Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor messenger RNA

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Object. The goal of this study was to evaluate the activity of certain hairpin ribozymes against deletion-mutant epidermal growth factor receptor (ΔEGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 801-bp deletion mutation associated with amplification of the EGFR gene is present in a large subgroup of primary GBMs and confers enhanced tumorigenicity in vivo. As a result of the deletion mutation, the fusion junction of the gene is created directly upstream of a GTA triplet, which is subsequently transcribed into a ribozyme target codon (GUA).

Methods. In attempts to intercept ΔEGFR gene expression at the mRNA level, the authors designed three different hairpin ribozymes derived from the negative strands of satellite RNAs in tobacco ringspot virus, chicory yellow mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV1, identified as the most active anti-ΔEGFR hairpin ribozyme motif, was cloned into the retroviral plasmid N2A+tRNAimet. High-titer recombinant retrovirus-containing supernatants (> 105 colony-forming units/ml) derived from an amphotropic GP+envAM 12 packaging cell line transfected with the N2A+tRNAimet-anti-ΔEGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.ΔEGFR glioblastoma cells, which overexpress exogenous ΔEGFR. Using a virus/target cell ratio of 40:1 in the absence of drug selection, the ribozyme transfer resulted in a greater than 90% reduction of ΔEGFR mRNA levels, a 69% inhibition of ΔEGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions in vitro; transfer of a control mutant ribozyme that was rendered incapable of cleaving its target yielded none of these effects.

Conclusions. These findings indicate that the anti-ΔEGFR-sCYMV1 hairpin ribozyme is capable of specifically inhibiting the expression of ΔEGFR and reversing the ΔEGFR-associated malignant phenotype of GBM cells. This strategy may constitute a promising gene therapy approach for a molecularly defined subgroup of GBMs.

Article Information

Address reprint requests to: Marc-Eric Halatsch, M.D., Department of Neurosurgery, Georg-August University, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. email: halatsch@aol.com.

© AANS, except where prohibited by US copyright law.

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Figures

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    A: Schematic representation of wt and ΔEGFR gene sequences. Italics, gene sequence subjected to deletion mutation; bold, newly generated codon; double underlining, target codon for ribozyme cleavage. B: Schematic representation of the molecular interaction between the sCYMV1-derived hairpin ribozyme and ΔEGFR mRNA. Lower-case letters, base exchanges; bold letters, additional bases (compared with the “standard” sTRSV-derived ribozyme); 5′-GGG triplets pertain to the T7 RNA polymerase promoter sequence.

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    Schematic representation of the Moloney murine leukemia virus—based retroviral vector N2A+tRNAimet expressing the anti-ΔEGFR-sCYMV1 hairpin ribozyme. Asterisk indicates the placement of the foreign gene outside of the retroviral transcriptional unit (“double copy” design).

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    Gel blots showing incubation time-dependent cleavage of ΔEGFR RNA by the sTRSV-, sCYMV1-, and sArMV-derived hairpin ribozymes (reaction conditions: 12 mM MgCl2, 1:1 ribozyme/substrate ratio, 37°C, pH 7.5). Only the substrate RNA was labeled using [α-32P]CTP.

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    A: Gel blot showing Mg++ concentration-dependent cleavage of ΔEGFR RNA by the sCYMV1-derived hairpin ribozyme (reaction conditions: 4 hours of incubation, 1:1 ribozyme/substrate ratio, 37°C, pH 7.5). B: Gel blot showing molar ribozyme/substrate ratio-dependent cleavage of ΔEGFR RNA by the sCYMV1-derived hairpin ribozyme (reaction conditions: 4 hours of incubation, 12 mM MgCl2, 37°C, pH 7.5).

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    Gel blots showing reduction of ΔEGFR mRNA levels in U-87MG.ΔEGFR glioblastoma cells by retrovirus-mediated transfer of the anti-ΔEGFR-sCYMV1 hairpin ribozyme. Total cellular RNAs were isolated as described in Materials and Methods and subjected to semiquantitative RT-PCR. The resulting products were analyzed using ethidium bromide—stained 1.8% agarose gels. Molecular weight marker, pBR322 DNA-MspI digest; internal control (reference gene), β-actin.

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    Graph showing growth inhibition of U-87MG.ΔEGFR glioblastoma cells under relative serum starvation in vitro after retrovirus-mediated transfer of the anti-ΔEGFR-sCYMV1 hairpin ribozyme (diamonds). Solid line with Xs, uninfected U-87MG.ΔEGFR cells; broken line with triangles, empty retroviral vector; dashed line with squares, disabled ribozyme; solid line with stars (bottom curve), uninfected U-87MG cells. Data points represent the mean values derived from independent quadruplicates (5.58% ≤ standard deviation ≤ 29.33%; error bars omitted for clarity).

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    Photomicrographs showing suppression of anchorage-independent growth of U-87MG.ΔEGFR glioblastoma cells in soft agar under relative serum starvation in vitro after retrovirus-mediated transfer of the anti-ΔEGFR-sCYMV1 hairpin ribozyme (C). Original magnification × 20. A: Uninfected U-87MG.ΔEGFR cells. B: Empty retroviral vector. D: Disabled ribozyme.

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