Affiliations of authors: M. Aghi, X. O. Breakefield, Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Boston, MA, and Program in Neurosciences, Harvard Medical School, Boston; C. M. Kramm, Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital.
Correspondence to: Xandra O. Breakefield, Ph.D., Molecular Neurogenetics Unit, Massachusetts General HospitalEast Bldg., Bldg. 149, 13th St., Charlestown, MA 02129.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Folates and some antifolates possess a single terminal benzoylglutamate and are converted
intracellularly from monoglutamates into polyglutamates by an enzyme, folylpolyglutamyl
synthetase (FPGS), that attaches up to six glutamyl groups in -peptide linkage to the
terminal benzoylglutamate. Polyglutamylation causes 1) intracellular folate and antifolate
accumulation because ionized polyglutamates cross cell membranes ineffectivelyefflux
of methotrexate polyglutamates out of cells is 70 times slower than that of the
monoglutamateand 2) enhanced affinity of folates for enzymes utilizing them as
cofactors, increased antifolate enzyme inhibition, and inhibition by antifolates of an expanded
range of enzymes (1,2).
Because methotrexate is polyglutamylated less efficiently than are folates, reductions in FPGS activity that do not alter folate polyglutamylation reduce methotrexate polyglutamylation and efficacy. FPGS mutations render human leukemias antifolate resistant (3-7). Human sarcomas are intrinsically methotrexate resistant due to low FPGS activity (8). The least toxic antifolates have a greater number of total and higher order polyglutamylates in tumors than in normal tissues (9).
Transfecting mutant cells that lack FPGS activity with an FPGS expression cassette increases their sensitivity to 4-hour methotrexate pulses in culture, a clinically relevant model system (10). However, the issue of whether tumor cells already expressing FPGS can be imbued with enhanced methotrexate sensitivity after FPGS gene delivery has not been addressed. Methotrexate's toxicity for gastrointestinal mucosa and bone marrow could be alleviated by a gene therapy that enhances the drug's tumor-specific toxicity.
Therefore, we conducted a preliminary evaluation of FPGS gene delivery followed by low-dose antifolate treatment as a cancer gene therapy. Antifolate pulse sensitivity of a glioma cell line transfected with an expression cassette containing the FPGS complementary DNA (cDNA) was evaluated in culture and in subcutaneous tumors grown in immunodeficient nude mice. The bystander effect of transfectants on nontransfectants and its possible mechanism were also studied, as were antifolate pulse sensitivities of cultured glioma cells transduced with a viral vector carrying the FPGS cDNA.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amethopterin (methotrexate; MTX), aminopterin, and leucovorin (an antifolate rescue agent)
were obtained from Sigma Chemical Co. (St. Louis, MO). Edatrexate
(10-ethyl-10-deazaaminopterin; EDX) (11) was provided by F. M.
Sirotnak (Memorial Sloan-Kettering Cancer Center, New York, NY). 2-Desamino-2-methyl-N10-propargyl-5,8-dideazofolate (DMPDDF) (12) was
provided by M. G. Nair (University of South Alabama, Mobile). J. Wright (Dana- Farber Cancer
Institute, Boston, MA) provided the nonpolyglutamylatable antifolate PT523
(N-4-amino-4-deoxypteroyl-N-
-hemiphthaloyl-L-ornithine) (13), a better control substance than trimetrexate due to closer
resemblance to methotrexate, and methotrexate (MTX) polyglutamate standards MTX-Glu1 to MTX-Glu5 (methotrexate itself has one glutamate; MTX-Glu1
through MTX-Glu5 represent methotrexate polyglutamates with one to five
additional glutamates attached).
Expression Plasmids
The human FPGS cDNA was provided by B. Shane (University of California, Berkeley). The plasmid FPGS/pCDNA3.1 was generated by cloning the human FPGS cDNA into the EcoRI site of the plasmid pCDNA3.1 (Invitrogen Corp., Carlsbad, CA) and placing it under the control of the cytomegalovirus immediate-early 1 (CMV IE 1) enhancer/promoter, with the gene for neomycin resistance under control of the simian virus 40 (SV40) early promoter. The M12Y vector was provided by Y. Saeki (Massachusetts General Hospital, Boston) and is a hybrid amplicon containing the following: 1) the herpes simplex virus-1 (HSV-1) origin of DNA replication, oriS, and DNA cleavage/packaging signal, pac, which allow the plasmid to replicate and be packaged into HSV-1 virions in cells cotransfected with a cosmid set containing the HSV-1 genome and deleted in pac signals (14); 2) the Epstein-Barr virus (EBV) oriP region and the EBNA-1 (Epstein-Barr nuclear antigen-1) cDNA under the control of the Rous sarcoma virus promoter, which allow episomal replication; 3) the green fluorescent protein (gfp) cDNA under the control of the HSV immediate-early 4/5 promoter; and 4) a cloned transgene of interest under the control of the CMV IE 1 promoter.
Tissues and Cell Lines
Human glioblastoma and astrocytoma tissues were obtained from D. Louis (Massachusetts General Hospital); normal human liver tissue and human liver tissue containing colon carcinoma metastases were obtained from K. Tanabe (Massachusetts General Hospital). All cell lines were grown at 37 °C in a 5% CO-95% air atmosphere in Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Co.) containing 10% fetal bovine serum (Sigma Chemical Co.), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma Chemical Co.). The rat 9L gliosarcoma cell line has been described previously (15). The cell line 9L/BAG expresses the histochemically detectable marker ß-galactosidase (LacZ) and was generated by infecting 9L with the BAG retrovirus (16). The human glioblastoma cell line U87MG (17) was purchased from the American Type Culture Collection (ATCC; Manassas, VA). The human glioblastoma cell line Gli36 was provided by D. Louis. The rat glioma line C6, derived from a nitrosomethylurea-induced brain tumor (18), was purchased from ATCC. The C6/13 cell line is a clone derived from lipofectamine-mediated transfection of C6 cells with connexin43 and guanine phosphoribosyltransferase (gpt) cDNAs and selecting cells for gpt expression (19); these cells were provided by C. C. G. Naus (University of Western Ontario, London, Canada). 9L/CD and 9L/TK (20) cell lines were developed in our laboratory and express Escherichia coli cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK), respectively. The J3T canine glioblastoma cell line derives from a spontaneous tumor in a beagle dog and was provided by M. Berens (Barrow Neurological Institute, Phoenix, AZ). The 2-2 cell line was generated by transfecting Vero African green monkey kidney cells with the HSV-1 ICP 27 gene; these cells were provided by Rozanne Sandri-Goldin (University of California, Irvine).
