Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance–associated ABC transporter BCRP/MXR/ABCG2

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Abstract

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. Cancer Gene Therapy (2001) 8, 185–192

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Correspondence to Hermann Lage.

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Kowalski, P., Wichert, A., Holm, P. et al. Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance–associated ABC transporter BCRP/MXR/ABCG2. Cancer Gene Ther 8, 185–192 (2001) doi:10.1038/sj.cgt.7700294

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Keywords

  • BCRP
  • MXR
  • ABCG2
  • ribozyme
  • atypical multidrug resistance
  • mitoxantrone

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