Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance-associated ABC transporter BCRP/MXR/ABCG2.
Breast cancer resistance protein (BCRP) is a recently identified new member of the superfamily of ATP-binding cassette transporters. BCRP is a "half transporter" that may homo- or heterodimerize to form an active transport complex. A considerable overexpression of BCRP was reported from various atypical multidrug-resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone. Thus, BCRP represents a very interesting candidate molecule for reversal of a drug-resistant phenotype. Six hammerhead ribozymes directed against the BCRP-encoding mRNA were designed and tested for their ability to cleave their target molecule. The anti-BCRP ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. BCRP-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using total RNA prepared from the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV exhibiting a high BCRP mRNA expression level. One anti-BCRP ribozyme was found to show a very high endoribonucleolytic cleavage activity at physiologic pH and temperature. This ribozyme was characterized in a cell-free system with regard to its specific kinetic parameters using large target molecules.
Kowalski
P
P
Institute of Pathology, Humboldt University Berlin, Germany.
Wichert
A
A
Holm
P S
PS
Dietel
M
M
Lage
H
H
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D006801:Humans; D007700:Kinetics; D008942:Mitoxantrone / Q000627:therapeutic use*; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000138:chemical synthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D008942:Mitoxantrone; D012321:DNA-Directed RNA Polymerases