Transfection of 9L, Gli36, U87, C6, and C6/13 cells with pCDNA3.1/FPGS was carried out by use of lipofectamine (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD), followed by cloning under selection in medium containing 1 mg/mL G418 (neomycin analogue; Life Technologies, Inc.), thereby generating 9L/FPGS, Gli36/FPGS, U87/FPGS, C6/FPGS, and C6/13/FPGS cell lines. All clones proliferated at rates comparable to those of the parental cell lines. Connexin43 expression in the C6, C6/FPGS, C6/13, and C6/13/FPGS cell lines was verified by use of immunocytochemistry, as described previously (19).
Cell Culture Survival Studies
Cells were plated in triplicate at 8 x 105 cells per 10-cm-diameter plate or 4 x 105 cells per well in six-well plates. For bystander effect studies, the same total number of cells was used, with varying percentages of 9L/FPGS cells mixed with 9L cells. Cells were allowed to adhere for 4 hours, after which the medium was replaced with medium containing methotrexate or edatrexate. After 4 hours of drug exposure, plates were washed three times with phosphate-buffered saline (PBS). Drug-free medium was then added, and 3 days later cells were trypsinized and counted on a model ZM Coulter apparatus. Dose-response curves were generated by use of SigmaPlot 3.0 (SPSS Inc., Chicago, IL), with curve-fitting parameters based on the Marquardt-Levenberg method (21) used to determine ED50 (drug concentration that produces 50% survival).
Bystander Effect Studies
The bystander effect was evaluated by calculating transmission efficiency (TE):
![]() |
The superscripts 0, N, and 100 designate the ED50 values for cultures containing 0%, N%, and 100% transfected cells. The transmission efficiency indicates how well the drug's effect is transmitted from transfected to nontransfected cells; i.e., a TE of 50% means the nontransfectants in the coculture experience half the concentration of the active drug that transfectants experience.
Conditioned medium was harvested from untreated 9L and 9L/FPGS cells and from 9L and 9L/FPGS cells that had been pulsed with 900 nM edatrexate for 4 hours followed by 3 days' growth in drug-free medium. Some conditioned medium was dialyzed overnight (Spectra/Por Cellulose Ester Membrane, 1000 molecular weight cutoff) at 4 °C. Nondialyzed conditioned medium was left overnight at 4 °C to control for product degradation. Conditioned medium (4 mL) (filtered through 0.2-µm sterile acrodisc filter) plus 2 mL of fresh medium was added to 10-cm-diameter plates containing 9L/BAG cells (1000 cells per dish, clonogenic efficiency approximately 20%) plated the previous day. Leucovorin (1 mM) was added to some plates. Six days later, the cells were fixed with 4% paraformaldehyde in PBS. Fixed cells were stained for ß-galactosidase by incubation overnight in a solution containing 35 mM potassium ferricyanide, 35 mM potassium ferrocyanide, 2 mM magnesium chloride, and 0.2% 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (Xgal; Fisher Scientific Co., Pittsburgh, PA) at pH 7.3 (23). The next day, blue colonies greater than 1 mm in diameter were counted.
The time and density dependence of bystander effects were studied by coculturing 80% 9L/BAG cells with 20% 9L/FPGS, 9L/TK, or 9L/CD cells (the latter two used for comparative purposes) in six-well plates so that the total number of cells was 5 x 105 (high densityall cells in contact with each other; 51 500 cells/cm2) or 5 x 103 (low densityno cell-to-cell contact; 515 cells/cm2). Cells were treated with 900 nM edatrexate (4-hour pulse followed by 3 days' growth in drug-free medium), 9 µg/mL (36 µM) ganciclovir (3-day continuous exposure; Cytovene; Hoffmann-La Roche Inc., Nutley, NJ), or 90 µg/mL (697 µM) 5-fluorocytosine (3-day continuous exposure; Sigma Chemical Co.). These concentrations represent the highest concentrations that are relatively nontoxic to 9L or 9L/BAG cells. Every 24 hours for 3 days, triplicate sets of untreated and drug-treated cocultures were fixed and stained for ß-galactosidase expression, as described above. Blue and white cells were counted under a light microscope (Nikon, Melville, NY). Cultures of 100% 9L, 9L/FPGS, 9L/CD, and 9L/TK cells with identical numbers of cells were treated with the same drug concentrations and counted under a microscope and using a ZM Coulter Counter. Ten fields were counted at 10x (low density) and 40x (high density). With the use of known dimensions of microscope fields and wells, total numbers of LacZ-positive and LacZ-negative cells in each well and percent survival were calculated for each drug at each time point for low- and high-density plates.
In another study, a Falcon inert micropore membrane with pore diameter of 0.4 µm (Fisher Scientific Co.) was used to generate upper and lower layers of cells in six-well plates that were physically separated but that exchanged media. Cells (5 x 105) were plated in each chamber and allowed to adhere for 4 hours. Wells contained all possible combinations of 9L and 9L/FPGS. Some cells were treated with 900 nM edatrexate for 4 hours, washed three times in PBS, grown for 3 days in drug-free medium, trypsinized, and counted on a ZM Coulter apparatus.
