A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (tmq), a potent non-classical folate antagonist inhibitor--I effect on dihydrofolate reductase and growth of rodent tumors in vitro and in vivo.

Seventeen non-classical 2,4-diamino-6-[(anilino)methyl]quinazoline antifolates were tested as inhibitors of dihydrofolate reductase from L1210 leukemia cells and from human leukemia cells (acute lymphocytic leukemia). Several potent inhibitors of this enzyme were found, some with I₅₀ values of 10^?9 M, thus displaying activity comparable to that of methotrexate. In general, the potency of dihydrofolate reductase inhibition correlated with the inhibition of cell growth in vitro against L1210 cells. Two of these compounds, compound 14 (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; TMQ, JB-11, NSC 249008) and compound 3 (2,4-diamino-5-chloro-6-[(3,4-dichloroanilino)methyl]quinazoline; NSC 208652), were further evaluated against murine tumors in vivo and both showed a broad spectrum of antitumor effects. The results of these studies encourage further evaluation of these compounds, in particular compound 14, as possible anti-neoplastic agents in the treatment of human disease.     

Identification of dihydrofolate reductase in rabbit brain

After partial purification, rabbit brain extracts were assayed for dihydrofolate reductase by spectrophometric, radiochemical and methotrexate binding assays. By these assays, the specific activity of rabbit brain dihydrofolate reductase was about 15 per cent that of liver. Both rabbit liver and brain dihydrofolate reductase activities were abolished by methotrexate and had comparable K_m (3 μM), pH maxima (4.8), and cofactor requirements (NADPH). In vivo, a small percentage of [³H]folic acid was reduced to [³H]methyltetrahydrofolate when injected into the left lateral ventricle. Thus, both in vitro and in vivo, the central nervous system has the ability to reduce oxidized folates.     

Growth inhibitory,transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K_m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

A comparison of dihydrofolate reductase activity in intact leukemia cells and squamous cell carcinoma

This report examines the intracellular activity of dihydrofolate reductase using an in situ assay designed to measure enzymatic activity in intact cells. The rate of uptake of folic acid exceeded the rate of in situ dihydrofolate reductase activity suggesting that the reduction of folate to dihydrofolate, rather than transport, was the rate limiting step. In situ dihydrofolate reductase activity varied linearly with cell number. A comparison of the in situ activity revealed that a squamous cell carcinoma selected for methotrexate (MTX) resistant (SCC-15R) had 100 times greater dihydrofolate reductase (DHFR) activity than L1210 leukemia. In agreement with this finding, the in situ DHFR activity in SCC-15R cells was 50-fold less sensitive to the inhibitory effects of MTX than the L1210 in situ DHFR activity (IC50 = 1.1 x 10(-5) M and 2.4 x 10.7(-7) M respectively). The inhibition of in situ dihydrofolate reductase activity by MTX was found to correlate with the inhibition of growth, DNA synthesis (CdR incorporation) and in situ thymidylate synthase activity.     

Regiospecific gamma-conjugation of methotrexate to poly(L-lysine). Chemical and biological studies

Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

The in vitro effect of trimethoprim on blastogenic transformation of human peripheral blood lymphocytes.

Trimethoprim (TP) at concentrations of 1 × 10^?6 to 1 × 10^?3 M inhibits the incorporation of thymidine, uridine, formate, and leucine by human lymphocytes undergoing blastogenic transformation induced by conconavalin A. TP does not alter cell viability, as measured by trypan blue exclusion, or cell numbers. An effect of TP on dihydrofolate reductase, such as is seen with methotrexate, is insufficient to explain the inhibitory effects of TP since an in vivo estimation of dihydrofolate reductase activity, utilizing deoxyuridine incorporation into deoxyribonucleic acid, is not inhibited. Also the observed inhibition of precursor incorporation is not fully reversed by either folate or folinate. TP did not alter the percentage of cells transforming but reduced the number of autoradiographically-labelled lymphoblasts. Immunosuppression by TP in vivo may be associated with these findings.     

Alpha-monoamides of methotrexate as potential prodrugs

alpha-Monoamides of methotrexate were evaluated for their potential as prodrugs. Studies on 11 alpha-monoamides and 5 gamma-monoamides of methotrexate showed that the gamma-monoamides were about as strong inhibitors of Lactobacillus casei dihydrofolate reductase as methotrexate, while I50 of the alpha-monoamides were 1-2 orders higher. The concentration for growth inhibition of murine L1210 cells for methotrexate gamma-propylamide and alpha-propylamide were respectively 1-2 and 2-3 orders higher than that of methotrexate. In contrast, only alpha-monoamides caused significant increase in life span of mice with transplanted L1210 leukaemia, the highest effect being given by the alpha-propyl and the alpha-butylamide. The possibility that the in vivo activity of the alpha-monoamides might be related to in vivo transformation to methotrexate was studied by HPLC analysis of mice serum after administration of the alpha- and gamma-propylamides.     

Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.     

In vivo inhibition of adenosine deaminase by 2'-deoxycoformycin in mouse blood and leukemia L1210 cells

Adenosine deaminase (ADA) activities in mouse whole blood, washed erythrocytes and L1210 cells were 0.48, 0.93 and 4.76 units/ml respectively. Methods were developed to determine the second-order association rate constant (k₁) of a tight-binding ADA inhibitor, deoxycoformycin (DCF), and ADA in mouse blood and L1210 cells in vivo. After i.v. injection of DCF, the inhibition of the enzyme was of a monophasic pseudo-first-order nature in blood and biphasic (with an initial lag of 3–5 min) in L1210 cells. In contrast, i.p. injection of DCF produced the opposite pattern, monophasic in L1210 cells and biphasic in blood. The apparent k₁ values determined from the linear portions of these curves were compared with the k₁ values obtained in vitro. The mean k₁ values in vivo were: 4.2 × 10⁴ and 1.4 × 10⁴M^?1 sec^?1 in blood after i.v. and i.p. injections, respectively, and 2.6 × 10³ and 2.2 × 10⁴ M^?1 sec^?1 in L1210 cells after i.v. and i.p. injections respectively. The k₁ values with either whole blood or L1210 in vitro (3.1 × 10⁴ and 5.5 × 10³ M^?1 sec^?1, respectively) were of the same order of magnitude as those obtained with these tissues in vivo. In contrast, the k₁ values were about 150 to 1400-fold higher when either blood hemolysates (4.8 x 10^?6M^?1 sec^?1) or homogenized L1210 cells (7.5 x 10^6?1 sec^?1) were used. The 150 to 1400-fold higher k₁ values for blood hemolysates and homogenized L1210 cells than for intact cell samples (whole blood or whole L1210 ascitic fluid) suggest that the cell membrane plays a role in the interaction of DCF and ADA in these cell lines. The similarity of the rates of association of DCF and ADA in vivo and in vitro for mouse blood and ascites L1210 cells suggests that data obtained in vitro may be used to estimate the k₁ values in in vivo conditions.     

Synthesis and Cytotoxic Action of 3,5-Isoxazolidinediones and 2-Isoxazolin-5-ones in Murine and Human Tumors

The 3,5-isoxazolidinediones and 2-isoxazolin-5-ones demonstrated potent cytotoxicity against the growth of human Tmolt₃ T cell leukemia, murine P388 and L1210 leukemias, as well as human HeLa-S³ uterine carcinoma and glioma tumor cell growth. The specificity of the 3,5-isoxazolidinedione and 2-isoxazoline-5-one derivatives as cytotoxic agents varied with the histological type of tumor cell. Selected compounds were active against solid HeLa uterine, KB nasopharynx, skin A431, SW-480 adenocarcinoma, osteosarcoma and glioma growth. Selected compounds demonstrated in vivo antineoplastic activity against Ehrlich ascites carcinoma growth. In L-1210 leukemia cells, the agents blocked DNA and protein synthesis at 25,50 and 100 μM over 60 min. The agents were effective in reducing rate limiting enzymes in the de novo purine and pyrimidine pathways. In addition they suppressed dihydrofolate reductase and ribonucleoside reductase activities with moderate inhibition of DNA and RNA polymerase activities. DNA itself was not a target of the agents.     

Further studies on the pharmacologic effects of the 7-hydroxy catabolite of methotrexate in the L1210 murine leukemia cell

This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 microM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 microM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (approximately 50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 microM 7-OH MTX on cell growth were reversed completely by 10 microM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.     

Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin

gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.     

Quinazoline antifolates inhibiting thymidylate synthase: benzoyl ring modifications

Four new analogues of the antifolate N10-propargyl-5,8-dideazafolic acid were prepared that were substituted in the benzoyl ring. The 2'-chloro and 2'-methyl analogues were prepared from the appropriately substituted p-nitrobenzoic acids. The route to the 3'-chloro and 3',5'-dichloro analogues was by chlorination of diethyl N10-propargyl-5,8-dideazafolate and diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate, respectively, using sulfuryl chloride. The compounds were tested for their inhibition of purified L1210 thymidylate synthase (TS), for their inhibition of purified L1210 dihydrofolate reductase (DHFR), and for their inhibition of the growth of L1210 cells in culture. The 2'-chloro substituent reduced the TS inhibition by twofold and the 2'-methyl substituent reduced it by 20-fold; the 3'-chloro and 3',5'-dichloro derivatives were very poor inhibitors. The substituents only slightly affected the DHFR inhibition. None of the compounds improved upon N10-propargyl-5,8-dideazafolic acid in inhibiting the growth of L1210 cells in culture.     

