Introduction

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MTAP (EC 24.2.28) catalyzes the phosphorolysis of the nucleoside MTA, a metabolite of SAM produced during the synthesis of the polyamines spermidine and spermine, to yield adenine and 5-methylthioribose-1-phosphate (1). Each of the products of the enzyme are reused: adenine is converted to adenine nucleotide pools via adenine phosphoribosyltransferase (2), and 5-methylthioribose-1-phosphate is metabolized to methionine and formate (3, 4). Thus, MTAP plays a crucial role in initiating the recycling the adenine and methylthio moieties of SAM back to the metabolites from which SAM is formed (i.e., ATP and methionine) (5). These salvage pathways are clearly active in that MTA can, in MTAP-containing cells, serve as a purine source in cells treated with purine de novo synthesis inhibitors (6-8) and as the sole methionine source for cells cultured in methionine-deficient media (9). Although virtually all normal tissues, including red blood cells, contain MTAP activity, many malignant cells lack MTAP activity (5, 6, 10-14). Cultured MTAP-deficient malignant cells do not metabolize MTA but instead simply excrete it (15).

The explanation for the selective loss of MTAP activity in malignant cells took a number of years to unravel. Seminal studies led by Diaz and Rowley (16) revealed that whole or portions of the IFN gene cluster undergoes frequent homozygous deletion in leukemia cell lines and certain primary leukemias and that in many cases, these deletions are associated with loss of MTAP activity (16). These findings suggested that the MTAP gene was located near the INF cluster, which had been mapped to the chromosome 9p21 region (17), and that loss of MTAP activity might be the result of a homozygous deletion of the gene. Deletion mapping studies using cell lines with chromosome 9p21 defects demonstrated that the minimal region of overlap of these deletions resides centromerically from the IFN cluster, at a site close to the suspected MTAP locus (18). Subsequently, Kamb et al. (19) used chromosome walking techniques to map and sequence this deleted region and discovered it contained the genes encoding p16^INK4 and p15^INK4B (p16/MTS-1 and p15/MTS-2, respectively), which, as inhibitors of cyclin-dependent kinase-4 and -6, are thought to have tumor suppressor activity (19-21). Homozygous deletion of p16 has now been reported in subsets of a variety of human malignancies (22). Detailed mapping of this portion of the 9p21 region revealed that MTAP resides only ~100 kb in the telomeric direction from p16 (23), accounting for the high incidence of codeletion of MTAP (>85%) in malignant cells bearing p16 deletions (23, 24).

It has been suggested that the selective loss of MTAP in malignant cells might be exploited chemotherapeutically (6, 14). Exogenous MTA can be used in vitro as a purine source to selectively rescue MTAP-containing malignant cells but not MTAP-deficient malignant cells from the antipurine actions of MTX and related antifolates (6, 14). However, the presence of MTAP in the serum of many mammals, including humans (25), might limit the effectiveness of this strategy in vivo. Recently, we demonstrated that the antipurine action of MTX is more pronounced against p16, MTAP pancreatic carcinoma cells than normal p16⁺, MTAP⁺ epithelial cells; it was suggested that the operation of the MTAP-dependent adenine salvage pathway in normal cells might render such cells less sensitive to antifolates whose mechanism of action involves inhibition of purine de novo synthesis (7). This same conclusion was reached in a study on a non-small cell lung carcinoma cell line transfected with MTAP cDNA (8).

To help determine the mechanism by which the MTAP-dependent salvage pathways might alter cell physiology and responsiveness to chemotherapeutic agents, we recently developed cell model systems in which MTAP cDNA was transfected and expressed in two human pancreatic carcinoma cell lines that are naturally MTAP deficient via homozygous deletion. Here, these model systems are used to determine whether reintroduction of MTAP activity in these deficient cell lines (i) restores the MTAP-dependent adenine and methionine salvage pathways, (ii) alters the rate of purine de novo synthesis, and (iii) decreases the sensitivity of these cells to the antiproliferative actions of agents that act as inhibitors of purine de novo synthesis.

	Experimental Procedures

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Materials. MTA, MTX, MTT, thymidine, and azaserine were purchased from Sigma Chemical (St. Louis, MO). [8-¹⁴C]MTA (55 mCi/mmol) was obtained from Moravek Biochemicals (Brea, CA). [(U)-¹⁴C]Glycine (106 mCi/mmol) was obtained from New England Nuclear Research Products (Boston, MA).