The role of gap junctions in the bystander effect was investigated by comparing the bystander effect caused by edatrexate pulsing of cocultures containing 20% C6/FPGS plus 80% C6 to that caused by pulsing a coculture containing 20% C6/13/FPGS plus 80% C6/13.
Viral Vector Generation
Helper virus-free stocks of M12Y-FPGS and M12Y amplicon vectors were generated by
lipofectamine-mediated cotransfection of 2-2 cells with amplicon DNA and a set of five
overlapping cosmids (C6a48
a) spanning the HSV-1 genome (24) with deleted pac sequences (14). Vector was harvested 72
hours after transfection by scraping the cells, repeated freezing and thawing, sonification, and
centrifugation at room temperature for 5 minutes at 800g to remove cellular debris.
Vector stocks were titered on the 2-2 cell line by counting gfp-positive cells 24 hours after
infection by use of a standard fluorescence microscope (#78947 fluorescence illuminator;
Nikon). Infection at a multiplicity of infection (MOI) of 1 transducing unit per cell (titered on
cells from the 2-2 line) produced 50% gfp-positive J3T and Gli36 cells. After cells
transduced at this MOI were allowed to replicate for 3 days in the absence of selection,
sensitivities of the transduced cells to the highest edatrexate pulses nontoxic to M12Y-transduced
cells (Gli361 nM; J3T5 µM) were measured.
Enzyme Assay
FPGS activity was measured by use of a modification of a previously described protocol (25). Cells were trypsinized, suspended in 25 mM Tris-HCl (pH 7.5), 5 mM 2-mercaptoethanol, 2 µg/mL aprotinin (Sigma Chemical Co.), and 1 µg/mL leupeptin (Sigma Chemical Co.), and then sonicated (550 Sonic Dismembrator; Fisher Scientific Co.). Sonicated cells were centrifuged at 4 °C at 10 000g for 3 minutes to remove cellular debris. Protein concentration was calculated by use of the Bradford assay (Bio-Rad Laboratories, Hercules, CA). After tumor margins were confirmed histologically, frozen tissue was ground into small pieces, suspended in lysis buffer (50 mM Tris [pH 7.4], 250 mM NaCl, 0.1% Nonidet P-40 [NP40], 5 mM EDTA, 2 µg/mL aprotinin, and 1 µg/mL leupeptin), homogenized (Brinkmann Instruments, Inc., Westbury, NY), and kept on ice for 1 hour to allow for completion of lysis. For the detection of the low FPGS activity in tissue, samples were concentrated by use of an Integrated Speed Vac (Savant Instruments, Inc., Farmingdale, NY), after which protein concentration was determined by use of the Bradford assay. The FPGS enzymatic assay reaction mixture consisted of 1 M Tris (pH 9.75), 50 mM glycine, 100 µM aminopterin, 1 mM glutamate, 50 µM [3H] glutamate (1 µCi/µL; 50 Ci/mmol; Du Pont NEN, Boston, MA), 5 mM adenosine triphosphate, 10 mM MgCl2, 20 mM KCl, 100 mM 2-mercaptoethanol, 0.1 µg/µL BSA, and 0.4-0.8 mg of cellular protein. The reaction was carried out in 500 µL at 37 °C for 2 hours and was terminated by the addition of 1.5 mL of ice-cold 30 mM 2-mercaptoethanol and 10 mM glutamate. Free glutamate was separated from glutamate bound to aminopterin by chromatography on a 1-1.5-mL bed volume minicolumn (Poly-Prep Chromatography Column; Bio-Rad Laboratories) containing DE52 DEAE cellulose (Whatman Inc., Clifton, NJ). The column was equilibrated with 5 mL of 10 mM Tris (pH 7.5) and 80 mM NaCl. The terminated reaction was then applied to the column. The column was washed with 5 mL of equilibration buffer to remove unincorporated glutamate. Glutamylated aminopterin was then eluted with 3 mL of 0.1 N HCl with the eluant collected in a scintillation vial containing 10 mL of scintillation fluid (ScintiVerse II; Fisher Scientific Co.) and subsequently counted in a scintillation counter.
Thin-Layer Chromatography of Methotrexate Polyglutamates
Cells (106 9L or 9L/FPGS) were plated in 6-cm-diameter dishes. After adhering for 4 hours, cells were incubated with 2 µM 3', 5',7-[3H] methotrexate (Moravek Biochemicals; Brea, CA). After 24 hours, cells were trypsinized, centrifuged at 4 °C for 10 minutes at 2000g, resuspended in 300 µL of deionized water, sonicated, and centrifuged at 4 °C for 5 minutes at 800g briefly to remove debris. Proteins were precipitated with 1 mL of methanol at -20 °C for 2 hours and removed by centrifugation at 4 °C at 20 000g for 30 minutes. The supernatant was concentrated to 30 µL under a stream of N2 at 37 °C on a Meyer N-Evap Analytical Evaporator (Organomation Associates Inc., Northborough, MA). From each sample, 1 µL was used for the Bradford protein assay and for scintillation counting. Counts per minute (cpm) were converted into moles of drug taken up by determining the cpm of a known quantity of [3H] methotrexate.
