Combined radiation and cytochrome CYP4B1/4-ipomeanol gene therapy using the EGR1 promoter

Anticancer Res. 2003 May-Jun;23(3B):2723-8.

Abstract

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.

Materials and 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.

Conclusion: 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.

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacokinetics*
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / toxicity
  • Aryl Hydrocarbon Hydroxylases / biosynthesis
  • Aryl Hydrocarbon Hydroxylases / genetics*
  • Aryl Hydrocarbon Hydroxylases / metabolism
  • Biotransformation
  • Cell Line
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Cell Survival / radiation effects
  • Combined Modality Therapy
  • DNA-Binding Proteins / genetics*
  • Early Growth Response Protein 1
  • Gene Expression Regulation, Enzymologic / radiation effects
  • Genetic Therapy / methods*
  • Glioma / enzymology
  • Glioma / genetics
  • Glioma / therapy
  • Humans
  • Immediate-Early Proteins*
  • Kidney / drug effects
  • Kidney / enzymology
  • Kidney / physiology
  • Promoter Regions, Genetic / radiation effects
  • Rabbits
  • Radiation Tolerance / physiology
  • Radiotherapy / methods*
  • Rats
  • Terpenes / pharmacokinetics*
  • Terpenes / pharmacology
  • Terpenes / toxicity
  • Transcription Factors / genetics*
  • Transfection
  • Transgenes
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Egr1 protein, rat
  • Immediate-Early Proteins
  • Terpenes
  • Transcription Factors
  • Aryl Hydrocarbon Hydroxylases
  • cytochrome P-450 CYP4B1
  • 4-ipomeanol