Cell culture and growth studies. The MiaPaCa-2 and PANC-1 wild-type lines were originally obtained from American Type Culture Collection (Rockville, MD). These cells were routinely cultured in DMEM supplemented with 50 units/ml penicillin G, 50 µg/ml streptomycin, 0.5 µg/ml Fungizone, 1 mM sodium pyruvate, and 10% fetal calf serum. Transfection of these cells with vectors containing the neo and/or MTAP sequences and selection of cell clones expressing these genes will be described elsewhere.¹ Cell lines expressing the neo genes, MiaPaCa-2/neo and PANC-1/neo, were grown in the above-mentioned DMEM medium containing 0.25 mg/ml geneticin (G418). Some MTAP-transfected cell lines, MiaPaCa-2/MTAP-AzG and PANC-1/MTAP-Az, were maintained in the the above-mentioned DMEM medium, except fetal calf serum was replaced with 10% donor horse serum and the medium was supplemented with 10 µM azaserine and 10 µM MTA. These conditions select for MTAP-expressing cells, which are able to use MTA as a purine source in the presence of the inhibitor of de novo purine biosynthesis, azaserine. Another set of MTAP-transfected cell lines, MiaPaCa-2/MTAP-G and PANC-1/MTAP-G, were cultured in methionine-deficient DMEM medium supplemented with 50 units/ml penicillin G, 50 µg/ml streptomycin, 0.5 µg/ml Fungizone, 1 mM sodium pyruvate, 1 mM glutamine, 3% donor horse serum, and 15 µM MTA. These conditions select for MTAP-expressing cells, which are able to grow using MTA as their sole methionine source.

Assessment of 9p21 markers and MTAP activity. All 9p21 markers except MTAP were assessed by polymerase chain reaction amplification of genomic DNA, as described previously(7, 24). MTAP activity was determined using the radiochemical assay reported previously (24).

Incorporation of [¹⁴C]MTA into purine nucleotides pools. Control or MTAP-transfected cell lines were harvested in trypsin-EDTA and washed in phosphate-buffered saline, and 6 × 10⁶ cells were plated onto 100-mm dishes in 6 ml of DMEM and 10% horse serum medium containing 5 µM [8-¹⁴C]MTA (0.15 µCi/dish). After a 6-hr period at 37° in a humidified 95% air/5% CO₂ incubator, the cells were harvested in trypsin-EDTA and pelleted, and the supernatant was removed. Nucleotides were extracted by the addition of 100 µl of 6% perchloric acid onto the cell pellet; the samples were then vortexed and placed on ice for 10 min. The extracts are brought to neutral pH with 8 N KOH and centrifuged to remove perchlorate salts, and 50 µl of the supernatant was analyzed for incorporation into purine nucleotide pools using a modification of a previously described anion-exchange high performance liquid chromatography technique (26). A Waters model 510 liquid chromatograph equipped with a Whatman (Clifton, NJ) Partisil 5 SAX column (25 cm) was used, and the purine nucleotides were separated using a programmed gradient of 1 mM potassium phosphate, pH 4.5, as the low concentrate eluent, and 500 mM potassium phosphate, pH 4.5, as the high concentrate eluent. The gradient profile consisted of a linear increase of high concentrate eluent of 0-100% over a 40-min period, followed by a 20-min isocratic period at 100% high concentrate, at a flow rate of 2 ml/min. Absorbance was monitored at 259 nm, and fractions were collected at 1-min intervals. Each fraction was added to 10 ml of EcoScint A (National Diagnostics, Atlanta, GA) and counted in a liquid scintillation counter (model LS 6500; Beckman Instruments, Columbia, MD). Authentic adenosine and guanosine nucleotide standards (Sigma Chemical) were used to identify peaks based on retention times.