Methotrexate polyglutamates were separated by ascending thin-layer chromatography (TLC) of samples on Baker Si250PA silica gel plates (Fisher Scientific Co.), as described previously (26,27). Plates were eluted with a chloroform-methanol-acetic acid mixture (2 : 2 : 1) containing 20 mg/mL cetyl trimethylammonium bromide (Sigma Chemical Co.). Following elution, the plates were placed under ordinary fluorescent light for 2 days, after which 20 nmol of unlabeled standards were visualized under long-wave UV light. Plates were run in duplicate and the average RF values of the standards were as follows: methotrexate = 0.66, MTX-Glu1 = 0.59, MTX-Glu2 = 0.44, MTX-Glu3 = 0.33, MTX-Glu4 = 0.27, and MTX-Glu5 = 0.25. Regions corresponding to where standards migrated were marked in the sample lane, scraped individually into scintillation vials, and counted. The amount of each radioactive metabolite in a sample lane was determined as a percentage of the total radioactivity recovered from that sample lane, after subtraction of the background counts obtained from lanes lacking radioactive sample.
In Vivo Experiments
Cells (106 9L or 9L/FPGS) or a mixture of 8 x 105 9L plus 2 x 105 9L/FPGS cells in 200 µL of DMEM were injected subcutaneously into the flanks of 6-week-old female nude mice (NCr/Sed, nu/nu, 20 g; Massachusetts General Hospital breeding colony). Animals were cared for in accordance with institutional guidelines. After 14 days, when the tumors had reached an average volume of 60 mm3, the mice were randomly divided into experimental groups with five mice per group. The maximum tolerated dose (MTD) of methotrexate is 50 mg/kg per day for humans, compared with 9 mg/kg per day for nude mice, corresponding to peak plasma levels of 0.1 and 0.03 mM, respectively (28,29). Therefore, nude mice were given intraperitoneal injections of 9 mg of methotrexate/kg body weight or 3 mg edatrexate/kg body weight dissolved in 200 µL of 0.9% NaCl administered daily (MTD) or every third day. Tumor size was measured weekly by use of calipers. Tumor volume was calculated as length x width x height, as described previously (20).
Statistical Analysis
All experiments were performed in triplicate, and comparisons of 9L to 9L/FPGS were made
by use of Student's t test. Error bars on graphs are 95% confidence
intervals and data points were fit to sigmoidal dose-response curves by use of SigmaPlot 3.0. In
Table 1, 95% confidence intervals are given. All P values
are two-sided, with statistical significance defined as P<.05.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human brain tumor tissues possess 10-fold less FPGS activity than
does colon carcinoma (Table 1), an antifolate-sensitive tumor
(30). Initially, 9L cells were transfected with FPGS because
they are readily transfectable, used in gene therapy studies
(20,31), and had the lowest FPGS activity of the glioma cell
lines tested (Table 1
). 9L cells and clones selected after transfection
with a plasmid containing the FPGS cDNA were treated with 0.3
µM methotrexate for 4 hours, followed by 3 days in
drug-free medium. One clone, 9L/FPGS, displayed 40% survival after
methotrexate pulse treatment compared with 90% survival for 9L.
The FPGS activity of 9L/FPGS cells was more than fourfold greater than that of 9L (Table
1). Evidence that increased FPGS activity enhanced the methotrexate
pulse sensitivity of 9L/FPGS came from the similar sensitivity of 9L and 9L/FPGS to pulses of
the nonpolyglutamylatable antifolate PT523 (data not shown).
Measuring Methotrexate Polyglutamates in 9L and 9L/FPGS
The FPGS enzyme assay quantifies drug converted into any of five polyglutamates. However, only MTX-Glu4 and MTX-Glu5 display increased retention and enzyme inhibition (1). Intracellular methotrexate levels after a 24-hour incubation with [3H] methotrexate were 6.3 (9L) and 12.7 (9L/FPGS) pmol/mg protein. TLC showed that 30% of the radioactivity in 9L cells represented methotrexate unaltered by FPGS compared with 10% of the radioactivity in 9L/FPGS. Only 14% of the radioactivity in 9L was associated with MTX-Glu4 and MTX-Glu5 compared with 31% in 9L/FPGS. Thus, 9L/FPGS formed more higher order methotrexate polyglutamates than 9L. Greater methotrexate polyglutamylation in 9L/FPGS lowers intracellular monoglutamate concentration, promoting methotrexate uptake.
Methotrexate Pulse Dose-Response of 9L and 9L/FPGS
9L and 9L/FPGS cells were treated with 4-hour pulses of varying
methotrexate concentrations (Fig. 1, A).
ED50 values of 2.9 µM (9L) and 101 nM
(9L/FPGS) were obtained (Table 1
), a 35-fold difference. In contrast,
ED50 values for these cells grown in methotrexate for 72
hours were 40 nM. Thus, 9L/FPGS cells display enhanced
sensitivity to methotrexate pulses but not to continuous drug exposure,
consistent with enhanced drug retention.
|
The methotrexate sensitivities of subcutaneous 9L and 9L/FPGS tumors
in nude mice were compared. Mice bearing 9L or 9L/FPGS tumors were
treated with methotrexate daily or every third day. Fold growth
relative to when treatment commenced was plotted versus time (Fig. 2,
A). 9L/FPGS tumors responded better than 9L tumors
to each methotrexate regimen at each time point. After 3 weeks, daily
methotrexate treatment produced 10-fold growth in 9L tumors and
fourfold growth in 9L/FPGS tumors, while methotrexate treatment every
third day yielded 26-fold growth in 9L tumors and ninefold growth in
9L/FPGS tumors.
|
Next, 9L and 9L/FPGS were pulsed with edatrexate, aminopterin,
and DMPDDF, drugs with higher FPGS affinity than methotrexate
[aminopterin > edatrexate = DMPDDF > methotrexate
(1,11,12,32)]. The separations between the dose-response
curves of 9L and 9L/FPGS cells pulsed with aminopterin or DMPDDF (not
shown) were comparable to those obtained with methotrexate. However,
edatrexate dose-response curves (Fig. 1, B) displayed greater
separation than methotrexate curves (Fig. 1,
A). ED50 values
for edatrexate pulses were 1.2 µM (9L) and 18 nM
(9L/FPGS), a 67-fold difference, approximately twice the methotrexate
ED50 enhancement (Table 1
). The cell lines responded
differently over a 10 000-fold range of edatrexate
concentrations, greater than the 100-fold range seen with methotrexate.
