Correspondence to: F. M. Sirotnak, Ph.D., Laboratory of Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail: sirotnaf{at}mskcc.org).
Effective cytotoxic action of classical folate analogues against
tumor cells (1,2) relies not only on their potent inhibition
of the primary intracellular targetanalogues have been tailored to
inhibit dihydrofolate reductase, thymidylate synthase, and glycinamide
ribonucleotide formyl transferasebut also on their efficiently
mediated internalization and subsequent conversion to
poly--glutamates. As with natural folates, internalization of
folate analogues by tumor cells [reviewed in (2)] is
mediated by a plasma membrane transporter encoded by the RFC-1 gene.
The enzymatic conversion of the internalized folate analogue to
poly-
-glutamates occurs by amide bond formation at the
-carboxyl group of the resident glutamyl moiety of the folate
analogue with the amino group of a second glutamate (1,3). A
series of these reactions, mediated by folylpolyglutamate synthetase
(FPGS) (4-6), results in the formation of
poly-
-glutamates incorporating one to several additional
glutamates. Poly-
-glutamates of folate analogues are retained more
readily in tumor cells as well as in other mammalian tissues than is
the parent analogue (7-9); as a result, poly-
-glutamates
produce a more prolonged inhibition of the target enzyme and
suppression of macromolecular biosynthesis. Also, poly-
-glutamates
of 4-amino folate analogues such as methotrexate are often more
effective as inhibitors of folate-dependent enzymes that are involved
in macromolecular biosynthesis (1,10,11)thus potentially
targeting these enzyme directly. Net intracellular accumulation of
poly-
-glutamate derivatives of folate analogues also reflects the
action of folylpolyglutamate hydrolase (FPGH; also known as
-glutamyl hydrolase) (12), which mediates the hydrolysis
of these compounds and their turnover following internalization into
lysosomes (13).
The importance of poly--glutamate formation to the cytotoxicity of folate analogues
was originally inferred from research (9) associating the degree of
cytotoxicity with the extent of poly-
-glutamylation of structurally different analogues by
various tumor cells. Further support for this notion was derived in other studies (14-16) showing that tumor cell variants with acquired resistance to folate analogues
often had low levels of FPGS activity or gene expression compared with drug-naive parental
cells or had high levels of FPGH (17). Additional evidence has
accumulated subsequently (18) and strongly suggests that the natural
refractoriness of some human cancers in a clinical setting to methotrexate may be due, in large
measure, to its accumulation as poly-
-glutamates at only low levels.
Taking advantage of this extensive background of information on folate analogues, in this
issue of the Journal, Aghi et al. (19) present studies that sought to
enhance by gene transfer the cytotoxic sensitivity to folate analogues of rat gliosarcoma cells and
other animal and human tumors originating in the central nervous system. The approach that they
used was to transfect these cells with a plasmid or viral construct encoding human FPGS in the
form of a complementary DNA (cDNA) prior to exposing them to a 4-aminofolate, dihydrofolate
reductase inhibitor. This approach was somewhat similar to attempts by these authors (20) and others (21,22) to sensitize tumor cells to
the cytotoxic action of pyrimidine and purine nucleosides by transfecting these cells with a viral
thymidine kinase or deoxycytidine kinase. However, in contrast to these approaches, which
attempt to enhance cytotoxicity by increased conversion to the proximate antimetabolite
(pyrimidine or purine nucleotide), the elevated FPGS expression in the transfected tumors
resulted in higher levels of intracellular accumulation and retention of the folate analogue as
poly--glutamate metabolites.
What do the authors' findings reveal? Other investigators (23) have already shown that vector-mediated transfection of FPGS cDNA can restore FPGS activity, reverse auxotrophy, and reintroduce cytotoxic sensitivity into variant hamster cells that express no endogenous FPGS activity. These results provided the most direct proof of the importance of FPGS as a determinant of cytotoxicity of classical folate analogues. What Aghi et al. (19) have done is to introduce the notion of vector-mediated gene therapy and to demonstrate in this context that elevation of FPGS activity beyond the endogenous level characteristic of most target tumor cells will augment their cytotoxic sensitivity. Moreover, the authors provide further support for this notion by also showing that the impact of transfection was greater in the case of edatrexate (24), a more efficient substrate for FPGS, but nonexistent in the case of PT523 (25), an analogue that is not a substrate for FPGS. Of interest, the authors go on to provide evidence for a bystander effect (26), seemingly related to the gradual and prolonged release of folate analogues into the extracellular environment, that was more pronounced in the case of edatrexate. They also present some results suggesting that the impact of FPGS gene transfer on cytotoxic sensitivity is also manifested as an increase in therapeutic responsiveness to these folate analogues in a corresponding in vivo model of gliosarcoma.
What is the significance of the studies by Aghi et al.? Their data that show enhanced
cytotoxic sensitivity of tumor cells to folate analogues following FPGS gene transfer
providewithin a limited contexta proof-of-principle for the validity of an
antifolate-based approach to gene therapy. Although these results are provocative and this
approach may ultimately have valuable application in the case of human cancer, the results must
be considered preliminary, as the authors have stated. Substantially more study will be required
before the true merit of this approach will be apparent. The authors' experiments were
carried out primarily with stably transfected, clonal cell lines, a far cry from the unselected
delivery of exogenous genes to target tumors that will be required in clinically relevant model
systems in vivo, let alone in human tumors in situ. The authors have taken
their studies one step further toward clinical relevance by providing evidence for the impact of
unselected transfer of the FPGS gene on cytotoxic sensitivity of a target tumor to edatrexate. In
addition, they provide some documentation for a favorable bystander effect in the case of both
selected and unselected gene transfer. This is an important prerequisite for success under
real-world conditions, where the transfer of genes to cells within a tumor is substantially less
than 100%. Given the usual strategic and tactical limitations associated with gene transfer,
it is unclear at this time to what extent the authors' approach will compare favorably with
other approaches that have a different pharmacologic focus and end point. The large body of
knowledge available pertaining to the cellular pharmacology of folate analogues [reviewed
in (1,2)] makes the authors' approach fundamentally
appealing and provides a substantial conceptual advantage. However, one highly probable
limitation that may present a major obstacle to widespread clinical application of this approach
comes to mind: Many tumors that respond poorly to folate analogues do so (27) because of the less than optimal level of internalization of these agents by the
RFC-1-encoded folate transporter. Since permeation of folate analogues into the cell by action of
this transporter controls the amount of substrate available for FPGS, increased activity of this
enzyme following FPGS gene transfer may have little impact on the ability of the tumor to
generate poly--glutamates. Furthermore, there is also the question of tumor-specific
delivery. How this will be achieved will be important to the eventual success of this therapy,
since normal, proliferative cells in the gastrointestinal tract and bone marrow are also capable, to
their detriment, of forming poly-
-glutamates from these analogues [reviewed in (1)]. Despite these potential limitations, an extension of the studies
of Aghi et al. appears warranted and, with time and reasonable effort, should give us some
indication of the intrinsic merit of this approach and its possible clinical applications.
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