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Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells
W B Parker1, P W Allan, S C Shaddix
1Southern Research Institute, Birmingham, AL 35205, USA. Parker@SRI.ORG
Abstract:
Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.
Insights
This study explores novel cancer suicide gene therapy using Escherichia coli purine nucleoside phosphorylase (PNP) to activate toxic purine analogs. These analogs, 2-fluoroadenine (F-Ade) and 6-methylpurine (MeP), show potent, distinct mechanisms against cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Suicide gene therapy for cancer shows promise.
- Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is a potential strategy.
- Mechanisms of toxic purine bases 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade) are not well understood.
Purpose of the Study:
- To investigate the mechanisms of action of F-Ade and MeP.
- To compare their efficacy and toxicity with existing anticancer agents.
- To evaluate their potential in cancer suicide gene therapy.
Main Methods:
- Cell viability assays (CEM, MRC-5, Balb 3T3 cells) with F-Ade, MeP, and 5-fluorouracil.
- Measurement of protein, RNA, and DNA synthesis inhibition.
- Intracellular conversion of F-Ade and MeP to phosphorylated forms (F-ATP, MeP-R-TP).
- Analysis of F-ATP, MeP-R-TP, and ATP half-lives.
- Assessment of MeP and F-Ade incorporation into cellular RNA and DNA.
Main Results:
- F-Ade and MeP demonstrated significantly higher potency (lower IC50 values) than 5-fluorouracil in inhibiting CEM cell growth.
- Both F-Ade and MeP were toxic to proliferating and quiescent cells.
- Treatment with F-Ade or MeP inhibited protein, RNA, and DNA synthesis.
- CEM cells converted F-Ade to F-ATP and MeP to MeP-R-TP.
- MeP-R-TP had a longer cellular half-life (approx. 48 hr) compared to F-ATP and ATP (approx. 5 hr).
- Both F-Ade and MeP were incorporated into cellular RNA and DNA.
Conclusions:
- F-Ade and MeP exhibit distinct mechanisms of action compared to other anticancer agents.
- Their generation by E. coli PNP offers potential advantages for cancer suicide gene therapy.
- These advantages include potent toxicity to both proliferating and non-proliferating tumor cells via a novel mechanism.
- The high potency during short-term treatment is a key benefit.