Correspondence to: Francis M. Sirotnak, Ph.D., Memorial Sloan-Kettering Cancer Center, Laboratory for Molecular Therapeutics, Box 79, 1275 York Ave., New York, NY 10021.
Jansen et al. raise an interesting pharmacologic question, but one that does not, however, appear to be immediately relevant to the report by Aghi et al. The notion that they propose, that complementary DNA (cDNA)-mediated elevation of folylpolyglutamyl synthetase (FPGS) can increase folate pools in target tumor cells as a result of folate polyglutamylation, is inconsistent with the enhanced cytotoxicity and therapeutic efficacy of folate analogues already observed in these cells by such transfection. Moreover, Jansen et al. themselves agree that these results represent a definite "proof-of-concept" for this approach to gene therapy. Although Aghi et al. achieved their results by enhancing FPGS gene expression exogenously, the idea that alterations in intracellular levels of FPGS will influence the cell's cytotoxic and therapeutic sensitivity to folate analogues is certainly not new. There is earlier documentation of this effect in the literature in the form of reports (1-3) of tumor cell variants with either decreased or increased levels of FPGS that display corresponding differences in resistance to folate analogues. Also, let us not forget the earlier study by Kim et al. (4), in which FPGS cDNA transfection restored cytotoxic sensitivity of an FPGS-deficient auxotroph to methotrexate. It is difficult to imagine how these consequences of altered FPGS gene expression could occur if there was a commensurate expansion in folate pools as a result of their polyglutamylation.
The evidence that Jansen et al. cite in support of their notion emanate from studies (5-7) that use tumor cell variants with alterations at the plasma membrane affecting either the mediated entry or egress of folates. These studies provided interesting conclusions and demonstrate the impact of expanded folate pools on the cytotoxicity to folate analogues. However, these alterations in folate pool sizes, in the main, would appear to reflect the changes in membrane transport of folates and less so folate polyglutamylation. For this reason, the bearing of these studies on that by Aghi et al. is questionable.
Given the information available on the relative saturability and capacity of mediated transport of folates and their analogues in tumor cells by means of the reduced folate carrier-1 encoded system, it is clear that the concentration of folates normally found in tissue culture medium or in plasma is severely limiting to folate polyglutamylation (folic acid is a very poor substrate and competitive inhibitor for FPGS as well). This is not the case for the folate analogues used in studies by Aghi et al. The pulse exposure in cell culture and bolus dosing in mice that were used result in the presentation of micromolar concentrations of these analogues to the target glioma cells. As a consequence, one would expect FPGS cDNA transfection to produce substantially greater enhancement of intracellular levels of methotrexate and edatrexate polyglutamates (and more so of ZD1694 and MTA polyglutamates) as compared with enhancement of folate polyglutamate levels. In light of these considerations, the concerns expressed by Jansen et al. would appear to be unfounded.
REFERENCES
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2
McClosky DE, McGuire JJ, Russell CA, Rowan BG, Bertino JR,
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Jansen G, Barr HM, Kathmann I, Bunni MA, Priest DG,
Noordhuis P, et al. Multiple mechanisms of resistance to polyglutamatable and lipophilic
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and intralysosomal drug sequestration. Mol Pharmacol 1999;55:761-9.
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Jansen G, Mauritz R, Drori S, Sprecher H, Kathmann I, Bunni
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