Rates of de novo purine biosynthesis. The rates of purine de novo biosynthesis in these cell lines were determined using a modification of a previously reported method (27). Here, de novo purine synthesis rates are assayed by measuring the incorporation of [(U)-¹⁴C]glycine into FGAR in the presence of azaserine, which inhibits the further metabolism of FGAR. Cultured cells were harvested in trypsin-EDTA and washed twice in glycine-deficient RPMI 1640 medium containing 10% horse serum, and a cell suspension containing 4 × 10⁶ cells in this medium was placed in a 37° shaking water bath. After a 15-min period to allow temperature equilibration, azaserine was added to final concentration of 0.6 mM and incubated for 15 min. At this time, glutamine was added to a final concentration of 2 mM, along with 5 µCi of [(U)-¹⁴C]glycine (106.8 mCi/mmol), and the cell suspension was brought up to a total volume of 1 ml. After 3 hr of incubation at 37°, the cells were pelleted by centrifugation at 4°, and the supernatant fluid was removed. The cell pellet was washed once with cold 2 ml of glycine-free medium, and the washed pellet was extracted in 1 ml of 6% ice-cold perchloric acid. After 15 min of incubation on ice, the extract was alkalinized with 2 N KOH. After centrifugation to remove percholate salts, 1.3 ml of the neutralized supernatant was applied to a column of AG-1 × 8 anion-exchange resin (200-400 mesh, BioRad). Columns were washed with 20 ml of 0.5 M formic acid, and labeled FGAR was eluted with 15 ml of 4 M formic acid collected in three 5-ml aliquots. Fifteen milliliters of scintillant (EcoScint A; National Diagnostics, Atlanta, GA) was added to each aliquot and counted in a Beckman LS 6500 Scintillation System.

	Results

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Expression of MTAP cDNA in p16, MTAP malignant cell lines. To develop model systems for studying the influence the MTAP-dependent salvage pathways have on cell physiology and drug sensitivity, MTAP cDNA was subcloned into the pSVL mammalian expression vector and transfected into two human pancreatic carcinoma cell lines, MiaPaCa-2 and PANC-1, both of which contain homozygous deletions of the p16 and MTAP genes, as well as other other loci in the chromosome 9p21 region (7). As will be described elsewhere,¹ selection of MTAP-expressing cell clones were carried out using three methods: (i) selection based on ability to grow in the presence of the purine de novo inhibitor azaserine, with MTA used as a purine source; clones selected in this way were given the suffix MTAP-Az; (ii) after cotransfection with MTAP and neo- containing vectors, selection based on the ability to grow in medium containing G418, azaserine, and MTA; clones selected by this method were labeled MTAP-AzG; and (iii) after cotransfection with MTAP and neo-containing vectors, selection based initially on G418 resistance, followed by selection in methionine-deficient medium containing MTA as the sole source of this amino acid; these clones were labeled with the suffix, MTAP-G. Cell clones selected in this manner, PANC-1/MTAP-Az, PANC-1/MTAP-G, MiaPaCa-2/MTAP-AzG, and MiaPaCa-2/MTAP-G, but not the corresponding neo-transfected or wild-type cells, expressed MTAP because extracts of these cell clones were capable of converting [8-¹⁴C]MTA to [8-¹⁴C]adenine and contained MTAP mRNA.² The MTAP activities in these MTAP-transfected cell lines were 0.1-0.3 nmol/min/mg of protein (Table 1), which is at the low to middle end of the range of MTAP activity found in a survey of malignant cell lines (24). These MTAP-transfected lines as well as their neo-transfected counterparts display the same pattern of homozygous deletion of chromosome 9p21 markers as the parental (wild-type) lines, verifying their pedigrees and confirming that transfection has not restored any of the other deleted loci (Table 1). Finally, the doubling times of these transfected cell lines were found to be generally comparable to, or slightly longer than, those of the parental, wild-type cell lines (Table 1). Thus, these panels of cell lines are suitable as model systems to determine the effect of MTAP activity on cell physiology and drug sensitivity.

