Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+.
BACKGROUND
Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37).


METHODS
To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase.


RESULTS
The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 "factory" cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing.


CONCLUSIONS
The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products.
Doloff
Joshua C
JC
Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA 02215, USA.
Su
Ting
T
Waxman
David J
DJ
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics / Q000628:therapy / Q000821:virology; D024201:Bystander Effect; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003520:Cyclophosphamide / Q000494:pharmacology*; D065702:Cytochrome P-450 CYP2B6; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D018316:Gliosarcoma / Q000235:genetics / Q000628:therapy / Q000821:virology; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000235:genetics / Q000628:therapy / Q000821:virology; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000378:metabolism; D011355:Prodrugs; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D018906:Antineoplastic Agents, Alkylating; C500695:CDCA5 protein, human; D018797:Cell Cycle Proteins; D011355:Prodrugs; D012333:RNA, Messenger; D003520:Cyclophosphamide; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D020169:Caspases