Bystander Effects
Therapeutic effectiveness of a vector-carried gene at low
transduction efficiencies requires bystander effects, enabling
transduced cells to confer cytotoxicity on nearby nontransduced cells
by transferring toxic metabolites (33,34). Dose-response
curves were generated for cultures of 9L cells, 9L/FPGS cells, and a
coculture (20% 9L/FPGS plus 80% 9L) of both cells treated with
antifolate pulses (Fig. 1, C and D). Based on the ED50 values
(Table 1
), the TEs in cocultures containing 20% 9L/FPGS were
50%
(methotrexate) and 87% (edatrexate). Cocultures containing 10% and
1% 9L/FPGS were treated with 4-hour edatrexate pulses. The resulting
ED50 values (Fig. 1,
D; Table 1
) gave
TEs of 64% (10%
9L/FPGS) and 33% (1% 9L/FPGS).
Bystander Effect Mechanistic Studies
Separation of transfectants and nontransfectants. Conditioned medium was harvested from 9L or 9L/FPGS cells that had been exposed to edatrexate for 4 hours, washed, and incubated for 3 days in drug-free medium. The clonogenicity of 9L/BAG in this conditioned medium was determined. Because of washes after pulsing, edatrexate in conditioned medium must derive from edatrexate that was retained intracellularly and released after pulsing. 9L/BAG cells exposed to conditioned medium from 9L/FPGS cells pulsed with edatrexate formed 64% as many colonies as those in conditioned medium from non-edatrexate-treated 9L/FPGS cells. Equal clonogenicity occurred with conditioned medium from edatrexate-treated or non-edatrexate-treated 9L. Conditioned medium from edatrexate-treated 9L cells was less toxic than that from edatrexate-treated 9L/FPGS cells (P<.005). Medium from edatrexate-pulsed 9L/FPGS cells was nontoxic until 72 hours of conditioning. Toxicity of the medium was reversed by the addition of leucovorin or by dialysis. Next, 9L and 9L/FPGS cells were cultured in wells in which a membrane prevents cell-to-cell contact but allows exchange of media. 9L cells sharing wells with 9L/FPGS cells displayed 60.9% survival after edatrexate pulsing, a statistically significant reduction (P<.01) compared with the 88.2% survival seen after pulsing 9L cells sharing wells with 9L cells. Thus, at least part of the bystander effect observed after edatrexate pulsing of cocultures results from the release of edatrexate by transfectants, followed by re-uptake by nontransfectants.
Bystander effect density and time dependence. High- and low-density cocultures
containing 80% 9L/BAG cells and 20% 9L/CD, 9L/TK, or 9L/FPGS cells were
pulsed (edatrexate) or treated continuously (5-fluorocytosine and ganciclovir) for 3 days. Every
24 hours, ß-galactosidase-positive (9L/BAG) and ß-galactosidase-negative
(drug-sensitive 9L) cells were counted. At high density, pulsing with edatrexate produced
20% (1 day) and 7% (3 days) 9L/FPGS survival, while cocultured 9L/BAG
experienced 48% survival after 3 days (Fig. 3). Similar results
were obtained with low-density 9L/BAG plus 9L/FPGS cocultures (Fig. 3)
. The 9L/TK and 9L/CD bystander effects exceeded those of 9L/FPGS (Fig. 3)
. Low density abrogated the HSV-TK bystander effect but not the CD and
FPGS bystander effects (Fig. 3)
. While FPGS bystander cells experienced
less toxicity than CD or HSV-TK bystander cells, drug administration killed 9L/FPGS cells more
quickly than it did 9L/CD or 9L/TK cells (Fig. 3)
. Thus, not only is
cell-to-cell contact not required for the FPGS bystander effect but also it does not enhance the
effect.
|
Transfecting and transducing human and canine cells with FPGS. Human
glioblastoma cell lines Gli36 and U87 were transfected with the FPGS cDNA, generating
Gli36/FPGS and U87/FPGS cell lines. Although Gli36 cells are very sensitive to edatrexate
pulses, Gli36/FPGS was sixfold more sensitive (Table 1).
Edatrexate-pulse sensitivity was also enhanced in U87/FPGS (Table 1
).
The FPGS/edatrexate bystander effect suggested that transducing a subpopulation of glioma cells
with a viral vector carrying the FPGS cDNA could enhance the edatrexate-pulse sensitivity of the
whole population. The M12Y/FPGS hybrid amplicon was packaged into HSV virions, which
were used to infect Gli36 and J3T at MOIs that produced 50% gfp-positive cells. Pulsing
cells transduced with M12Y/FPGS with the highest edatrexate concentration that was nontoxic to
cells transduced with M12Y alone resulted in 65.2% (Gli36) and 42.9% (J3T)
survival. J3T responded better than Gli36 (P<.02 for Gli36; P<.005 for
J3T), presumably reflecting lower pre-existing FPGS activity and edatrexate sensitivity.
