Affiliations of authors: M. Aghi, X. O. Breakefield, Molecular Neurogenics Unit, Department of Neurology, Massachusetts General Hospital, Boston, MA, and Program in Neurosciences, Harvard Medical School, Boston; C. M. Kramm, Molecular Neurogenics Unit, Department of Neurology, Massachusetts General Hospital.
Correspondence to: Xandra Breakefield, Ph.D., Massachusetts General Hospital and Harvard Medical School, Molecular Neurogenics Unit, Department of Neurology, Bldg. 149, 13th St., Charlestown, MA 02129-2000.
The issue of whether or not enhanced polyglutamylation of natural folates counteracts enhanced polyglutamylation of antifolates is one that we considered during the study of folylpolyglutamyl synthetase (FPGS) gene transfer. This issue was not addressed in detail in our initial report (1), but our thoughts on the matter are presented here.
Jansen et al. (2) point out that the antifolate thymidylate synthetase inhibitors ZD1694 and LY231514 exhibit marked loss of their growth inhibitory effects when administered to human leukemia cell lines that have been mutated to overexpress the RFC1 gene. These studies differ from our work in that they involve increasing folate uptake, while our studies involved increasing polyglutamylation. The distinction is important because it has been shown that, at physiologic concentrations of folate (200 nM), folates are close to being maximally polyglutamylated, with FPGS active sites readily available for additional folate. In the 4-hour pulse protocol used in our study, most antifolates were employed at doses at which they are not close to being maximally polyglutamylated (3,4). Therefore, increasing the transport of folate, as was done in the ZD1694 and LY231514 studies (2), would provide for the continuous presence of additional intracellular folate that could be polyglutamylated by free FPGS active sites, which would, in turn, increase levels of the folate polyglutamates that can displace antifolates from their targets. On the other hand, increasing FPGS expression provides extra FPGS enzyme, which has a minimal effect on the already close to maximally polyglutamylated folate pools but can enhance the polyglutamylation of the submaximally polyglutamated antifolate pools during the brief 4-hour pulse. In fact, it has been shown (3) that using transfection to increase FPGS activity over a 10-fold range has minimal effect on the folate polyglutamate distribution at physiologic folate concentrations, with most folates having four to eight glutamates attached, regardless of FPGS activity. Over the same range of FPGS activities, methotrexate polyglutamate pools increase significantly after a 24-hour drug exposure (4).
In addition, as was mentioned in the original report (1), some
antifolates would be suboptimal for therapeutic use with FPGS gene transfer. The best antifolates
for use with FPGS gene transfer are probably substrates that have an intermediate affinity for
FPGS. Drugs that are very good substrates for FPGS may be close to being maximally
polyglutamylated at physiologic levels of FPGS activity, and drugs that are poor substrates for
FPGS would not experience greater polyglutamylation following the expansion of FPGS activity
by the degree achieved through transfection. Studies (5,6) of antifolates as
FPGS substrates have produced the results shown in Table 1. Thus, it
appears that edatrexate possesses the desirable intermediate kinetics sought in a therapeutic
substrate of FPGS because it experiences greater increases in polyglutamylation from FPGS gene
transfer than do weaker FPGS substrates, such as methotrexate, or better substrates, such as
ZD1694 and LY231514.
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REFERENCES
1
Aghi M, Kramm CM, Breakefield XO. Folylpolyglutamyl
synthetase gene transfer and glioma antifolate sensitivity in culture and in vivo. J
Natl Cancer Inst 1999;91:1233-41.
2
Jansen G, Mauritz R, Drori S, Sprecher H, Kathmann I, Bunni M,
et al. A structurally altered human reduced folate carrier with increased folic acid transport
mediates a novel mechanism of antifolate resistance. J Biol Chem 1998;273:30189-98.
3
Lowe KE, Osborne CB, Lin BF, Kim JS, Hsu JC, Shane B.
Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of
folylpoly-gamma-glutamyl synthetase substrate specifity and level on folate metabolism and
folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J
Biol Chem 1993;268:21665-73.
4
Lin BF, Huang RFS, Shane B. Regulation of folate and
one-carbon metabolism in mammalian cells: IV. Role of folylpoly-gamma-glutamate synthetase in
methotrexate metabolism and cytotoxicity. J Biol Chem 1993;268:21680-5.
5 Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL, et al. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 1997;57:1116-23.[Abstract]
6 Rumberger BG, Barrueco JR, Sirotnak FM. Differing specificities for 4-aminofolate analogues of folylpolyglutamyl synthetase from tumors and proliferative intestinal epithelium of the mouse with significance for selective antitumor action. Cancer Res 1990;50:4639-43.[Abstract]
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