Abstract
Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neor) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain 2 < fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.
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