Bystander effect in vivo. Subcutaneous tumors in nude mice formed from 9L cells, 9L/FPGS cells, or a mixture containing 20% 9L/FPGS and 80% 9L cells were treated with edatrexate daily. After 2 weeks, 9L tumors grew eightfold, 9L/FPGS tumors grew slightly less than fourfold, and mixed tumors grew slightly more than fourfold. After 3 weeks, 9L tumors grew 10-fold, 9L/FPGS tumors doubled in volume, and mixed tumors grew fivefold. The comparable growth of treated mixed and 9L tumors after 3 weeks might reflect 9L/FPGS death in the mixed tumor during the first 2 weeks, leaving behind a 9L percentage higher than 80%.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The original prodrug-activating enzymes, HSV-TK and CD, were prokaryotic. Interest has developed in mammalian enzymes that activate chemotherapy drugs, such as cytosine arabinoside (Ara-C; cytarabine) phosphorylation by deoxycytidine kinase (dCK) (31). Ara-C requires enzymatic modification for toxicity, making it a prodrug. However, tumors express dCK normally and are somewhat Ara-C sensitive without gene therapy. Therefore, the chemosensitivity achieved after delivery of genes like dCK tends to be less than that of foreign transgenes like HSV-TK and CD.
Because antifolates are toxic without enzymatic modification, they are not prodrugs. However, FPGS and prodrug-activating enzymes are all "chemosensitizing." In fact, although the 67-fold-enhanced edatrexate sensitivity in 9L/FPGS is less than the 500-fold enhancement in 9L/TK, it is near the 80-fold enhancement in 9L/CD (20) and exceeds the ninefold enhancement obtained when 9L expressed dCK (31), a ubiquitous enzyme, like FPGS.
Advantages of FPGS gene therapy include the following: 1) Tumor cells that express foreign enzymes may generate an immune response before the cells are able to mediate an effective bystander effect, while increased FPGS expression should elicit no response, and 2) lost transgene expression renders ganciclovir or 5-fluorocytosine ineffective, while antifolates remain somewhat effective. In addition, FPGS gene transfer chemosensitized cells with high pre-existing FPGS activity or antifolate sensitivity.
The faster replication and somewhat greater FPGS activities of tumor compared with normal cells create some antifolate therapeutic window. Unfortunately, bone marrow and gastrointestinal epithelium replicate rapidly and are methotrexate sensitive, and liver FPGS activity confers susceptibility in that organ (1). Intratumoral FPGS gene transfer enhances the disparity between tumor and normal cells in an antifolate susceptibility criteria. In this report, 300-900 nM edatrexate pulses killed most 9L/FPGS cells while preserving most 9L cells. Because doses nontoxic to tumor cells prior to gene delivery should be nontoxic against normal tissues, FPGS gene transfer could reduce antifolate toxicity.
9L/FPGS tumors responded as well to 3 mg of methotrexate/kg per day (not shown) or 9 mg/kg every third day as 9L tumors did to 9 mg/kg per day. The response of FPGS transfectants to lower doses reflects the greater inhibitory effect of antifolate polyglutamates, while the response to lower treatment frequencies reflects polyglutamate retention. Reducing dose frequency may be more beneficial than reducing dose because prolonged drug exposure increases the chance of normal cells entering S phase during drug exposure (1). The response of 9L/FPGS to treatment every third day supports studies showing that, after a single methotrexate injection, tumors with FPGS activity that is comparable to that measured in 9L/FPGS cells retain sufficient polyglutamates to occupy all DHFR sites for 48 hours (35). The lack of tumor regression observed in mice might not occur in humans because of the latter's higher MTD. While tumors often survive human antifolate doses, combining the human MTD protocol with FPGS gene transfer will hopefully permit regression.
Leucovorin displaces methotrexate from DHFR, rescuing cells from methotrexate-induced cytotoxicity. Leucovorin does not rescue tumors, however, because cancer cells have somewhat higher FPGS activities than do normal cells. Leucovorin cannot displace polyglutamates, which have greater DHFR affinity than monoglutamates (36). FPGS gene transfer should enhance leucovorin's differential rescue by increasing tumoral antifolate polyglutamylation.
Another way to improve FPGS gene delivery is mutating FPGS to preferentially polyglutamylate antifolates instead of folates. A similar study in which HSV-TK mutagenesis was performed to increase enzyme affinity for gancyclovir compared with thymidine produced encouraging results (37). Antifolate design could improve the effectiveness of FPGS gene transfer. Antifolates are usually designed to possess either high FPGS affinity or toxicity without glutamylation. However, FPGS gene transfer requires drugs with intermediate FPGS affinity (e.g., edatrexate), to maximize the polyglutamylation caused by the modest increases in FPGS activity achievable through gene transfer.
Although a bystander effect, important because of low transduction efficiencies (38), seems unlikely with FPGS gene transfer because the strategy enhances
intracellular drug retention, earlier studies support the possibility. Intracellular antifolate
polyglutamates bind DHFR or are unbound/free in the cytoplasm. The latter can be degraded into
monoglutamate by lysosomal -glutamyl hydrolase (2). Once
extracellular methotrexate drops after the pulse, intracellular monoglutamylated methotrexate
effluxes down its concentration gradient. Decreased intracellular monoglutamylated methotrexate
stimulates unbound/free polyglutamate degradation over polyglutamylation. Six hours after
methotrexate removal, 43% of methotrexate polyglutamates have left the cell as
monoglutamate after cleavage (2).
Thus, an FPGS bystander effect could arise for the following reasons: 1) if antifolate is retained intracellularly after polyglutamylation by transduced cells; 2) when extracellular drug concentration drops after the pulse, unbound polyglutamates inside surviving transduced cells are cleaved into monoglutamate; 3) if monoglutamate effluxes down its concentration gradient out of transduced cells; and 4) if drug released from transduced cells is taken up by nontransduced cells and given a second opportunity, at a lower concentration, to kill nontransduced tumor cells that survived the pulse. The first two steps have been described (2), and the last two are supported here. The sensitivity of cells in this study to continuous antifolate exposure suggests that even a small amount of antifolate released after pulsing can be toxic because of its continuous presence. The FPGS bystander effect was less than for other enzymes. Cocultures in which 5% of 9L cells express CD display a TE of 87% (Aghi M, Breakefield XO: unpublished observation). However, the CD bystander effect has produced therapy-related deaths in rodents (39).
