Adenovirus carrying cytochrome P450 4B1 (CYP4B1) under an EGR1 promoter combined with 4-ipomeanol (4-IM) prodrug and radiation

Abstract

Purpose: To test the in vitro efficacy of the chemotherapeutic alkylating agent prodrug 4-ipomeanol (4-IM) combined with ionizing radiation (IR) in cells transduced with an adenoviral vector carrying a chimeric fusion protein of rabbit cytochrome CYP4B1 and enhanced green fluorescent protein (EGFP) under the control of the radiation-inducible EGR1 promoter. Materials and Methods: Cassette effect was tested by comparing survival fractions determined by low-input MTT assay for HEK293 cells stably transfected with the prodrug-activating fusion protein CYP4B1-EGFP under the control of the EGR1 promoter with that of control HEK293 cells stably transfected with EGFP alone, 8 days after treatment with 4-IM and IR. Radiosensitization by activated 4-IM was tested by comparing survival fractions from clonogenic assays of 9L cells transfected with the CYP4B1-EGFP cassette or EGFP alone, both under a constituitive CMV promoter, after 24 hours of 4-IM exposure. The EGR-4B1-EGFP cassette was inserted into the E1A region of an adenoviral vector, and efficacy compared to adenoviral vector carrying CMV-EGFP (MOI = 100), was tested in U87-MG cells using MTT assay 3 days after treatment with 4-IM and IR. In each case, cells were irradiated at doses of 0, 2, and 4 Gy on day 1, following 4-IM treatment at concentrations of 0, 2.5 and 5.0 μg/ml. Results: MTT assays demonstrated a decrease in the relative survival fractions (survival with 4-IM/survival without 4-IM) of EGR1-CYP4B1-EGFP transfected cells with increasing radiation dosage, but not of the control cells: 88%, 57%, 39% at 0, 2 and 4 Gy for the EGR1-CYP4B1-EGFP cells, and 96%, 87%, 96% at 0, 2 and 4 Gy for the control cells (2.5 μg/ml 4-IM). Clonogenic assays for radiosensitization demonstrated that survival fractions decreased with increasing radiation doses and 4-IM concentrations (for the CYP4B1 bearing cells), but no significant differences in D0 were seen with 4-IM. Of relevance, a similar lack of sensitization was seen in the same model using rat cytochrome P450 2B1 (CYP2B1) to activate cyclophosphamide, but a pronounced effect was seen when this combination was used in a replication competent HSV-1 vector (rRp450). 4-IM (5.0 μg/ml) reduced the relative survival of cells treated with adenovirus carrying EGR-4B1-EGFP to 66 +/− 11% with the addition of 2 Gy IR and to 57 +/− 10% with 4 Gy IR. No effect was seen in cells treated with adenovirus carrying CMV-EGFP. Conclusion: IR potentiates the cytotoxic activity of the EGR1/CYP4B1/4-IM transgene system. Cytochrome P450 activated alkylating agent prodrugs do not appear to themselves sensitize cells to radiation in the models tested, but significant augmentation of IR efficacy can be obtained when these are incorporated into appropriate transgene and vector systems. There may be additional variability between agents and cell lines, as has been reported for temozolomide. We are proceeding to test these gene therapy vectors in murine models.