Expression of MTAP cDNA in p16, MTAP malignant cell lines: restoration of MTAP-dependent salvage pathways. Our initial question was whether the expression of MTAP activity in these malignant cells bearing homozygous deletions of the MTAP locus would restore the salvage pathways that depend on this phosphorylase. If transfection and expression of MTAP restored the operation of the MTAP-dependent adenine salvage pathway in these cell clones, then they should be able to convert exogenous [8-¹⁴C]MTA into their purine nucleotide pools. To test this, each of these MTAP-transfected cell lines and their sham-transfected counterparts were incubated for 6 hr in the presence of [8-¹⁴C]MTA, the cells were extracted, and the nucleotide pools were analyzed by anion-exchange high performance liquid chromatography. As shown in Fig. 1A, cell lines in which the expression of MTAP was restored by transfection (e.g., PANC-1/MTAP-G and MiaPaCa-2/MTAP-AG) incorporate labeled MTA into both adenine and guanine nucleotide pools, whereas the corresponding cells transfected with neo-containing vectors alone show virtually no incorporation. Additional evidence for restoration of the MTAP-dependent adenine salvage pathway is based on the fact that the MTAP-transfected cell lines, but not the sham-transfected or wild-type cell lines, are able to grow using MTA as a purine source in the face of a blockade of purine de novo synthesis effected by high concentrations (10⁶ M) of MTX, in the presence of thymidine (Fig. 1B). The responsiveness of MTAP-transfected cell lines to the MTA analog 5-dFAdo is further evidence for restoration of the adenine pathway. 5-dFAdo is an alternative substrate of MTAP; the product of this reaction, 2-fluoroadenine, is converted to cytotoxic 2-fluoroadenosine-containing nucleotides via adenine phosphoribosyltransferase and the enzymes of adenine nucleotide salvage and is ~2 orders of magnitude more growth inhibitory to MTAP-containing leukemia cell lines than MTAP-deficient lines (28). In line with this, we found that expression of MTAP activity in the MiaPaCa-2 and PANC-1 cell lines markedly increased their sensitivity (ID₅₀ = 0.2-0.9 µM) to the antiproliferative actions of 5-dFAdo relative to the corresponding MTAP-deficient parental and neo-transfected lines (ID₅₀ = 13-236 µM; Fig. 1C).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Evidence for the restoration of the MTAP-dependent adenine salvage pathway in MTAP-deficient MiaPaCa-2 and PANC-1 human pancreatic carcinoma cell lines transfected with MTAP cDNA. A, Incorporation of [8-14C]MTA into the purine nucleotide pools of MTAP-transfected and control cell lines derived from the PANC-1 (top) and MiaPaCa-2 (bottom) human pancreatic carcinomas. Cells were incubated with [8-14C]MTA, cell extracts were obtained, purine nucleotides were separated by high performance liquid chromatography, and counts were assessed by liquid scintillation counting as described in the text. Authentic adenosine and guanosine nucleotide standards (Sigma Chemical) were used to identify purine nucleotide peaks on the basis of their retention times. B, Ability of MTAP-transfected and control MiaPaCa-2 and PANC-1 cell lines to grow using MTA as a purine source in the face of a blockade of purine de novo synthesis by MTX. Cells were plated onto 12-well dishes (20,000 cells/well) in DMEM supplemented with 10% horse serum, 20 µM thymidine, and with or without 1 µM MTX and/or 10 µM MTA. Cells were incubated at 37° in a 95% air/5% CO2 atmosphere for either 5 days (MiaPaCa-2-derived cell lines) or 7 days (PANC-1-derived cell lines), and the cell number was determined using the MTT-based assay. Values are mean ± standard deviation of four independent determinations. *, Data are significantly different from samples treated with 1 µM MTX alone using a two-tailed paired t test (p < 0.05). C, Dose-response curves for the effect of the adenine-substituted MTA analog 5-dFAdo on the growth of MTAP-transfected and control cells derived from the MiaPaCa-2 (top) and PANC-1 (bottom) human pancreatic carcinoma cell lines. Cells (20,000/well) were plated in DMEM containing 10% donor horse serum with the indicated concentration of 5-dFAdo and incubated at 37° for 7 days. Cell number was determined using the MTT-based assay system (see Experimental Procedures). Values are mean ± standard deviation of three independent determinations. Top, MiaPaCa-2 wild-type,  (control cell doublings, 5.91 ± 0.03); MiaPaCa-2/neo,  (control cell doublings, 5.52 ± 0.13); MiaPaCa-2/MTAP-AG,  (control cell doublings, 4.55 ± 0.14); and MiaPaCa-2/MTAP-G,  (control cell doublings, 4.40 ± 0.10). Bottom, PANC-1 wild-type,  (control cell doublings, 4.05 ± 0.20); PANC-1/neo,  (control cell doublings, 3.91 ± 0.22); PANC-1/MTAP-Az,  (control cell doublings, 3.08 ± 0.15); and PANC-1/MTAP-G,  (control cell doublings, 4.49 ± 0.10).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Ability of MTA to serve as a methionine source for MTAP-transfected and control cell lines. Cultured MTAP- or sham-transfected Mia PaCa-2 cells (left) and MTAP- or sham-transfected PANC-1 cells (right) were harvested using trypsin-EDTA, washed twice in phosphate-buffered saline, and plated in 0.5 ml methionine-deficient (Met)MEM medium (made from Selectamine kits, GIBCO-BRL) supplemented with 6% undialyzed donor horse serum (20,000 cell/well, 12-well dishes); after a 4-6 hr period to allow cell attachment, an additional 0.5 ml of serum-free methionine-deficient medium was added to each well (final horse serum concentration, 3%), containing varying concentrations of MTA (top) or methionine (bottom). Cells were incubated 7 days at 37° in a humidified incubator in a 95% air/5% CO2 environment. Cell counts were determined using the MTT cytotoxicity assay (7). Values are mean ± standard deviation of four independent determinations. A and C, , MiaPaCa-2 wild-type cells; , MiaPaCa-2/neo cells; , MiaPaCa-2/MTAP-AzG cells; and , MiaPaCa-2/MTAP-G. B and D, , PANC-1 wild-type cells; , PANC-1/neo; , PANC-1/MTAP-Az; and , PANC-1/MTAP-G.

