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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to DegradeO 6-Methylguanine DNA Methyltransferase mRNA

Qiwei Zhang, David W. Ohannesian, Emiko L. Kreklau and Leonard C. Erickson
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 141-147;
Qiwei Zhang
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David W. Ohannesian
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Emiko L. Kreklau
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Leonard C. Erickson
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Abstract

O6-Methylguanine DNA Methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of the DNA alkylating agent 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to deplete MGMT activity have been developed. In the present study, a molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. The ribozymes were designed against eight GUC sites within the MGMT mRNA. cDNAs of these ribozymes were cloned into an expression vector and then all eight vectors were pooled and stably transfected into HeLa cells. Several HeLa/Rz clones sensitive to a sublethal dose of BCNU were identified using a short-term cell proliferation assay. The ribozyme inserts were amplified from genomic DNA by polymerase chain reaction and sequenced in the BCNU-sensitive clones. The ribozyme inserts Rz161, 178, and 212, targeted against nucleotide 161, 178, and 212, respectively, in the MGMT mRNA, were found to be present in these clones. MGMT activity, Western, and Northern blot analyses revealed that two of the HeLa/Rz clones contained very low levels of MGMT activity, protein, and mRNA. Investigation of CpG methylation within the MGMT promoter indicated that the lack of MGMT expression in these HeLa/Rz clones was not likely due to methylation silencing of the MGMT gene. By colony formation, the cell killing induced by 100 μM BCNU was increased by 2 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells.

Footnotes

  • This work was supported by National Cancer Institute Grants CA 45628 (to L.C.E.), CA 81683 (to D.W.O.), CA 86405 (to E.L.K.), and a Fellowship from the Indiana University Department of Pharmacology and Toxicology (to Q.Z.).

  • Abbreviations:
    CENU
    chloroethylnitrosourea
    BCNU
    1,3-bis-(2-chloroethyl)-1-nitrosourea
    MGMT
    O6-methylguanine DNA methyltransferase
    EtOH
    ethanol
    Rz
    ribozyme(s)
    PBS
    phophate-buffered saline
    PCR
    polymerase chain reaction
    HEX
    5′-hexachloro-fluorescein phosphoramidite
    bp
    base pair
    • Received March 2, 2001.
    • Accepted April 9, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 298 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 298, Issue 1
1 Jul 2001
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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to DegradeO 6-Methylguanine DNA Methyltransferase mRNA

Qiwei Zhang, David W. Ohannesian, Emiko L. Kreklau and Leonard C. Erickson
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 141-147;

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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to DegradeO 6-Methylguanine DNA Methyltransferase mRNA

Qiwei Zhang, David W. Ohannesian, Emiko L. Kreklau and Leonard C. Erickson
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 141-147;
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