Gliomas were chosen for this preliminary investigation because of their use in cancer gene therapy (20,31,33,40) and because most of these tumors possess relatively low FPGS activity. It is unclear why methotrexate glioma treatment has proven disappointinghypotheses include the following: that a low replicating fraction confers resistance to S-phase-specific drugs; that methotrexate penetrates spheroid tumors poorly; and distinct glioma enzyme (e.g., FPGS) expression (41,42). The hydrophilicity of methotrexate does not entirely prevent blood-brain barrier penetration, since high-dose methotrexate treats central nervous system lymphoma (43). The blood-brain barrier near gliomas is usually disrupted or can be disrupted chemically (44-46). If the low replicating fraction in gliomas confers methotrexate resistance, increasing tumoral FPGS activity might allow nonreplicating cells to retain drug until they resume replication. If low FPGS activity in gliomas causes methotrexate resistance, FPGS gene transfer can address this limitation.
FPGS gene transfer could be applied to other cancers investigated in gene therapy trials (40). Some are methotrexate sensitive, and FPGS gene therapy could enhance methotrexate efficacy; others are like gliomas in that methotrexate is not standard therapy but could become an option after FPGS gene delivery.
This report describes the novel finding that FPGS gene transfer into several glioma cell lines enhances antifolate sensitivity and creates a bystander effect in culture and in vivo. The magnitudes of the chemosensitivity enhancement and the bystander effect were close to those of prodrug-activating enzymes. Given the advantages of a nonimmunogenic enzyme-enhancing sensitivity to already moderately effective anticancer agents, FPGS gene transfer merits further investigation.
![]() |
NOTES |
---|
Supported by Public Health Service grant P01CA69246 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by the Giovanni Armenise-Harvard Foundation.
We thank Dr. Barry Shane for the folylpolyglutamyl synthetase (FPGS) complementary DNA and for technical assistance regarding the FPGS enzymatic assay; Dr. Joel Wright for methotrexate polyglutamate standards, technical assistance regarding thin-layer chromatography resolution of polyglutamates, and the drug PT523; Dr. Francis Sirotnak for edatrexate; M. S. Esteves and S. Camp for assistance in transfection of 2-2 cells; Dr. David Louis for glioblastoma tissue and the Gli36 cell line; Dr. Bruce Chabner for valuable advice; Dr. Yoshi Saeki for M12Y; Dr. Christian Naus for C6/13; and Dr. Michael Berens for J3T cells.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Chu E, Allegra C. Antifolates. In: Chabner BA, Longo DL, editors. Cancer chemotherapy and biotherapy: principles and practice. Philadelphia (PA): Lippincott-Raven; 1996. p. 109-48.
2 Balinska M, Galivan J, Coward JK. Efflux of methotrexate and its polyglutamate derivatives from hepatic cells in vitro. Cancer Res 1981;41:2751-6.[Medline]
3 Pizzorno G, Mini E, Coronnello M, McGuire JJ, Moroson BA, Cashmore AR, et al. Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug. Cancer Res 1988;48:2149-55.[Abstract]
4
Roy K, Mitsugi K, Sirlin S, Shane B, Sirotnak FM. Different
antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate
synthetase gene expression at the level of mRNA transcription. J Biol Chem 1995;270:26918-22.
5
Roy K, Egan MG, Sirlin S, Sirotnak FM. Posttranscriptionally
mediated decreases in folylpolyglutamate synthetase gene expression in some folate
analogue-resistant variants of the L1210 cell. Evidence for an altered cognate mRNA in the
variants affecting the rate of de novo synthesis of the enzyme. J Biol Chem 1997;272:6903-8.
6 Takemura Y, Kobayashi H, Miyachi H, Furihata K. Altered expression of folylpolyglutamate synthetase (FPGS) gene in human leukemia cells with defective polyglutamation of raltitrexed (ZD1694) [abstract]. Br J Cancer 1997;75(suppl 1):31.
7 Rodenhuis S, McGuire JJ, Narayanan R, Bertino JR. Development of an assay system for the detection and classification of methotrexate resistance in fresh human leukemic cells. Cancer Res 1986;46:6513-9.[Abstract]
8 Li WW, Lin JT, Tong WP, Trippett TM, Brennan MF, Bertino JR. Mechanisms of natural resistance to antifolates in human soft tissue sarcomas. Cancer Res 1992;52:1434-8.[Abstract]
9 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]
10
Kim JS, Lowe KE, 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.
11 Sirotnak FM, DeGraw JI, Moccio DM, Samuels LL, Goutas LJ. New folate analogs of the 10-deaza-aminopterin series. Basis for structural design and biochemical and pharmacologic properties. Cancer Chemother Pharmacol 1984;12:18-25.[Medline]
12 Patil SD, Jones C, Nair MG, Galivan J, Maley F, Kisliuk RL, et al. Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazofolic acid and related compounds. J Med Chem 1989;32:1284-9.[Medline]
13
Rosowsky A, Bader H, Cucchi CA, Moran RG, Kohler W,
Freisheim JH. Methotrexate analogues. 33. N--Acyl- N
-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity. J
Med Chem 1988;31:1332-7.[Medline]
14 Fraefel C, Song S, Lim F, Lang P, Yu L, Wang Y, et al. Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J Virol 1996;70:7190-7.[Abstract]
15 Weizsaecker M, Deen DF, Rosenblum ML, Hoshino T, Gutin PH, Barker M. The 9L rat brain tumor: description and application of an animal model. J Neurol 1981;224:183-92.[Medline]
16 Scharf JM, Boviatsis EJ, Fleet C, Breakefield XO, Chiocca EA. Genetically modified rat 9L gliosarcoma cells facilitate detection of infiltrating tumor cells in a rat model of brain neoplasms. Transgenics 1994;1:219-24.
