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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2356-2363
NEOPLASIA
From the Division of Experimental Hematology and the Departments of
Molecular Pharmacology and Biochemistry, St. Jude Children's Research
Hospital, Memphis, TN.
Antifolate drugs such as methotrexate are commonly used in cancer
chemotherapy. It may be possible to increase the antitumor activity of
antifolates by the coadministration of drugs that inhibit nucleoside
transport, thereby blocking the capacity of tumor cells to salvage
nucleotide precursors. An important limitation of this approach is
severe myelosuppression caused by many of these drug combinations. For
this reason, we have developed a gene therapy strategy to protect bone
marrow cells against combined treatment with antifolates and
nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of the
es nucleoside transporter. A retroviral vector (MeiIRG) was
constructed that expressed the NBMPR-insensitive ei
transporter, hypothesizing that transduced bone marrow cells would
survive drug treatment because of the preservation of nucleoside
salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG
vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing
this system in vivo, decreased myelosuppression was observed in mice
transplanted with MeiIRG-transduced bone marrow cells and subsequently
treated with trimetrexate and NBMPR-P. In these mice, significant
increases were noted in absolute neutrophil count nadirs, reticulocyte
indices, and the numbers of myeloid progenitors in the bone marrow.
Furthermore, a survival advantage was associated with transfer of the
MeiIRG vector, indicating that significant dose intensification was
possible with this approach. In summary, the MeiIRG vector can decrease
the toxicity associated with the combined use of antifolates and
NBMPR-P and thereby may provide a strategy for simultaneously
sensitizing tumor cells while protecting hematopoietic cells.
(Blood. 2000;95:2356-2363)
Antifolate drugs have been used for many years for the
treatment of neoplastic and viral disorders. In addition to the
well-established agents methotrexate (MTX) and 5-fluorouracil (5-FU),
many novel antifolates are under evaluation for clinical
efficacy.1 These compounds exert their cytotoxic effects by
targeting key enzymes such as dihydrofolate reductase (DHFR),
thymidilate synthetase (TS), and glycinamide ribonucleotide
formyltransferases (GARFT) in the de novo biosynthesis of purine and
pyrimidine nucleotides.2
There are many forms of acquired and inherent resistance to antifolate
drug therapies that are well characterized, such as decreased
expression of the reduced folate carrier, mutation or amplification of
the DHFR gene, and increased expression of the TS gene.2-4
We and others3-8 have postulated that another possible mechanism of resistance to antifolate drugs results from the ability of
tumor cells to salvage extracellular nucleosides. Many malignant cells
are able to use membrane-spanning nucleoside transporter proteins to
salvage extracellular nucleosides and thereby circumvent antifolate
toxicity by bypassing the de novo nucleotide synthesis pathway. To
date, 5 such transport proteins have been characterized in mammalian
cells.5,6 Three of these are sodium-dependent transporters
and are predominantly expressed in normal tissues.6,7 The
other 2 are equilibrative and are subdivided into 2 classes, es
and ei (equilibrative sensitive and equilibrative insensitive, respectively), based on their relative sensitivity to the nucleoside analog NBMPR. Low levels of ei are seen in most normal tissues and some tumor cell lines, whereas es is the major nucleoside transport activity expressed in both normal and malignant
tissues.6,7 The high levels of es transporter
expression in tumor cells suggests that NBMPR could be used to increase
the antitumor effect of antimetabolite drugs by blocking salvage
activity. Accordingly, cultured cell lines have been shown to be
greatly sensitized to the cytotoxic effects of MTX, 5-FU, and PALA
(N-phosphonoacetyl-L-aspartate) in vitro by the addition of nucleoside
transport inhibitors such as NBMPR or dipyridamole.2,9-12
NBMPR-P, a 5'-monophosphate pro-drug of NBMPR, has been
demonstrated to block nucleoside transport in vivo in mice treated with
lethal doses of tubercidin, a cytotoxic adenosine
analog.13,14 However, it is not clear whether tumor cells
can be sensitized to antifolates in vivo using nucleoside transport
inhibitors. In clinical trials in which patients were treated with
dipyridamole and 1 or more antifolate drugs, any potential clinical
benefit was masked by severe myelosuppression and, in some instances,
mild to moderate gastrointestinal toxicities.15-21 The
gastrointestinal toxicities noted are unlikely to result from the
addition of NBMPR or dipyridamole because the major nucleoside transporters in gastrointestinal epithelial cells are known to be
sodium dependent6,7 and therefore are not inhibited by either of these compounds. However, the severe myelosuppression resulting from the drug combination can be explained by the fact that
immature bone marrow (BM) cells predominantly express the es
transporter7 and are very sensitive to the combined effects of antifolate drugs and NBMPR.22
A wide body of evidence has accumulated that suggests that the
transient myelosuppression associated with antifolate chemotherapy, in
the absence of a nucleoside transport inhibitor, results from toxicity
at relatively late stages of hematopoietic
development.22-26 The severe myelotoxicity seen with a
combination of antifolate and nucleoside transport inhibitors results
from the sensitization of primitive hematopoietic precursor cells
that express the es transporter.22 The resultant
myelosuppression is more pronounced because the combination
therapy effectively depletes the more primitive progenitor and stem
cells.22,26,27 Therefore, protection of hematopoietic cells
will be necessary to treat patients effectively with a combination of
antifolates and nucleoside transport inhibitors.