Influence of operation of the MTAP-dependent adenine salvage pathways on the rates of purine de novo synthesis. The cell model systems in which the MTAP-dependent salvage pathways have been restored or remain inoperative enabled us to study the influence these pathways might have on aspects of cellular metabolism. One metabolic pathway that might be affected by the salvage of adenine from MTA, for example, is purine de novo synthesis. Preformed purines dampen the rate of purine de novo synthesis by mechanisms that involve a competitive consumption of precursors such as 5-phosphoribosyl-1-pyrophosphate and feedback inhibition by purine nucleotides of the early steps in the de novo pathway (29). Thus, restoration of the MTA-to-adenine nucleotide salvage pathway in MTAP-deficient malignant cells might decrease their purine de novo synthesis rates. To test this, we studied the rates of purine de novo synthesis in the MTAP-transfected versus wild-type or sham-transfected pancreatic carcinoma cell lines, using an assay system that measures the incorporation of [(U)¹⁴C]glycine into the purine nucleotide precursor FGAR during a blockade of phosphoribosylformylglycineamidine synthetase by azaserine. As shown in Fig. 3, the two MiaPaCa-2 cell lines in which the MTA-to-adenine nucleotide salvage pathway has been restored, MiaPaCa-2/MTAP-AzG and MiaPaCa-2/MTAP-G, displayed statistically significant decreases in their rates of de novo purine synthesis (34-47%) relative to the corresponding MTAP-deficient lines. The rates of incorporation of [(U)¹⁴C]glycine into FGAR in the wild-type or sham-transfected PANC-1 cell types were lower than that of the comparable MiaPaCa-2-derived cell lines, presumably due to their longer doubling times. The MTAP-transfected PANC-1 lines had de novo purine synthesis rates that were 18-36% lower than the corresponding MTAP-deficient PANC-1 lines, although this difference was statistically significant only when compared with the values for the PANC-1 wild-type cells.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Effect of restoration of the MTAP-dependent adenine salvage pathway in MTAP-deficient cells on the rate purine de novo synthesis. Purine de novo synthesis was determined by measuring the cellular rate of incorporation of [(U)-14C]glycine into the purine nucleotide precursor, FGAR, in the presence of azaserine, as described in the text. Values are mean ± standard deviation of three to six independent determinations. **, Significantly different from the corresponding wild-type (WT) or sham-transfected cell lines, as determined by a two-tailed paired t test (p < 0.05). *, Significantly different (p < 0.05) only from those of the corresponding wild-type cell lines, as determined by a two-tailed paired t-test.

Role of the MTAP-dependent salvage pathways in determining sensitivity to purine de novo synthesis inhibitors. Part of the mechanism of action of MTX involves the inhibition of several folate-requiring steps of purine de novo synthesis (e.g., GARFT) by polyglutamylated forms of MTX and/or dihydrofolate, the latter accumulating as results of inhibition by MTX of dihydrofolate reductase (30, 31). Recently, we showed that the antipurine actions of MTX are more efficacious in MTAP pancreatic carcinoma cell lines, including PANC-1 and MiaPaCa-2, than MTAP⁺ normal epithelial cells or pancreatic carcinoma lines; it was postulated that in cells in which the MTAP-dependent adenine salvage pathway is operational, the efficient recycling of purine moieties might render these cells less sensitive to the purine de novo synthesis inhibitory effects of MTX (7). If so, reintroduction of MTAP activity via transfection should decrease the sensitivity of such originally MTAP-deficient cells to the antipurine effects of MTX and other agents that inhibit de novo purine synthesis.

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View larger version (21K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for the antipurine-related growth-inhibitory effects of MTX (A and B) or azaserine (C and D) in MTAP-transfected versus control PANC-1- and MiaPaCa-2-derived cell lines. Experiments were carried out in DMEM containing 10% horse serum in 12-well dishes (20,000 cells/well). For experiments involving MTX, the medium was supplemented with 20 µM thymidine to eliminate the thymidine nucleotide-depleting actions of this antifolate. Cells were incubated at 37° in a humidified incubator with a 95% air/5% CO2 atmosphere for 4 days (MiaPaCa-2-derived cells) or 7 days (PANC-1-derived cells), and cell numbers were assessed using the MTT-based assay. A, Dose-response curves for the antipurine-related growth-inhibitory actions of MTX on MiaPaCa-2/neo cells (, control cell doublings, 4.65 ± 0.06) and MiaPaCa/MTAP-G cells (, control cell doublings, 2.47 ± 0.20). B, Dose-response curves for the antipurine-related growth-inhibitory actions of MTX on PANC-1/neo cells (, control cell doublings, 4.93 ± 0.02) and PANC-1/MTAP-G cells (, control cell doublings, 3.73 ± 0.14). C, Dose-response curves for the growth-inhibitory actions of azaserine on MiaPaCa-2/neo cells (, control cell doublings, 4.63 ± 0.25) and MiaPaCa-2/MTAP-G cells (, control cell doublings, 3.00 ± 0.21). D, Dose-response curves for the growth-inhibitory actions of azaserine on PANC-1/neo cells (, control cell doublings, 5.13 ± 0.05) and PANC-1/MTAP-G cells (, control cell doublings, 3.81 ± 0.17). Values are mean ± standard deviation of three independent determinations. Curve fitting by nonlinear regression was carried out using InPlot (GraphPAD, San Diego, CA).

	Discussion

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In previous work, it was demonstrated that the antipurine-related growth-inhibitory action of the antifolate agent MTX was more pronounced in subsets of pancreatic carcinoma cell lines that were p16, MTAP than in pancreatic carcinomas or normal keratinocyte epithelial cells that were p16⁺, MTAP⁺ (7). Furthermore, it was shown that the coaddition of an inhibitor of MTAP enhanced both the potency and efficacy of the antipurine-related growth-inhibitory actions of MTX in MTAP⁺ but not MTAP cell lines (7). These data were consistent with the hypothesis that operation of the adenine salvage pathway in MTAP-containing cells, including normal cells, decreases their dependence on de novo purine synthesis and permits the maintenance, to some degree, of purine pools even in the face of a pharmacological blockade of purine de novo synthesis. Thus, MTAP⁺ cells are less sensitive to the inhibitory effects that antifolates such as MTX have on purine de novo synthesis. In contrast, MTAP-deficient malignant cells, which are unable to recycle the purine moiety of MTA, are more dependent on purine de novo biosynthesis and are more sensitive than MTAP-containing cells to the antipurine actions of antifolates. In the current study, we demonstrated that reexpression of MTAP in p16, MTAP malignant cells, via transfection, (i) restores their MTAP-dependent salvage pathways, (ii) decreases their rates of purine de novo synthesis, and (iii) decreases their sensitivity to the antipurine actions of MTX and azaserine, all of which are predicted by the original hypothesis. These concepts are supported by a recent study by Hori et al. (8), who found that transfection of MTAP cDNA into the MTAP non-small cell lung carcinoma cell line A549 renders these cells less responsive to antifolates such as 5,10-dideaza-5,6,7,8-tetrahydrofolate, which acts as a specific inhibitor of GARFT of the purine de novo synthetic pathway (8).

