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.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Transport of methotrexate in L1210 cells: Effect of ions on the rate and extent of uptake

Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport (K_t values) vary from 0.9 μm in cells suspended in potassium-Hepes buffer containing Mg²⁺ (Hepes-Mg), to 10 μm in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity (V) regardless of the buffering medium. The increased K_t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate (K_i = 0.87 mM) and Cl^? (K_i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl^?, Ca²⁺, and Na⁺. Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.     

Determination of Concentration of Methotrexate Enantiomers in Intracellular and Extracellular Fluids of HepG2 Cells by Liquid Chromatography–Tandem Mass Spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG₂ cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1 % formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4 mL min^?1. The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 → m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL^?1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15 %. The mean recovery of (+)-MTX and (?)-MTX in the extracellular fluid of HepG₂ cells were 95.30 and 96.53 %, respectively, and the mean recovery of (+)-MTX and (?)-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12 %, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG₂ cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of (?)-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and (?)-MTX was (+)-MTX > (?)-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro.     

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells)

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.     

The role of glutamine in regulation of ammonium transport in Azotobacter vinelandii

Under N₂-fixing conditions, Azotobacter vinelandii expresses a specific transport system for methylammonium (ammonium) [E. M. Barnes, Jr. and P. Zimniak (1981) J. Bacteriol. 146, 512–516]. This activity is decreased markedly by culture of cells in the presence of 10 mm ammonium or 2 mm methylammonium; in both cases, the V_max values for methylammonium uptake were 25% of those of N₂-fixing cells. Mixing experiments with assay medium indicate that transport activity is controlled by intracellular rather than extracellular metabolites. Glutamine synthetase activity of cells cultured with ammonium was 33% that of N₂-fixing cultures, but activity was unaffected by incubation with methylammonium. Thus ammonium transport and ammonium fixation are regulated independently. When ammonium was removed from the medium, cells recovered over 90% of the initial transport activity after 1 h; this recovery was not affected by addition of chloramphenicol. The loss of uptake activity in cells incubated with ammonium or methylammonium correlated with over sixfold increases in intracellular levels of glutamine and γ-glutamylmethylamide, respectively. Recovery of transport was accompanied by similar reductions in pools of these compounds. Over one-half of methylammonium transport activity could be blocked by direct addition of 10 mm glutamine or γ-glutamylmethylamide to transport assays; these concentrations were similar to those observed in vivo. The glutamine analog, 6-diazo-5-oxo-l-norleucine, was the most potent inhibitor found (68% inhibition at 10 μm). These results indicate that the regulation of ammonium transport by ammonium and methylammonium is due to inhibition of the transporter by intracellular γ-glutamyl amides rather than by repression of transporter synthesis.     

Modeling mechanisms of in vivo variability in methotrexate accumulation and folate pathway inhibition in acute lymphoblastic leukemia cells

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.     

Folylpolyglutamate synthetase activities of Neurospora

The folylpolyglutamate synthetase (FPGS) activities of Neurospora crassa, wild type (FGSC 853) and two polyglutamate-deficient mutants, met-6,35809 (FGSC 1330) and mac, 65108 (FGSC 3609), were examined after growth in defined media. Extracts of the wild type produced H₄PteGlu₆ (60 %), H₄PteGlu₃ (35 %) and H₄PteGlu₂ (15 %). Met-6 extracts formed H₄PteGlu₂ but lacked the ability to utilize H₄PteGlu₄ or H₄PteGlu₅. The mac mutant failed to catalyse glutamate addition to H₄PteGlu but H₄PteGlu₂ was an effective substrate for tri- and hexaglutamate synthesis. These polyglutamates were also formed by reaction systems containing mixtures of met-6 and mac protein or heterokaryon protein derived from mycelial fusions of met-6 and mac. Extract fractionations and heat treatments provided evidence for more than one FPGS activity in the wild type. A mitochondrial FPGS catalysed the H₄PteGlu₂ → H₄PteGlu₃ reaction but a cytosolic fraction synthesized di-, tri- and hexaglutamates when incubated with H₄PteGlu and glutamate. The latter system contained a temperature-sensitive diglutamate-forming activity and a relatively stable H₄PteGlu₂ → H₄PteGlu₆ activity. Polyglutamate synthesis in N. crassa appears to involve more than one step, H₄PteGlu → H₄PteGlu₂ followed by H₄PteGlu₂ → H₄PteGlu₆, in addition to the mitochondrial activity. These partial activities are lacking in mac and met-6 respectively. Consequently, these mutants are unable to form the folylhexaglutamates that predominate the folate pool of the wild type.     

Evidence for the facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Basic properties and specificity for polyglutamate chain length.

Evidence is presented outlining basic properties of a previously undescribed facilitative transport system mediating transfer of methotrexate (MTX) polyglutamates from the cytoplasmic to the lysosomal compartment of the cell. These experiments were conducted using purified lysosomes prepared from murine S180 cells, and a model substrate ([3H]MTX + G1; methotrexate with 1 additional glutamyl residue) to examine biological aspects as well as pharmacological significance of this process in a tumor cell model. The data, expressed as a function of latent beta-hexosaminidase activity, a measure of lysosomal integrity, show that [3H]MTX + G1 uptake in lysosomes is temperature-dependent, is stimulated specifically by magnesium chloride and potassium chloride with maximal enhancement observed in the presence of both agents together, exhibits Michaelis-Menten saturation kinetics with Km and Vmax values of 346 +/- 39 microM and 2.8 +/- 0.3 pmol/min/unit of beta-hexosaminidase activity, respectively, and is competitively inhibited by longer chain polyglutamates with increasing effectiveness as shown by Ki values of 334 +/- 19, 201 +/- 16, 106 +/- 13, and 42 +/- 8 microM, for MTX + G1, MTX + G2, MTX + G3, and MTX + G4, respectively. In addition, uptake is inversely related to medium osmolarity indicating that the phenomenon we observe represents internalization of the [3H]MTX + G1 and not adsorption to a possible surface binding site. As a whole, the data are consistent with a single mediated transport system shared by all MTX polyglutamates for entry into lysosomes. It is our view that this transport system represents the initial step in the degradation of polyglutamates in the cell. In addition, based on a comparative analysis of the kinetics for hydrolysis and transport, we suggest that it is also the limiting step in this process and, as such, regulates the extent of degradation of the free cellular pools of these compounds.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H₄PteGlu_n has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD–serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD–serine is quantified by comparison with standards. This method was used to determine the K_m and k_cat values for 5,10-CH₂–H₄PteGlu₅ of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH₂–H₄PteGlu₅ for an SHMT from any organism.     

Selective protection against the cytotoxicity of methotrexate and methotrexate-poly(lysine) by thiamine pyrophospate,heparin and Leucovorin

The cytotoxicity of both methotrexate (MTX) and methotrexate-poly(L-lysine) conjugate (MTX-poly(lys)) on L929 mouse fibroblasts can be prevented by the simultaneous administration of Leucovorin. Thiamine pyrophosphate, an inhibitor for MTX-transport, protects cells from the toxicity of MTX, but not of MTX-poly(lys). Heparin at low concentration (1 μg/m1) completely abolishes the toxic effect of MTX-poly(lys), but not of MTX. These results suggest that although MTX and MTX-poly(lys) share the same mode of drug action inside the cell, they possess completely different transport mechanisms at the cell surface. The protection of cells by heparin against MTX-poly(lys) is biphasic, i.e. at higher heparin concentration (>10 μg/m1) the cytotoxicity can be partially restored. This observation suggests that this polyanion may have cellular effects other than neutralizing the positive charges on the drug carrier poly(lysine).     

Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

L1210 cells transport P_i in the absence of added Na⁺. Uptake shows saturation kinetics (K_t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P_i, and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P_i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K_t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P_i uptake component. It is concluded that methotrexate and P_i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P_i. A principal energy source for active transport of methotrexate might then be the concentration gradient for P_i that is maintained by the Na⁺-dependent, P_i transport system of these cells.     

The kinetics of methotrexate polyglutamation in human breast cancer cells

The polyglutamation kinetics of methotrexate (MTX) in MCF-7 human breast cancer cells have been formulated mathematically. The model takes account of glutamation and hydrolysis kinetics up through the pentaglutamate level, increased synthesis of dihydrofolate reductase following exposure to drug, reversible tight-binding to reductase, and membrane transport of polyglutamates. The glutamation, hydrolysis, and efflux parameters have been determined from fits to experimental MTX polyglutamate uptake and efflux data. The preferred substrate for folypolyglutamyl synthase in the intact cell appears to be MTX diglutamate, on average being two to three times as reactive as either the parent drug or the triglutamate. Hydrolysis rate constants range from 0.03 to 0.19 h-1, but no clear trend with chain length is observable given the large uncertainty of each parameter estimate. However, the efflux of MTX polyglutamates from MCF-7 cells does show a trend with chain length decreasing with increasing length as expected. The best characteristic time of MTX diglutamate efflux is 4.1 h, about one-third that of the higher polyglutamate species, in agreement with observations on the MDA.MB.436 breast cancer cell line. The model shows quantitative agreement with the fraction of MTX polyglutamates found still to be bound to reductase in MCF-7 cells following 24 h of efflux, and qualitative agreement with the time dependence of bound MTX-polyglutamate concentration profiles obtained on the ZR-75 breast cancer line.     

Inactivation by chloroquine of alpha-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

Liposomal formulation of a methotrexate diglyceride conjugate: Activity toward a culture of methotrexate-resistant leukemia cells

We have recently synthesized a lipid conjugate of the anticancer agent methotrexate (MTX-DG) and showed that the conjugate is quantitatively included in the lipid bilayer of liposomes prepared by a standard extrusion technique from an 8:1:1 (mol) egg phosphatidylcholine-yeast phosphatidylinositol-MTX-DG mixture. Both the size of liposomes (126 ± 30 nm) and the MTX-DG concentration (4.4 mM) are relevant for systemic injections in mammals. The liposomal formulation of MTX-DG was shown to overcome the resistance of tumor cells in vitro to methotrexate: the cytotoxic activities (IC₅₀) of MTX in cultures of the human T-lymphoblastic leukemia cell line CEM-CCRF and the MTX-resistant subline CEM/MTX were 0.075±0.005 and 16.4±4.9 μM, respectively, while, in the case of liposomes loaded with MTX-DG, the IC₅₀ values were much closer: 0.77±0.06 and 3.8±1.9 μM.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate