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Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines
P Musk1, A Stowers, P G Parsons
1Queensland Institute of Medical Research, Herston, Australia.
Abstract:
Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.
Insights
Antimetabolite drugs like methotrexate inhibit viral replication in melanoma cells. Viral capacity assays predict drug sensitivity better than cellular DNA synthesis, aiding cancer treatment development.
Area of Science:
- Oncology
- Virology
- Molecular Biology
Background:
- Antimetabolites are crucial in cancer therapy.
- Drug resistance is a major challenge in melanoma treatment.
- Understanding drug mechanisms is key to improving efficacy.
Purpose of the Study:
- To evaluate the efficacy of methotrexate, 6-thioguanine, and cytosine arabinoside against adenovirus replication in human melanoma cells.
- To compare the predictive value of viral capacity inhibition versus cellular DNA/RNA synthesis inhibition for drug sensitivity.
- To investigate the role of de novo nucleotide synthesis and DNA repair in drug sensitivity.
Main Methods:
- Treatment of drug-sensitive and resistant human melanoma cell lines with antimetabolites (methotrexate, 6-thioguanine, cytosine arabinoside).
- Assessment of adenovirus replication inhibition (viral capacity).
- Measurement of cellular DNA and RNA synthesis.
- Analysis of DNA fragmentation and dihydrofolate reductase activity.
Main Results:
- Methotrexate, 6-thioguanine, and cytosine arabinoside inhibited viral capacity more effectively in drug-sensitive cell lines.
- Inhibition of viral capacity was a better predictor of cellular sensitivity than short-term inhibition of cellular DNA/RNA synthesis.
- Methotrexate was most effective when administered before viral infection, targeting de novo nucleotide synthesis.
- MTX-sensitive cell lines showed DNA fragmentation and were linked to DNA repair defects (Mer- phenotype).
Conclusions:
- Inhibition of viral capacity serves as a comprehensive cytotoxicity assay for antimetabolites.
- The viral capacity assay can predict cellular sensitivity to methotrexate, 6-thioguanine, and cytosine arabinoside, with potential for clinical application in primary tumor cultures.
- Methotrexate-sensitive cells and adenovirus replication depend on de novo nucleotide synthesis, suggesting a link between this pathway and DNA repair defects in Mer- cells.