17 Ponten J, Macintyre EH. Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 1968;74:465-86.[Medline]
18 Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differentiated rat glial cell strain in tissue culture. Science 1968;161:370-1.[Medline]
19 Zhu D, Caveney S, Kidder GM, Naus CC. Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc Natl Acad Sci U S A 1991;88:1883-7.[Abstract]
20
Aghi M, Kramm CM, Chou TC, Breakefield XO, Chiocca EA.
Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine
deaminase gene therapies. J Natl Cancer Inst 1998;90:370-80.
21 Marquardt DW. An algorithm for least square estimate of non-linear parameters. SIAM J Appl Math 1963;11:431-41.
22 Friedlos F, Denny WA, Palmer BD, Springer CJ. Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy. J Med Chem 1997;40:1270-5.[Medline]
23 Price J, Turner D, Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A 1987;84:156-60.[Abstract]
24 Cunningham C, Davison AJ. A cosmid-based system for constructing mutants of herpes simplex virus type 1. Virology 1993;197:116-24.[Medline]
25
Egan MG, Sirlin S, Rumberger BG, Garrow TA, Shane B,
Sirotnak FM. Rapid decline in folylpolyglutamate synthetase activity and gene expression during
maturation of HL-60 cells. Nature of the effect, impact on folate compound polyglutamate pools,
and evidence for programmed down-regulation during maturation. J Biol Chem 1995;270:5462-8.
26
Wright JE, Rosowsky A, Waxman DJ, Trites D, Cucchi CA,
Flatow J, et al. Metabolism of methotrexate and -tert-butyl methotrexate by human
leukemic cells in culture and by hepatic aldehyde oxidase in vitro. Biochem
Pharmacol 1987;36:2209-14.[Medline]
27
Ellenberger TE, Wright JE, Rosowsky A, Beverley SM.
Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates. J Biol Chem 1989;264:15960-6.
28 Stoller RG, Hande KR, Jacobs SA, Rosenberg SA, Chabner BA. Use of plasma pharmacokinetics to predict and prevent methotrexate toxicity. N Engl J Med 1977;297:630-4.[Abstract]
29 Inaba M, Kobayashi T, Tashiro T, Sakurai Y, Maruo K, Ohnishi Y, et al. Evaluation of antitumor activity in a human breast tumor/nude mouse model with a special emphasis on treatment dose. Cancer 1989;64:1577-82.[Medline]
30 Peters GJ, van der Wilt CL, Cloos J, Pinedo HM. Development of a simple folylpolyglutamate synthetase assay in tissues and cell lines. Adv Exp Med Biol 1993;338:651-4.[Medline]
31 Manome Y, Wen PY, Dong Y, Tanaka T, Mitchell BS, Kufe DW, et al. Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo. Nat Med 1996;2:567-73.[Medline]
32 Moran RG, Colman PD, Jones TR. Relative substrate activities of structurally related pteridine, quinazoline, and pyrimidine analogs for mouse liver folylpolyglutamate synthetase. Mol Pharmacol 1989;36:736-43.[Abstract]
33 Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, et al. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 1993;53:5274-83.[Abstract]
34 Huber BE, Austin EA, Richards CA, Davis ST, Good SS. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci U S A 1994;91:8302-6.[Abstract]
35 Fry DW, Anderson LA, Borst M, Goldman ID. Analysis of the role of membrane transport and polyglutamation of methotrexate in gut and the Ehrlich tumor in vivo as factors in drug sensitivity and selectivity. Cancer Res 1983;43:1087-92.[Medline]
36 Matherly LH, Barlowe CK, Goldman ID. Antifolate polyglutamylation and competitive drug displacement at dihydrofolate reductase as important elements in leucovorin rescue in L1210 cells. Cancer Res 1986;46:588-93.[Abstract]
37
Black ME, Newcomb TG, Wilson HM, Loeb LA. Creation of
drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc
Natl Acad Sci U S A 1996;93:3525-9.
38 Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med 1997;3:1354-61.[Medline]
39 Dong Y, Wen P, Manome Y, Parr M, Hirshowitz A, Chen L, et al. In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine. Hum Gene Ther 1996;7:713-20.[Medline]
40
Roth JA, Cristiano RJ. Gene therapy for cancer: what have we
done and where are we going? J Natl Cancer Inst 1997;89:21-39.
41 Shapiro WR. High-dose methotrexate in malignant gliomas. Cancer Treat Rep 1977;61:753-6.[Medline]
42 Terzis AJ, Fiskerstrand T, Refsum H, Ueland PM, Arnold H, Bjerkvig R. Proliferation, migration, and invasion of human glioma cells exposed to antifolate drugs. Int J Cancer 1993;54:112-8.[Medline]
43 Cher L, Glass J, Harsh GR, Hochberg FH. Therapy of primary CNS lymphoma with methotrexate-based chemotherapy and deferred radiotherapy: preliminary results. Neurology 1996;46:1757-9.[Abstract]
44 Yamada K, Ushio Y, Hayakawa T, Kato A, Yamada N, Mogami H. Quantitative autoradiographic measurements of blood-brain barrier permeability in the rat glioma model. J Neurosurg 1982;57:394-8.[Medline]
45 Neuwelt EA, Barnett PA, McCormick CI, Frenkel EP, Minna JD. Osmotic blood-brain barrier modification: monoclonal antibody, albumin, and methotrexate delivery to cerebrospinal fluid and brain. Neurosurgery 1985;17:419-23.[Medline]
46 Inamura T, Nomura T, Bartus RT, Black KL. Intracarotid infusion of RMP-7, a bradykinin analog: a method for selective drug delivery to brain tumors. J Neurosurg 1994;81:752-8.[Medline]
Manuscript received December 8, 1998; revised May 4, 1999; accepted May 21, 1999.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |