Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of γ-glutamyl hydrolase activity

A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates² using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme γ-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu₄) is achieved within 10 min using a 75 μm I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5×10⁵ eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.     

Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis

The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly. 4-Aminofolates are metabolized to retainable forms at a slow rate compared to folates. Although folate accumulation by cells is not very responsive to changes in folylpolyglutamate synthetase levels and cellular glutamate concentrations, cellular accumulation of anti-folate agents would be highly responsive to any factor that changes the expression of folylpolyglutamate synthetase activity.     

Epigenetic regulation of human gamma-glutamyl hydrolase activity in acute lymphoblastic leukemia cells

Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.     

Radiation induction of drug resistance in RIF-1 tumors and tumor cells

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.     

Phenotypic modification of SV40-transformed hamster lymphoid cells in vivo.

Explants of simian virus 40 (SV40)-induced lymphoid tumors yield SV40-T-antigen-positive derivatives that differ from GD248 lymphocytes propagated in suspension culture, (or in vivo), in the following respects: polygonal shape, adhesion to culture substrates in vitro, phagocytic capacity, lack of immunoglobulin and a chromosome complement at least twice that of GD248 lymphocytes. When GD-248 lymphocytes are propagated as suspension in vitro, no such adherent variants can be detected. However, sequential in vivo passage of GD248 lymphocytes obtained from the suspension-culture lines also yield adherent cell lines upon explanation in vitro. Injection of adherent cells into hamsters produces tumors with histological features of reticulum cell sarcoma.     

Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds.

Transport and metabolic turnover of methotrexate (MTX) polyglutamates were examined in lysosomes derived from S180 cells. These studies extend prior work from this laboratory (Barrueco, J. R., and Sirotnak, F. M. (1991) J. Biol. Chem 266, 11732-11737) which described basic properties of a facilitative transport system in lysosomes capable of mediating intralysosomal accumulation of MTX polyglutamates. In the present report, we show that the rate of turnover of MTX polyglutamates in lysosomes, which releases MTX in the extralysosomal space, is limited by the extent of mediated intralysosomal accumulation of the polyglutamate and reduced sulfhydryls that activate the enzyme folylpolyglutamate hydrolase. Evidence is presented that cysteine functions as the naturally occurring reduced sulfhydryl compound in lysosomes being equipotent to 2-mercaptoethanol as an activator of folylpolyglutamate hydrolase. Folylpolyglutamate hydrolase in permeabilized lysosomes from S180 cells exhibited a low pH optimum characteristic of a lysosomal enzyme, was activated at concentrations of reduced sulfhydryl at 0.1 mM and above, and exhibited Km values in the range of 0.2-3 microM that decreased with increase in polyglutamate chain length. Values for Km for MTX polyglutamates of folylpolyglutamate hydrolase activity were 100-200-fold lower than values for Km or Ki for facilitated intralysosomal transport, whereas capacities for both processes were similar. This relationship between the kinetic properties of each process ensures efficient hydrolysis of MTX polyglutamates within the lysosome.     

Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates

We have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major. These cells readily hydrolyzed MTX to N-10-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 microM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA. MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h. Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX. In contrast to MTX, folate is extensively polyglutamylated in L. major (Santi, D. V., Nolan, P., and Shane, B. (1987) Biochem. Biophys. Res. Commun. 146, 1089-1092). MAPA was found to be 190-fold less potent than MTX as an inhibitor of leishmanial growth and to bind less tightly than MTX to leishmanial dihydrofolate reductase-thymidylate synthase. We therefore examined the possibility that MTX resistance is mediated by increased MTX hydrolysis to MAPA in drug-resistant Leishmania. However, enzymatic assays show that the rate of MTX hydrolysis was unaltered in the MTX-resistant R1000-3 line and the primaquine-resistant PQ-R30 line (which is 24-fold cross-resistant to MTX). In addition to MAPA, several minor unidentified metabolites were observed in the LT252, R1000-5B, and PQR30 cells but no consistent differences in the amounts of these metabolites were evident among these lines. These data indicate that alterations in the rate of MTX hydrolysis in vitro, or in the characteristics of MTX metabolites formed in vivo, do not underly the MTX resistance displayed by the H region-amplified R1000-5B and PQ-R30 lines.     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users.     

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH₂-10-CH₃PteGlu₄). When the time-dependent synthesis of MTX polyglutamates (4-NH₂-10-CH₃PteGlu₂ and higher) at extracellular concentrations of 10 and 100 μm methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μm is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μm intracellular methotrexate, respectively, after 24 h in the presence of 100 μm extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μm with both cell types which corresponded to 10 to 12 μm intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu_4–5) are more avidly retained by the cells than the shorter ones (Glu_2–3).     

Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.     

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.     

Cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines: effects of methotrexate treatment

The cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines were determined by autoradiography (PLM method) as follows: DT-5: generation time (TC), 15.2 h; pre-synthetic gap phase (TG1), 3.2 h; DNA-synthetic phase (TS), 8.2 h; post-synthetic gap ph se (TG2), 3.3 h; visible mitotic phase (TM), 0.5 h. 11028: TC, 13.6 h; TG1, 1.9 h; TS, 7.7 h; TG2, 3.4 h; TM, 0.6 h. 11028+FeLV (11028 productively infected with feline leukemia virus): TC, 11.2 h; TG1, 0.2 h; TS, 8.3 h; TG2, 2.1 h; TM, 0.6 h. Exposure of the lymphoma cell lines to methotrexate (MTX) in vitro produces dose-related increases in cellular volume, associated with reductions in cellular proliferation. The relative sensitivities of these cell lines to MTX, measured by the ID50 MTX concentrations for DT-5, 11028, and 11028+FeLV are 118 nM, 122 nM, and 28 nM respectively. The cell kinetic effects of the ID50 MTX concentrations added to cultures of lymphoma cells pulse-labeled with tritiated thymidine are an approximately 2-h prolongation of TC, attributable to a lengthening of TS, with other cell cycle phases not significantly altered. These cell lines are highly tumorigenic when transplanted into the cheek pouches of immunosuppressed hamsters, with inocula of 10(4) cells producing rapidly growing, well vascularized tumors.     

Synthesis and biological evaluation of five-membered heterocycles fused to cyclopenta[c]thiophene as new antitumor agents

A series of 10 derivatives 2-6 issued from the fusion of various five-membered heterocycles to cyclopenta[c]thiophene were evaluated for potential anticancer activity in the NCI's in vitro human disease-oriented tumor cell line screening panel that consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse histologies. Among these tested compounds, four were found to be cytotoxic allowing us to point out some structure-activity relationships. The oxazolidinone derivatives 2a-c displayed further in vivo antitumor activity in the hollow fiber assay and standard xenograft testing developed at the NCI.     

Synthesis and biological evaluation of a series of flavone derivatives as potential radioligands for imaging the multidrug resistance-associated protein 1 (ABCC1/MRP1)

Multidrug resistance (MDR) is one of the major problems affecting the treatment of cancer. In vivo visualization and quantification of MDR proteins would be of great value to better select the therapeutic strategy. Six flavone-based compounds were synthesized and evaluated for their cytotoxic activity and MDR-reversing capacity using hMRP1 or hMDR1 overexpressing cell lines for in vitro assays. All the flavone derivatives were highly selective for hMRP1-expressing cell lines. These derivatives each used at 4muM (a non-cytotoxic concentration) enhance significantly the sensitivity of hMRP1-mediated MDR cell line toward doxorubicin toxicity. Their MDR-reversing capacity suggests that, in particular, the 4'-fluoroalkyloxy and 4'-iodo apigenin derivatives are potential new radiopharmaceuticals to visualize in vivo MRP1-mediated MDR phenomenon by PET or SPECT.     

Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines

Determinants of methotrexate (MTX) resistance in cell lines resistant to short, but not continuous, MTX exposure were investigated since such lines may have relevance to clinical resistance. CCRF-CEM R30dm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant subline of the CCRF-CEM human leukemia cell line), had growth characteristics similar to CCRF-CEM. R30dm was resistant to a 24-h exposure to levels as high as 300 microM MTX but was as sensitive as CCRF-CEM to continuous MTX exposure. MTX resistance of R30dm was stable for greater than 68 weeks in the absence of selective pressure. Initial velocities of MTX transport were comparable for R30dm and CCRF-CEM, as were dihydrofolate reductase specific activity and MTX binding. A 2-fold thymidylate synthase activity decrease for R30dm from that of CCRF-CEM was not a significant factor in R30dm MTX resistance. Decreased MTX poly(gamma-glutamate) synthesis resulted in lower levels of drug accumulation by R30dm. Decreased polyglutamylation was attributable to folylpolyglutamate synthetase (FPGS) activity in R30dm extracts which was 1, 2, and less than or equal to 10% of CCRF-CEM extracts with the substrates MTX, aminopterin, and naturally occurring folates, respectively. Comparison of cell lines with varying levels of resistance to short term MTX exposure indicated that the extent of MTX resistance was proportional to the reduction of FPGS activity. The evidence supported decreased FPGS activity as the mechanism of resistance to short MTX exposure in the cell lines investigated.     

