Low Concentrations Of Methotrexate Research Articles

Low concentration of methotrexate causes toxicity against neuron during synapse formation

Low concentration of methotrexate causes toxicity against neuron during synapse formation

Combined toxicity of therapeutic pharmaceuticals to duckweed, Lemna minor

Pharmaceuticals, which are designed to be biologically active at low concentrations, are found in surface waters, meaning aquatic organisms can be exposed to complex mixtures of pharmaceuticals. In this study, the adverse effects of four pharmaceuticals, 17α-ethynylestradiol (synthetic estrogen), methotrexate (anticancer drug), diclofenac (nonsteroidal anti-inflammatory drug) and fluoxetine (antidepressant), and their binary mixtures at mg/L concentrations were assessed using the 7-day Lemna minor test, with both apical and biochemical markers evaluated. The studied biochemical markers included chlorophyll a, chlorophyll b, carotenoids and oxidative stress enzymes catalase, glutathione-S-transferase and glutathione reductase, with effects compared to solvent controls. The adverse effects on Lemna minor were dose-dependent for frond number, surface area, relative chlorophyll content and activity of glutathione S-transferase for both individual pharmaceuticals and binary mixtures. According to the individual toxicity values, all tested pharmaceuticals can be considered as toxic or harmful to aquatic organisms, with methotrexate considered highly toxic. The most sensitive endpoints for the binary mixtures were photosynthetic pigments and frond surface area, with effects observed in the low mg/L concentration range. The concentration addition model and toxic unit approach gave similar mixture toxicity predictions, with binary mixtures of methotrexate and fluoxetine or methotrexate and 17α-ethynylestradiol exhibiting synergistic effects. In contrast, mixtures of diclofenac with fluoxetine, 17α-ethynylestradiol or methotrexate mostly showed additive effects. While low concentrations of methotrexate are expected in surface water, chronic ecotoxicological data for invertebrates and fish are lacking, but this is required to better assess the environmental risk of methotrexate.

Mechanism and pathways underlying the self-sensitized photodegradation of methotrexate under simulated solar irradiation.

Methotrexate, a chemotherapeutic agent, was found to undergo self-sensitized photodegradation in aqueous environments. As the initial concentration increased, methotrexate was able to enhance its own direct photolysis reaction not only in DI but also in natural waters. The methotrexate degradation rate increased through the production of singlet oxygen (1O2), the triplet excited state of methotrexate (3MTX*), and the triplet excited state of the pteridine structure (3PT*) from the phototransformation byproducts. At low methotrexate concentrations (<20 ppb), 1O2 played an important role, whereas at higher methotrexate concentrations (>2000 ppb), the presence of oxygen decreased the overall methotrexate degradation rate by physically quenching 3MTX* and 3PT*. The cleavage of the CN bond resulted in a significant amount of byproducts: pteridine derivatives and N-(4-aminobenzoyl)-l-glutamic acid (yields: 13.5 ± 0.6% and 32.3 ± 2.2% for 10 ppm and 500 ppb MTX, respectively). The reactivity of the phototransformation byproducts and the substructures of methotrexate were investigated to help elucidate the proposed self-sensitized pathways. The results indicated that methotrexate as well as compounds containing a pteridine structure will generate pteridine byproducts during photodegradation and 3PT* is the primary triplet excited species that can cause self-sensitized photodegradation.

Application of microRNA Targeted 3'UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools.

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

Low dose MTX, HCQ and CTX can increase the expression of GRα mRNA in vitro

Objective To investigate the effects of methotrexate (MTX), hydroxychloroquine (HCQ), cyclophosphamideon (CTX) and active metabolite of Leflunomide (LEF)-A771726 on the expression of glucocorticoid receptorα (GRα) mRNA in human monocyte cell line human myeloid leukemia mononuclear cells (THP)-1. Methods Cultured THP-1 cells were stimulated by MTX, HCQ, CTX, and A771726 with different concentrations, with dexamethasone (DEX) as positive control, after 36 h the GRα mRNA expressions were examined by real-time polymerase chain reaction (PCR). Data analysis was performed with Kruskal-Wallis H test and Nemenyi test. Results After 36 h, DEX decreased significantly the expression of GRα mRNA in THP-1 cells (P 0.05); high and medium concentration of MTX, HCQ and CTX decreased the expression of GRα mRNA in THP-1 cells (P>0.05). Compared to the blank control, low concentration of MTX, HCQ, and CTX increased the expression of GRα mRNA in THP-1 cells (P>0.05); Compared to the positive control, low concentration of MTX, HCQ, and CTX increased the expression of GRα mRNA in THP-1 cells (P<0.05). Conclusions Low dose MTX, HCQ, and CTX-increased GRα mRNA may be one of the mechanism of action of the treatment of autoimmune diseases. Key words: Methotrexate/AD/PD; Hydroxychloroquine/AD/PD; Cyclophosphamide/AD/PD; Receptors, glucocorticoid/ME/DE; Monocytes/ME

Experience of the concentration of methotrexate evaluation in children with juvenile idiopathic arthritis

Juvenile idiopathic arthritis - one of the most common rheumatic disease in children (1:1000) [Timothy Beukelman et all, 2011]. The most effective basic drug is methotrexate, an initial dose of 15 mg/m2/week. Our study to determine the concentration of methotrexate in serum aims to develop a test system to clarify the reasons for lack of efficacy of methotrexate in some patients. The study currently includes 46 children (19 boys (41,3%), 27 girls (58.7%)) with different forms of JIA, according to the classification of ILAR. Systemic arthritis – 9 children (20%), arthritis oligoarticular – 9 (20%), polyarticular arthritis – 15 (34%), psoriatic arthritis – 1 (2%), arthritis entesitis – 11 (24%). Of the 46 children noone is in clinical remission off medication. 26 (56.5%) children met the criteria of inactive disease. Results of determining the concentration of methotrexate in children with JIA were distributed as follows - 0-1 umol/l - 2,18%; 1-1,5 umol/l - 34.78%; 1,5-2 umol/l - 17.39 % 2-2,5 umol/l - 32.61%; 2,5-3 umol/l - 8.69% above 3 umol/l - 4,35%. Among children with the results of the concentration of methotrexate up to 1.5 umol/l - 88.24% have signs of active disease (elevated ESR / CRP high / dysproteinemia / active joints). Low concentrations of methotrexate in 73.3% are associated with high levels of ESR and CRP. Among children with the results of methotrexate concentration above 1,5 umol/l signs of active disease manifestations are much rarer in 48,28% of cases.

The plastidial folylpolyglutamate synthetase and root apical meristem maintenance

Folylpolyglutamate synthetase (FPGS) catalyzes the attachment of glutamate residues to the folate molecule in plants. Three isoforms of FPGS have been identified in Arabidopsis and these are localized in the plastid (AtDFB), mitochondria (AtDFC), and cytosol (AtDFD). We recently determined that mutants in the AtDFB (At5G05980) gene disrupt primary root development in Arabidopsis thaliana seedlings. Transient expression of AtDFB-green fluorescent protein (GFP) fusion under the control of the native AtDFB promoter in Nicotiana tabacum leaf epidermal cells verified the plastid localization of AtDFB. Furthermore, low concentrations of methotrexate (MTX), a compound commonly used as a folate antagonist in plant and mammalian cells induced primary root defects in wild type seedlings that were similar to atdfb. In addition, atdfb seedlings were more sensitive to MTX when compared to wild type. Quantitative (q) RT-PCR showed lower transcript levels of the mitochondrial and cytosolic FPGS in roots of 7 day old atdfb seedling suggesting feedback regulation of AtDFB on the expression of other FPGS isoforms during early seedling development. The primary root defects of atdfb, which can be traced in part to altered quiescent center (QC) identity, pave the way for future studies that could link cell type specific folate and FPGS isoform requirements to whole organ development.

Bioassay for detection of methotrexate in serum

Objective: A bioassay is developed for the measurement of methotrexate (MTX) in serum.Methods: The assay is based on MTX inhibition of the proliferation of hypoxanthine‐guanosine phosphoribosyl transferase (HGPRT) negative mouse B‐cells (B9.H). HGPRT negative cells cannot use the salvage pathway of nucleotide synthesis to overcome inhibition by MTX.Results: When B9.H cells are cultured with serial dilutions of serum, inhibition of proliferation is a measure of the amount of MTX in the serum. Circulating folates do not interfere with the assay.Conclusion: This simple assay can detect low concentrations of MTX in serum: it is therefore useful for following the pharmacodynamics of functional MTX after low‐dose MTX treatment.

Methotrexate suppresses the interleukin-6 induced generation of reactive oxygen species in the synoviocytes of rheumatoid arthritis.

Various cytokines and reactive oxygen species (ROS) play a fundamental role in the inflammatory and immunologic processes of rheumatoid arthritis (RA). Methotrexate (MTX) is one of the disease-modifying anti-rheumatic drugs and its effect may be partly due to the modulation of immunologic or inflammatory reactions by some cytokines. In the present study, we investigated the effects of MTX on the gene expression and synthesis of interleukin-6 (IL-6), and the proliferative activity and the production of ROS in the fibroblast-like synoviocytes (FLSs) obtained from the patient of RA. The expression or production of IL-6 was induced spontaneously, and augmented by the addition of recombinant human IL-6 or recombinant human IL-1 beta and TNF-alpha in FLSs. These spontaneous and augmented IL-6 expressions or productions were suppressed by treatment with low-concentration of MTX (1 microg/ml). Also, IL-6 stimulated the proliferation of FLSs, and this IL-6 driven proliferation was inhibited with the treatment of MTX or N-acetylcysteine (NAC, 1 mM). Furthermore, ROS production in FLSs was increased significantly by IL-6, and its effect was also abrogated in the presence of MTX or NAC. These results suggest that inflammatory reaction in the synovium of RA patients could be augmented by the autocrine or other cytokine-induced production of IL-6 with subsequent generation of ROS in the synoviocytes, and the modulations of IL-6 synthesis and ROS production may contribute to the therapeutic effects of MTX for RA.

Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification.

Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.

DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine.

Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death.

Construction of a dominant selectable marker using a novel dihydrofolate reductase

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu 22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978–1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu 22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38–48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539–1546]. Transfection of DHFR − Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR + phenotype with equal efficiency (i.e., > 2.5 × 10 −3 ), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR + cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of > 8 × 10 −4 . Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (88 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.

Simultaneous changes in various mechanisms that mediate the cell incorporation of folate compounds account for low levels of resistance to methotrexate.

Changes in the mechanisms of folate incorporation were studied in cells treated with low concentrations of methotrexate in order to evaluate their contribution to the development of resistance to antifolate drugs. The uptake of methotrexate via reduced-folate system, the membrane-associated high-affinity folate binding capacity and the activity, levels and affinity for methotrexate of dihydrofolate reductase were measured in L5178 murine leukemic lymphoblasts and in a subline, MTX/R16, 16 times more resistant to methotrexate which was isolated after a short exposure to the antifolate. Various simultaneous changes were characterized in MTX/R16 cells which co-participated in the development of resistance: a decreased affinity of the carrier for methotrexate uptake via the reduced-folate system of entry, the increase of dihydrofolate reductase activity and levels and a two-fold increased expression of a membrane-associated high-affinity folate-binding protein (mFBP). The increase of the mFBP expression, besides ensuring the growth of resistant cells by its contribution to the reduced folate intake, also participates in the methotrexate resistance by the internalization of folate cofactor which would compete with methotrexate hindering the effective inhibition of dihydrofolate reductase by the antifolate.

Comparative effect of 6S, 6R and 6RS Leucovorin on methotrexate rescue and on modulation of 5-fluorouracil

The comparative efficacy of the pure diastereoisomers of leucovorin, the natural (6S) and the unnatural (6R) forms was compared to the racemic form (6RS). A protective effect in methotrexate-treated CCRF-CEM cells was obtained with 6S at concentrations 100-fold higher than those of methotrexate and with 6RS at concentrations 2-fold greater than those of 6S; however, at low concentrations of methotrexate, 6S was more effective than 6RS in preventing the cytotoxicity of methotrexate; on the opposite, 6R exhibited a protective effect at concentrations 10(4) higher than those of methotrexate. On the same cell line, 6S was shown to enhance the cytotoxic effect of 5 Fluorouracil exactly as 6RS while 6R did not exhibit any enhancing effect on cells exposed to 5 Fluorouracil.

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate.

In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM-1 microM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h 51Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette+ (E+) population, but not in E-rosette- (E-). In addition, when E+ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E+CD16+ cell population was mainly involved in this augmentation. Positively sorted E+CD16+ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E+CD16+ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene.

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort

Methotrexate pharmacokinetics in age-fractionated erythrocytes

Age fractionation of erythrocytes is useful for further studies of the pharmacokinetics of methotrexate (MTX) in red blood cells. We separated erythrocytes from five blood donors and four patients at different time points after MTX infusions, using discontinuous Percoll gradients consisting of four solutions with a difference of 3% in density among them. The procedure yielded five distinct fractions of erythrocytes of increasing mean cell age as judged by declining reticulocyte enrichment and erythrocyte aspartate aminotransferase activity among the five fractions. MTX concentrations of the erythrocytes were measured at different times in connection with five 24-h MTX infusions (0.7-4 g/m2) on 14 occasions. Two days after completion of MTX infusion, no MTX was detected in the youngest erythrocyte population in two patients. Seven days after the infusion, the highest MTX concentrations were found in the youngest red blood cells. Ten to fourteen days following the MTX treatment, considerably lower MTX concentrations were found in the young red blood cells, and the MTX-containing erythrocytes seemed to have moved down the gradient. Just before the next MTX infusion (after 28 days) no MTX could be detected in the young erythrocytes. The MTX concentrations at that time were highest in the oldest erythrocyte fractions. This study shows more directly that MTX is incorporated in the red cell precursors of the bone marrow. The pharmacokinetics demonstrated correspond to a maturation time of the erythroblasts of about 7 days.

Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow.

Immature myeloid precursor cells were preferentially selected from normal human bone marrow by using immune rosette techniques that employed monoclonal antibodies against mature granulocytes, monocytes, T and B lymphocytes, and erythroid precursors (Mo5, M3, OKT3, B1, and EP1, respectively). We examined the formation, retention, and cytotoxic effects of methotrexate (MTX) polyglutamates (MTX-PGs) in these purified myeloid precursor cells. After 1- and 24-h exposures to MTX, with thymidine and deoxyinosine as rescue, the intracellular MTX-PG profile was examined by high-pressure liquid chromatography. Efflux patterns of MTX-PGs were also studied after additional 1- and 24-h incubations in drug-free media. Cytotoxic effects of retained MTX-PGs on bone marrow myeloid precursors were examined by colony formation in drug-free semisolid agar. Normal myeloid precursor cells converted MTX to MTX-PGs in a concentration- and time-dependent manner, preferentially retaining MTX-PGs with three to five glutamyl moieties. At low concentrations of MTX (1 microM), MTX-PG formation was insufficient to maintain saturation of the target enzyme dihydrofolate reductase after removal of drug from the incubation medium, and there was no decrease in myeloid colony formation. At higher concentrations of MTX (10 microM), formation of higher molecular weight polyglutamates was sufficient to allow for 24-h saturation of intracellular binding capacity after removal of extracellular drug and resulted in a 35% reduction in the formation of colony-forming units in culture. Comparison of MTX metabolism in normal bone marrow cells and the MTX-sensitive HL-60 human leukemia cell line showed twofold greater PG formation by these tumor cells after 24-h exposure to 1 or 10 microM MTX, and a marked (greater than 30-fold) increase in cytotoxicity for the HL-60 cells as compared with normal myeloid precursors, suggesting that the MTX polyglutamation may be important to its selective antitumor action.

Isolation and Serial Cultivation of Rabbit Skin Epithelial Cells

A method to isolate and to serially cultivate rabbit skin epithelial cells from adult trunk skin has been developed. Using a collagen gel as substrate and trypsin and EDTA to dissociate cells, nonproliferative primary cultures of rabbit cells may be converted to proliferative populations, and at least 3 serial passages achieved. In the presence of large concentrations of methotrexate (up to 1000 microgram/ml), epithelial cells in primary culture show no decrease in their ability to attach, spread, or keratinize. Following conversion to proliferative populations by trypsin and EDTA low concentrations of methotrexate (1 microgram/ml) are strongly cytotoxic. When the incorporation of 32PO4(3-) and 3H-thymidine into DNA of proliferative and nonproliferative cells is compared, the incorporation of 32PO4(3-), but not that of 3H-thymidine, correlates with changes in cell number and DNA content. In both primary and serially cultivated cells, L-serine is required for optimal growth.