Transfer of drug-resistant genes to hematopoietic stem cells has shown
success as an experimental strategy for protecting hematopoiesis
against drug-induced myelosuppression.28 This approach has
been validated in preclinical model systems using a variety of
drug-resistant genes and cytotoxic drug combinations.29 With regard to antifolates, drug-resistant variants of DHFR gene have
been shown to protect mice from MTX and TMTX-induced
myelosuppression.30-32 One disadvantage with DHFR vectors
is that the protection is limited to antifolate drugs that only target
the DHFR enzyme, not drugs such as 5-FU and Tomudex, which are TS
inhibitors, or LY231514, a multi-targeting drug that inhibits DHFR, TS,
and GARFT. We hypothesized that transfer of a nucleoside transporter
gene would confer protection against a broad spectrum of antifolate
drugs by allowing transduced cells to salvage preformed nucleotide
precursors from the serum, thereby bypassing the antifolate-induced
block in de novo synthesis. Because we intend to use NBMPR to sensitize
tumor cells to antifolate toxicity, an NBMPR-insensitive nucleoside
transporter gene (ei)33 was selected for
hematopoietic protection. In the current study, we show that
retroviral-mediated transfer of the ei gene protects bone
marrow cells from antifolates when used together with NBMPR, both in
vitro and in transplanted mice in vivo.
Materials
MeiIRG retroviral vector and ecotropic producer cells
Functional characterization of MeiIRG vector 2.0 × 105 BALB/3T3 clone A31 cells were transduced with supernatant from MeiIRG producer cell line for 48 hours until 100% of cells were GFP positive, as determined by FACS analysis. 3H thymidine (Thd) uptake was measured in wild-type or MeiIRG-transduced 3T3 cells, in the presence or absence of nucleoside transport inhibitors NBMPR (100 nmol/L) or dipyridamole (10 µmol/L). 3H Thd (1 µmol/L at 4 µCi/mL) influx was measured over 10 seconds in sodium-free buffer containing 120 mmol/L N-methyl-D-glucamine (MGA), 20 mmol/L Tris-HCl, 3 mmol/L K2HPO4.3H2O, 10 mmol/L glucose, 1 mmol/L MgCl2, and 1 mmol/L CaCl2. A sodium-free buffer was used to eliminate nucleoside uptake by any endogenous sodium-dependent transporters in A31 3T3 cells.Retroviral-mediated bone marrow cell transduction Bone marrow cells were harvested from tibia and femurs of untreated female C57BL/6J mice (for in vitro myeloid progenitor cells assays) or for transplant studies 48 hours after treatment with 150 mg/kg 5FU (Sigma) given intraperitoneally. Retroviral-mediated gene transfer was carried out by coculture of harvested bone marrow cells on producer cells as previously described.30,37 Briefly, bone marrow cells (1 × 106/mL) were prestimulated for 48 hours in DMEM medium supplemented with 20 ng/mL murine IL-3, 50 ng/mL human IL-6, 50 ng/mL rat stem cell factor, and 20% FBS (Hyclone). Prestimulated BM cells were subsequently cocultured for 48 hours with either MeiIRG, MGirDHFR (L22Y/F31R) or irGFP ecotropic retroviral producer cell lines, rendered replication defective by irradiation (11 Gy), with the above cytokines and 6 µg/mL polybrene. After transduction, nonadherent cells were harvested and resuspended in either DMEM plus 15% dialyzed FBS (Hyclone) for in vitro myeloid progenitor cell assays or PBS containing 2% FBS plus 20 U/mL heparin for transplantation. Dialyzed FBS was purchased as a single lot that contained undetectable thymidine and hypoxanthine as determined previously.22In vitro drug sensitivity assays Bone marrow cells were harvested from tibia and femurs of untreated C57Bl/6J mice and transduced with the MeiIRG, MGirDHFR(L22Y/F31R) or irGFP vectors as described above. Nonadherent, transduced BM cells were the collected and cultured in 6-well plates (Corning, Corning, NY) at 1.0 × 106 cell/mL in growth media (DMEM) containing 15% dialyzed FBS, cytokine cocktail (as described above), 1 µmol/L or 100 nmol/L Thd, and 100 µmol/L hypoxanthine. Transduced BM cells were treated in suspension cultures for 4 days at varying concentrations of TMTX, Tomudex, or LY231514 in the presence or absence of NBMPR and dipyridamole. After treatment, nonadherent and adherent BM cells were extensively washed to remove residual drugs and were resuspended in equal volumes of PBS. The volume equivalent of 3.0 × 104 BM cells from drug-free cultures were resuspended in 3 mL methylcellulose semisolid culture media (MethoCult M3530) supplemented with 3 U/mL recombinant human erythropoietin. The progenitor colony numbers were determined 7 days after plating by scoring all colonies consisting of more than 50 cells. A stock solution of NBMPR (10 mmol/L) was prepared in dimethyl sulfoxide before the addition to culture medium, TMTX was dissolved in sterile water and Tomudex and LY231514 were dissolved in 0.4 mol/L sodium bicarbonate, and all 3 were diluted in DMEM before use.Transplantation Transduced or untransduced control bone marrow cells (2 to 4 × 106) were transplanted through lateral tail veins into lethally irradiated (11 Gy at 1.24 Gy/minute) B6.C-H1b/ByJ (also referred to as HW80) recipients. Mice were placed on chlorinated drinking water and filter top cages 2 days before and for 4 weeks after transplantation.GFP flow cytometric analysis Whole blood obtained from the retro-orbital sinus 10 weeks after transplantation was diluted 100-fold in PBS and used to determine GFP expression in RBCs and platelets, which were gated based on forward and side light scatter as previously described.37 Peripheral blood leukocytes were depleted of RBCs by incubation for 10 minutes on ice in RBC lysis buffer (0.9% ammonium chloride), and then viable (based on propidium iodine exclusion) cells were analyzed for GFP expression.Drug treatment of mice Control and ei transplanted mice were treated with a combination of TMTX and NBMPR-P for 5 consecutive days 10 (cohort 1) and 25 (cohort 2) weeks after transplantation. TMTX glucuronate, provided by the Drug Synthesis and Chemistry Branch at the NCI, was used to treat mice in cohort 1 at a dose of 100 mg/kg. Neutrexin (trimetrexate glucuronate), purchased from US Bioscience, was found to be more active than the NCI TMTX and was given to cohort 2 at a dose of 80 mg/kg. Both the TMTX salt and Neutrexin were dissolved in sterile water and delivered as subcutaneous injections. NBMPR-P was synthesized as previously described39 and dissolved in sterile water, and 20 mg/kg was administered as intraperitoneal injections.Hematology Peripheral blood was obtained from the retro-orbital sinus with microcapillary tubes after Metofane anesthesia and was used to determine standard hematologic parameters. White blood cell counts were performed on erythrocyte-depleted blood diluted 1000-fold in Isoton using a Coulter (Hialeah, FL) counter analyzer. The hematocrit was determined by reading the height of packed red cells. Differentials were performed manually on Wright Giemsa-stained whole blood films.Progenitor cell counts Bone marrow was harvested from femurs of mice transplanted with untransduced or ei transduced BM cells 24 hours after final drug treatment and resuspended in 1 mL PBS per femur. Immature progenitor cell colonies were assessed as described above and expressed as clonogenic colonies per volume of femur relative to untreated control animals.Statistical methods The probability of statistical differences between experimental groups was determined by a 2-tailed Student t test using Instat 2.03 software for Macintosh (Apple, Cupertino, CA).
MeiIRG vector The MeiIRG vector was constructed by cloning the open reading frame of the ei gene33 into the MSCV retroviral backbone.34 An IRES-GFP fragment was inserted downstream of the ei gene to facilitate detection of transduced cells.37 Stable ecotropic GP+E86 producer cells36 were made as previously described.37 A polyclonal population with a titer of 5 × 105 on NIH 3T3 cells was used for all subsequent experiments. Southern blot analysis of transduced 3T3 and murine bone marrow cells showed that the proviral genome was transmitted unrearranged to these target cells.Thymidine uptake in MeiIRG-transduced BALB/3T3 clone A31 cells Functional activity of the MeiIRG retroviral vector was determined by comparing the influx of 3H thymidine (Thd) in untransduced cells and MeiIRG-transduced BALB/3T3 (A31) cells. A31 cells were transduced by incubation with supernatant from the MeiIRG GP+E86 producer cells until more than 98% of the cells were GFP positive. Influx of 3H Thd was measured at 24°C over a 10-second interval in sodium-free MGA buffer in the presence or absence of 100 nmol/L NBMPR or 10 µmol/L dipyridamole (Figure 1). The major nucleoside transporter in WT A31 cells is es (J.A.B., unpublished data) consistent with our observation that the addition of 100 nmol/L NBMPR diminishes Thd influx in the untransduced cells down to background levels. In A31 cells transduced with MeiIRG construct, influx of Thd was approximately 3-fold greater than in untransduced cells. Furthermore, this activity was not inhibited by NBMPR, consistent with the known resistance of the ei transporter to NBMPR. Both the endogenous es and the retroviral transduced ei activities were significantly inhibited in the presence of 10 µmol/L DIP, a nucleoside transport inhibitor known to inhibit both es and ei transport activities.5,6
NBMPR potentiates cytotoxicity of Tomudex and LY231514 in control murine progenitor cells in vitro Salvage of extracellular nucleotide precursors by nucleoside transporter proteins has been postulated as a mechanism of resistance to antimetabolite drugs in certain tumor cell lines and in undifferentiated BM progenitor cells.4,7,15,22 These studies predict that NBMPR should sensitize both tumor cells and bone marrow cells to a variety of antifolate drugs. To test whether this was true for bone marrow cells, the cytotoxic effects of Tomudex, a thymidylate synthase (TS) inhibitor,1,40 and LY231514, a multi-targeting antifolate that inhibits DHFR, TS, and GARFT1,41 were evaluated in vitro in the presence or absence of 1 µmol/L NBMPR. Murine bone marrow cells were transduced with the control irGFP vector and treated with varying concentrations of Tomudex (0 to 500 nmol/L) or LY231514 (0 to 1000 nmol/L) in suspension cultures containing 1 µmol/L Thd and 100 µmol/L hypoxanthine for 4 days. Figure 2A shows that in the absence of NBMPR, no significant reduction in progenitor colony number was observed at the highest concentration of Tomudex (500 nmol/L) tested. However, when the BM cells were treated with a combination of Tomudex and 1.0 µmol/L NBMPR, the number of progenitor colonies fell below 50% at Tomudex concentration of less than 40 nmol/L (IC50 = 36 nmol/L). Similar observations were noted with LY231514, a multi-targeting antifolate drug. When BM cells were treated with LY231514 alone, the progenitor colony number remained unaffected even at 1000 nmol/L. However, when the antifolate was tested in combination with 1.0 µmol/L NBMPR, the number of progenitor cell colonies again dropped to below 50% at LY231514 concentrations less than 35 nmol/L (IC50 = 31 nmol/L). The findings from these in vitro studies show that NBMPR markedly sensitizes murine progenitor cells to the cytotoxic effects of multi-targeting antifolate drugs.
MeiIRG vector confers antifolate resistance in murine progenitor cells in vitro Having shown that NBMPR sensitizes murine progenitor cells to the cytotoxic effects of antifolate drugs, we examined whether the MeiIRG vector could protect murine BM cells against antifolate/NBMPR drug combinations. Murine BM cells were transduced with retroviral vectors irGFP, MGirDHFR(L22Y/F31R), or MeiIRG, expressing either GFP alone, a TMTX-resistant DHFR variant, or the ei gene, respectively. Transduced cells were treated for 4 days with TMTX, Tomudex, or LY231514 in suspension cultures in the presence or absence of NBMPR or DIP. All drug treatments were carried out in dialyzed FBS supplemented with 100 nmol/L Thd, which approximates levels measured in human serum,42 and 100 µmol/L hypoxanthine as indicated. The irGFP and MeiIRG-transduced progenitor cells were highly sensitive to 150 nmol/L TMTX when treated in the absence of exogenous Thd and hypoxanthine, whereas the addition of 100 nmol/L Thd and 100 µmol/L hypoxanthine resulted in complete rescue (Figure 3A). When NBMPR was added to the latter culture conditions, myeloid progenitor colonies in irGFP-transduced cells were reduced to 1% of that seen in TMTX-free cultures, whereas BM cells transduced with the MeiIRG vector or the TMTX resistant MGirDHFR(L22Y/F31R) vector were completely protected. As expected, when dipyridamole was added, a drug that inhibits both es and ei transporters, BM cells transduced with the MeiIRG vector were entirely sensitized to TMTX toxicity whereas the MGirDHFR(L22Y/F31R)-transduced BM cells continued to be protected.