Quantitative correlations of biological activities of dactinomycin analogs and methotrexate derivatives with van der Waals volume

The biological activities namely the ability to bind with DNA base pairs and the in vitro inhibition of human lymphoblastic leukemia cells of certain C-7- and N2-substituted dactinomycin (actinomycin D, AMD) analogs, and the growth inhibitory activity of a series of side chain substituted methotrexate (MTX) derivatives against L 1210 mouse leukemia cells in culture and their binding affinity for dihydrofolate reductase (DHFR) enzyme extracted from this system and L. casei are found to be significantly correlated with van der Waals volume of the substituents. In case of MTX derivatives, not only the side chain substituted analogs but certain ring substituted analogs, too, are shown to have their different activities dependent upon the Vw of the substituents. In case of side chain substituted analogs, however, it is found that the size of the substituent of alpha-position produces a greater effect on the activity than that of gamma-position. Based on the correlating equations obtained it is assumed that in these cases the drug-receptor interaction might be involving either hydrophobic interaction or the van der Waals type of interaction.     

Compartmentation of intracellular folates. Failure to interconvert tetrahydrofolate cofactors to dihydrofolate in mitochondria of L1210 leukemia cells treated with trimetrexate

Following exposure of L1210 leukemia cells to antifolates, tetrahydrofolate-dependent purine and pyrimidine biosyntheses are blocked despite the presence of the major portion of tetrahydrofolate cofactors. Previous studies from this laboratory demonstrated that this cannot be due to direct inhibition of thymidylate synthase by dihydrofolate polyglutamates or other endogenous folates and suggested that this phenomenon is due to compartmentation of tetrahydrofolate cofactors unavailable for interconversion and/or oxidation when dihydrofolate reductase activity is abolished by antifolates. The present paper evaluates the possibility that tetrahydrofolate cofactors in subcellular organelles, in particular, mitochondria, are unavailable for oxidation by thymidylate synthase. Particulate and cytosolic fractions were obtained from L1210 cells following homogenization and differential centrifugation. The crude mitochondrial fraction contained 20.1% of the total folate pool and included 5-formyltetrahydrofolate, 10-formyltetrahydrofolate and tetrahydrofolate in proportions similar to intact cells. The cytosolic fraction had an increased proportion of tetrahydrofolate and decreased proportions of 5-formyl- and 10-formyltetrahydrofolate relative to intact cells or the particulate fraction. Exposure of cells to 10 microM trimetrexate for 30 min produced approximately 45% interconversion of tetrahydrofolate cofactors to dihydrofolate in the cytosolic fraction, a level much greater than that observed in whole cell extracts (25-30%), but had no effect on folate pools in the crude mitochondrial fraction. These data indicate that subcellular compartmentation accounts, in part, for the failure to oxidize tetrahydrofolate cofactors to dihydrofolate in the presence of antifolate levels that abolish dihydrofolate reductase activity.     

Modification of in vivo methotrexate antitumor effect in L1210 leukemia by induced impairment of purine salvage

Studies with murine cells have shown that the antitumor action of methotrexate (MTX) may be through a purineless mechanism. If the MTX effect depends, in part, on inhibition of de novo purine synthesis, then the ability of tumor cells to salvage available purine precursors could reduce the cell kill. In the present study, we produced L1210 murine leukemia mutants with impaired purine salvage to determine whether this would affect responsiveness to MTX. Mutant lines L1210/MP, L1210/FAMP, and L1210/555 were produced by developing resistance to the purine analogs 6-mercaptopurine (6-MP), 6-MP + 2-fluoroadenine (2-FA), and 6-MP + 2-FA + 6-methylmercaptopurine riboside respectively. The purine salvage capability of the cell lines was confirmed in vitro by testing the ability of various purines to reverse the growth inhibitory and biochemical effects of MTX in the presence of thymidine. Dose-response curves demonstrated identical in vitro MTX sensitivity for L1210/MP, L1210/FAMP, and the parent line, L1210/S. Despite identical in vitro MTX sensitivity, the cell lines L1210/MP and L1210/FAMP displayed increased sensitivity to the biochemical effects of MTX in an in vivo model, and this was translated into enhanced sensitivity as measured by survival experiments in tumor-bearing mice. The results indicate that impairment of purine salvage sensitizes cells to the antitumor effect of MTX in vivo. This has implications for the clinical use of MTX in view of the variety of rescue techniques that is available.     

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, Raji and TK^? (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK^?. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log id₅₀ (median inhibitory dose) for L1210 cell growth, on the one hand, and log id₅₀ for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log id₅₀ values for the two human lymphoblast cell lines. However, there was no significant correlation (r < 0.40) either between the log id₅₀ for the TK^? Raji cells and the parental TK⁺ Raji cells, or between the log id₅₀ for the TK^? L1210/BdUrd cells and the parental TK⁺ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.