Previous studies on MTX have demonstrated that there are a number of biochemical factors that can affect the responsiveness of a cell to this agent. The activities of two distinct carrier-mediated active transport systems, the reduced folate carrier and the high affinity folate binding protein (human folate receptor), are important factors in determining the intracellular levels of MTX within a given tissue; in addition, both the activity of FPGS, which traps MTX in the cell by catalyzing its polyglutamation, and DHFR, which is one of the initial targets of MTX and its polyglutamated forms, are also determinants of MTX efficacy (32). Interestingly, in patients with acute lymphoblastic leukemias, p53 mutations have been associated with DHFR amplification and consequent MTX resistance (33). The current study, which used syngeneic cell model systems in which the above-mentioned determinants of MTX action (e.g., folate carrier, FPGS and DHFR activities, and p53 status) are presumably equivalent, have demonstrated that MTAP status and operation of its attendant salvage pathways can also be determinants of MTX efficacy, at least under controlled conditions. The finding that a purine salvage pathway can influence the action of antifolates is not unprecedented: Sano et al. (34) showed that cultures of HL-60 promyelocytic leukemia cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase are 3-fold more sensitive to the antiproliferative actions of MTX than are wild-type cells. These findings support the postulate that loss of a purine recycling mechanism renders cells more dependent on purine de novo synthesis during proliferation and hence more vulnerable to inhibitors of the de novo pathway. Operation of the MTA-to-methionine pathway may also be important in conferring a decreased responsiveness to the actions of MTX. MTX, at relatively high concentrations (>10⁶ M), has been shown to block the cellular uptake of methionine (35); thus, MTAP-expressing cells, which have the ability to convert endogenously generated MTA to methionine, might be less prone to the growth-inhibitory consequences of this blockade.

There are a number of potential implications of these findings for clinical cancer chemotherapy. If normal cells, which are uniformly MTAP positive (5), are intrinsically less sensitive to the antipurine actions of antifolates such as MTX, then this might in part be the basis for the relatively selective action these agents have against certain malignancies. If the corollary is correct that MTAP-deficient malignant cells are especially sensitive to certain antifolates that act in whole or in part by inhibiting purine de novo synthesis, one would theoretically be able to obtain an improved therapeutic index by identifying the subset of patients with MTAP-deficient malignancies and treating them with such agents. From this point of view, it is not surprising that the more MTX-responsive malignancies, such as T cell acute lymphocytic leukemias, happen to have high incidences of 9p21 deletions, including the MTAP locus (36). The development of methodologies for detecting MTAP in clinical samples (e.g., those based on in situ hybridization or immunohistochemistry) is a priority.

These considerations may also influence the design of clinical trials of some of the new antifolates currently under development. Of particular interest are third-generation antifolates such as LY309887; this thienyl derivative of 5,10-dideaza-5,6,7,8-tetrahydrofolate acts as a potent and selective GARFT inhibitor and requires relatively low polyglutamation for activation (37). One would predict that this compound, which exclusively targets purine de novo synthesis, might be especially efficacious against MTAP-deficient malignancies, regardless of their FPGS activity: indeed, among the xenografted tumors that were reported to be highly responsive to LY309887 are the MTAP pancreatic carcinomas PANC-1 and BxPC-3 (37). This hypothesis is being investigated. In any case, the fact that MTAP deficiency in malignant cells can alter their sensitivity to particular antineoplastic agents may have had an impact on past drug development efforts. Two of the early model systems used for screening the activity of anticancer drugs, the L1210 and P388 murine leukemias, are MTAP deficient (10); this may explain in part their high degree of responsiveness to antifolates such as methotrexate. One can only speculate whether the use of these MTAP model systems might have favored the development, for example, of particular antifolates with strong antipurine activity. Even among the current cell models for drug screening of the National Cancer Institute (includes 60 cell lines), 47% have been found to bear homozygous deletions of p16 (38); presumably a high percentage of this subset also bear homozygous deletions of MTAP. Having both MTAP⁺ and MTAP cell lines within a drug screening system is not disadvantageous and can in fact be advantageous with awareness of this genotypic/phenotypic difference so data are appropriately interpreted.