; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014700:Verapamil / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D014747:Vinblastine; D004317:Doxorubicin; D014700:Verapamil