Combined radiation and cytochrome CYP4B1/4-ipomeanol gene therapy using the EGR1 promoter.
BACKGROUND
Cytochrome p450 isozyme CYP4B1 converts the inert prodrug 4-ipomeanol (4-IM) into toxic alkylating metabolites. Induction of cytotoxicity by 4-IM combined with ionizing radiation (IR) in cells transfected with a fusion protein of rabbit cytochrome CYP4B1 under control of the radiation inducible EGR1 promoter was investigated. The capability of activated 4-IM to sensitize cells to IR was also assessed.


METHODS
Survival fractions of cells, determined by MTT assays, stably transfected with EGR1-CYP4B1 were compared with that of cells transfected with a control plasmid after IR followed by 4-IM. Radiosensitization was tested by comparing clonogenic survival curves of cells transfected with the CYP4B1 cassette under a CMV promoter instead of EGR-1, irradiated with or without 4-IM.


RESULTS
MTT assays for cytotoxicity indicated a decrease in relative survival fractions (survival with 4-IM/survival without 4-IM) of the EGR1-CYP4B1 transfected cells with increasing radiation dosage, but not of control cells. Clonogenic assays revealed decreased survival fractions with increasing radiation doses (CYP4B1 transfected and control cells) and 4-IM concentrations (CYP4B1 transfected cells), but showed no significant differences in slope of survival curves with 4-IM.


CONCLUSIONS
The results indicate IR potentiates the cytotoxic activity of the EGR1-CYP4B1/4-IM transgene system, but activated 4-IM does not sensitize cells to IR. Thus, the EGR1-CYP4B1/4-IM system is a viable radiation-gene therapy system that may allow for improved spatial and temporal control of cytotoxicity by therapeutic radiation fields.
Hsu
H
H
Gene Therapy Program, Translation Cancer Research Program, UCSD Cancer Center, Univ. of California at San Diego, San Diego, CA, USA.
Rainov
N G
NG
Quinones
A
A
Eling
D J
DJ
Sakamoto
K M
KM
Spear
M A
MA
D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D003131:Combined Modality Therapy; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects; D015316:Genetic Therapy / Q000379:methods*; D005910:Glioma / Q000201:enzymology / Q000235:genetics / Q000628:therapy; D006801:Humans; D017874:Immediate-Early Proteins; D007668:Kidney / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D011817:Rabbits; D011836:Radiation Tolerance / Q000502:physiology; D011878:Radiotherapy / Q000379:methods*; D051381:Rats; D013729:Terpenes / Q000493:pharmacokinetics* / Q000494:pharmacology / Q000633:toxicity; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D013729:Terpenes; D014157:Transcription Factors; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; C001470:4-ipomeanol