TMTX- and NBMPR-P-induced myelosuppression is abated in MeiIRG-transplanted mice We next tested the protective properties of the ei gene in vivo using a mouse model for stem cell gene transfer. Briefly, primary BM cells from C57BL/6J mice were transduced with the MeiIRG vector and transplanted into lethally irradiated recipient mice as previously described.44 Control mice were transplanted with fresh untransduced BM cells from C57BL/6J mice. The BM transduction efficiency of the MeiIRG vector ranged from 62% to 91%, as determined by GFP expression in peripheral blood leukocytes, red blood cells, and platelets in 15 mice evaluated 10 to 25 weeks after transplantation (Table 1). Transplanted mice were treated with 100 mg/kg TMTX daily for 5 days, together with 20 mg/kg NBMPR-P. Peripheral blood was collected from the retro-orbital sinus 24 hours after the fifth day of treatment and the absolute neutrophil count and reticulocyte index were determined. Figures 4A and 4B showed that both the absolute neutrophil count and the reticulocyte index fell to zero in all but 1 of the control mice (N). In comparison, the absolute neutrophil count and the reticulocyte indices were significantly higher in all ei-transplanted mice. These findings were reproduced with a second cohort of control and ei-transplanted mice treated for 5 consecutive days with 80 mg/kg trimetrexate glucuronate (Neutrexin; US Bioscience) and 20 mg/kg NBMPR-P 25 weeks after transplantation (data not shown). In the second experiment, the Neutrexin and NBMPR-P doses used resulted in a 50% (5 of 10) loss of mice in the control group on day 6 after the start of treatment, whereas all 12 of the ei-transplanted mice survived the treatment. Indeed, other than the 5 mice used for BM progenitor cell counts, the remaining 7 were still alive 14 weeks after treatment. BM cells from 5 ei-transplanted and the 5 surviving control mice were harvested 24 hours after the fifth treatment, and the progenitor CFU-C was determined as clonogenic cells per volume of femur relative to untreated animals. Figure 5 shows that the myeloid progenitor CFU-C was conserved in 4 of 5 of the ei-transplanted mice, whereas only 1 of the control transplanted mice showed any surviving progenitors (4%) after 5 consecutive days of drug treatment. The protection of BM progenitors in ei-transplanted mice was statistically significant when compared to control transplanted mice (P < .02).
The inherent or acquired resistance to cytotoxic drugs in certain tumor cells is a major concern in the treatment of neoplastic disorders.2-4 The conventional mechanisms of resistance, such as p53 status, gene amplification, mutation of target genes, and altered drug transport, have been well characterized.4 More recently some of these resistance mechanisms, initially considered to be an impediment, have been used in gene therapy approaches for the protection of normal tissues from the cytotoxic effects of chemotherapeutic agents. A number of chemoresistance genes have been identified for potential bone marrow protection, including P-glycoprotein (MDR-1),45,46 aldehyde dehydrogenase (ADH),47 DHFR,30-32 TS,48,49 O6-methylguanine DNA methyltransferase (MGMT),46,50,51 glutathione-S transferase (GST).52 Some of these enzymes, either wild-type or mutated forms, have already been demonstrated to protect murine and human BM cells in vitro and in vivo. In the current study we report the use of a nucleoside transporter gene for the protection of murine BM cells against the cytotoxic effects of a broad spectrum of antifolate drugs and in doing so are introducing a new class of drug-resistant genes for hematopoietic protection.
Submitted September 2, 1999; accepted November 19, 1999.
Supported in part by the ASSISI Foundation of Memphis grant 94-00, National Heart, Lung and Blood Institute Program Project grant P01 HL 53749, Cancer Center Support grant P30 CA 21765, National Cancer Institute research grant RO1-CA55056, and the American Lebanese Syrian Associated Charities.
Reprints: Divyen H. Patel, Division of Experimental Hematology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105; e-mail: divyen.patel{at}stjude.org.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Introduction