Folylpolyglutamate synthase and gamma-glutamyl hydrolase regulate leucovorin-enhanced 5-fluorouracil anticancer activity

Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2′-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU.     

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Non-classical antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines and 2,4-Diamino-6(5H)-oxopyrimidines,synthesis and antitumor studies

A series of non-classical antifolates, namely 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines (25a-i) and 2,4-diamino-(N-phenylpyrrolidin-3-yl)-6(5H)-oxopyrimidines (26a,b,c,f,h,i) was synthesized and evaluated for their in vitro cytotoxicity. Reacting aniline derivatives with 1,4-dibromo-2-butanol gave 1-phenyl-3-pyrrolidinols (19a--i), which were oxidized to pyrrolidin-3-ones (20a-i). The Knoevenagel reaction of 20a-i with malononitrile or ethyl cyanoacetate gave 3-(dicyanomethylene)- (21a-i) and 3-[cyano(ethoxycarbonyl)methylene]-pyrrolidines (22a,b,c,f,h,i), respectively, which were subsequently reduced to the corresponding 3-(dicyano)methyl- or 3-[cyano(ethoxycarbonyl)methyl)]pyrrolidines (23a-i and 24a,b,c,f,h,i, respectively). Condensation of either 23a-i or 24a,b,c,f,h,i with guanidine afforded the target compounds. The cytotoxicity of these compounds was evaluated based on their ability to inhibit various human tumors (human colon adenocarcinoma COLO 205, lung carcinoma H23 and its adriamycin resistant cell line H23/0.3, T-cell leukemia MOLT-4, promyelocytic leukemia HL-60, and T-cell acute lymphocytic leukemia CCRF-CEM) cell growth in culture. These studies revealed that the 2,4,6-triaminopyrimidine derivatives were more cytotoxic than the 2,4-diamino-6(5H)-oxopyrimidine counter parts, in which the latter was inactive in all testing systems. The 2,4,6-triaminopyrimidine derivatives bearing halogen substituent on the phenyl ring (25f,h,i) were cytotoxic in all cultured leukemia cell growth. Among these compounds, 5-(4-fluoro and 4-chlorophenyl)-2,4,6-triaminopyrimidines (25e and 25h, respectively) were more potent than methotrexate (MTX) in inhibiting of H23/0.3 cell growth. These compounds inhibit the folate metabolic pathways as indicated by tritium release from [5-3H]deoxyuridine in MTX sensitive human fibrosarcoma HT-1080 cells. Dihydrofolate reductase is the major target for 25f,h,i, as shown by leucovorin (LV) rescue of MTX cytotoxicity.     

Immunological and morphological properties of fibroblasts transformed in vitro. II. The change in the susceptibility of FC3H3 cells to lymphocytes--natural killers and macrophages during their selection for malignancy

The previous report contained the information on obtaining in vitro continuous cell lines of embryonic fibroblasts of mice of 4 genotypes, including C3H. In this paper, data on the degree of lysis of some derivatives of FC3H3 cells by natural killers and macrophages are given. It was shown that normal cells were not lysed by these effectors unlike cells which overcame Hayflick's limit and got minimum malignancy in vitro. However, it was established that the in vivo selection of cells for high tumorigenicity resulted in the formation of tumor cells resistant to lymphocytes--natural killers and macrophages.     

Studies of methotrexate-induced limb dysplasias utilizing a 51chromium release assay

The folate antagonist methotrexate (MTX), widely used in chemotherapy, is a well-documented teratogen. However, the mechanism by which it exerts its effects is still unclear. Specifically, we have examined the cytotoxicity of MTX in vivo and in vitro and have looked at the relationship between cytotoxicity and teratogenesis. The chick embryo was utilized to examine the effects of the drug administered to carefully staged embryos. Embryos were exposed at stages 18-22 and examined on day 11 of incubation. Wings were malformed in a stage-dependent manner while legs were affected similarly at each stage used. A modification of the 51chromium-release assay was used to test the toxicity of MTX to limb cells in vitro. None of the tissues tested showed measurable toxicity in vitro even though the drug kills cells in vivo, thereby suggesting that MTX may be metabolized differently in vitro. Malformations induced by MTX do not seem to be due to changes in the amount of cell death taking place in the limb but may be caused by a transient inhibition of cell division.