title: New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2-aminoanthracene/4-ipomeanol.
abstract: Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.
authors.affiliation: Molecular Neurogenetics Unit, Neurology Service, Massachusetts General Hospital, Harvard Medical School, Boston 02129, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Rainov
authors.forename: K U
authors.initials: KU
authors.lastname: Dobberstein
authors.forename: M
authors.initials: M
authors.lastname: Sena-Esteves
authors.forename: U
authors.initials: U
authors.lastname: Herrlinger
authors.forename: C M
authors.initials: CM
authors.lastname: Kramm
authors.forename: R M
authors.initials: RM
authors.lastname: Philpot
authors.forename: J
authors.initials: J
authors.lastname: Hilton
authors.forename: E A
authors.initials: EA
authors.lastname: Chiocca
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D017077:Culture Media, Conditioned; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000187:drug effects*; D015316:Genetic Therapy / Q000379:methods*; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018316:Gliosarcoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010957:Plasmids / Q000235:genetics; D011355:Prodrugs / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000873:Anthracenes; D000970:Antineoplastic Agents; D017077:Culture Media, Conditioned; D004273:DNA, Neoplasm; D011355:Prodrugs; D013729:Terpenes; C006593:2-anthramine; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; D005978:Glutathione; C001470:4-ipomeanol