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J Jolivet

Publications and source records attributed to J Jolivet.

At least 55 records · Page 3Linked to original sources
PubMed1985

Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.

We report the enhanced inhibitory potency of methotrexate (MTX) polyglutamates and dihydrofolate pentaglutamate on the catalytic activity of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase purified from MCF-7 human breast cancer cells. In the present work, MTX (4-amino-10-methylpteroylglutamic acid) and dihydrofolate, both monoglutamates, were found to be weak competitive inhibitors of AICAR transformylase with Kis of 143 and 63 microM, respectively, and their inhibitory capacity was largely unaffected by the glutamated state of the folate cosubstrate. In contrast, MTX polyglutamates were found to be potent competitive inhibitors, with an approximately 10-fold increase in inhibitory potency with the addition of each glutamate group up to four (i.e., the pentaglutamate derivative). MTX tetra-and pentaglutamates were the most potent, with equivalent Kis of 5.6 X 10(-8) M or 2500-fold more potent than MTX. Dihydrofolate pentaglutamate was as potent an inhibitor as MTX pentaglutamate, with a Ki of 4.3 X 10(-8) M. The potent inhibitory effects demonstrated by the polyglutamate compounds when tested against the folate monoglutamate substrate were sharply curtailed when folate pentaglutamate was used as the substrate. MTX and dihydrofolate pentaglutamates were only 7- and 25-fold more potent than their monoglutamate counterparts under these conditions. A model depicting these complex interactions is postulated. These findings have significant implications regarding the mechanism of action of MTX.

Acyltransferases↗
PubMed1985

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

We have characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with less than 0.1 microM MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 microM) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 microM) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 microM drug. Two cell lines with resistance to 10 microM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 microM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.

Biological Transport, Active↗
PubMed1985

Prevention of methotrexate cytotoxicity by asparaginase inhibition of methotrexate polyglutamate formation.

Escherichia coli asparaginase (Asnase) pretreatment of Asnase-sensitive L5178Y cells in vitro is thought to antagonize methotrexate (MTX) cytotoxicity through nonspecific inhibition of protein synthesis and MTX uptake. We have reexamined the mechanism of this interaction in view of recent data demonstrating the importance of MTX metabolism to polyglutamate derivatives (MTXPGs) in the cytotoxic effects of the antifolate. After a 3-hr exposure to 0.5 microM MTX, 67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2-5), and cloning efficiency in drug-free medium was only 7% of untreated control. After a 3-hr pretreatment with E. coli Asnase (0.1 unit/ml), [3H]thymidine incorporation dropped by 29%, MTXPG formation during subsequent MTX exposure decreased by more than one-half (MTX-Glu2 unchanged; MTX-Glu3 and 4 decreased to 51.7 and 18.5% of levels achieved in cells not pretreated with Asnase; no MTX-Glu5 formed), and cloning efficiency increased to 71% of untreated control. This effect was not due to decreased MTX uptake into L5178Y cells or to decreased intracellular free L-glutamate or L-glutamine levels. A 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an essential amino acid for cell growth, prior to MTX exposure inhibited [3H]thymidine incorporation by 37%, decreased subsequent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels. Decreased MTXPG formation was responsible for the prevention of MTX cytotoxicity seen after both pretreatments. Unmetabolized MTX rapidly left L5178Y cells after removal of extracellular MTX. Consequently, lower levels of unbound intracellular drug, a prerequisite of drug activity, were maintained in pretreated than in control cells after passage in drug-free medium. Asnase pretreatment protects L5178Y cells from the cytotoxic effects of MTX, possibly through inhibition of cell growth which nonspecifically decreases MTXPG formation.

Animals↗
PubMed1984

A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates.

Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.

Breast Neoplasms↗
PubMed1984

Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture.

Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were established in culture by progressive dose escalation. The biochemical basis of resistance was studied. The line with the lowest resistance (R1) had a normal dihydrofolate reductase (DHFR) content but showed decreased MTX transport and polyglutamation. Lines of intermediate resistance (R2 and R3) showed an increased DHFR content and DHFR gene copy number and a defect in MTX transport. The line with the greatest resistance (R4) showed increased DHFR content and gene copy number but nearly normal MTX transport. These results demonstrate that multiple mechanisms of MTX resistance occur in human epithelial cells in culture. We also find evidence of alterations in DHFR gene expression. The MTX-resistant cells were either not cross-resistant or only partly cross-resistant to two lipophilic MTX ester derivatives. These compounds are of potential therapeutic interest for the treatment of MTX-resistant tumors.

Biological Transport↗
PubMed1983

Synthesis, binding and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells.

Synthesis, binding, and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells were investigated by gel filtration and high-pressure liquid chromatography to separate methotrexate from its metabolites. MCF-7, ZR-75-1, and MDA-231 human breast cancer cells were found to readily convert methotrexate to higher polyglutamates during a 24-hr incubation period, although at differing rates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase revealed that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. Similarly, methotrexate polyglutamates accumulated free in the cytosol and when cells were suspended in drug-free medium, were retained intracellularly both bound to dihydrofolate reductase and in the unbound fraction, indicating their slow passage through the cell membrane. Studies of methotrexate polyglutamate binding to purified bacterial dihydrofolate reductase revealed high affinity binding for compounds with up to 6 additional glutamyl residues. These studies demonstrate that methotrexate polyglutamates are readily formed in human breast cancer cells, bind intracellularly to dihydrofolate reductase, and are selectively retained both bound to the enzyme and free in the cell cytosol.

Binding Sites↗
PubMed1983

Test dose for predicting high-dose methotrexate infusions.

Eighteen evaluable patients were studied to determine whether individual methotrexate (MTX) kinetics, determined by test-dose bolus injection, could be used to predict plasma drug concentrations during and after high-dose infusion. Small nontoxic doses of MTX (10 mg/m2) was given to patients who were followed for 12 to 24 hr and the kinetic data were used to predict subsequent kinetic behavior of moderate- and high-dose methotrexate infusions (150 to 1500 mg/m2 over 12 to 18 hr). After test-dose injection, MTX clearance varied from 36 to 138 ml/min/m2 and decreased with advancing age (r = -0.49, P less than 0.05). MTX clearance varied from 24 to 100 ml/min/m2 after high-doses. Although there was a trend to decreasing clearance with advancing age, this was not as clear as with the test dose (r = -0.42, P greater than 0.05). There was no correlation between MTX clearance and creatinine clearance in this group of patients in whom creatinine clearance varied from 32 to 63 ml/min/m2. When the kinetic parameters derived from the test-dose data were used, accurate predictions could be made of the infusion plateau (r = 0.89, P less than 0.001) and 24-hr (r = 0.92, P less than 0.001) MTX concentrations after high-dose infusions. Our results indicate that test-dose MTX kinetics may serve as a guide to dose modification of MTX infusions in some high-risk patients.

Adult↗
PubMed1983

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

Methotrexate (MTX-Glu1) exerts its antitumor effects through its potent inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for maintaining the cellular pool of reduced folates. Since the drug-enzyme complex (bound drug) is slowly dissociable, an excess of drug (unbound or free drug) above that required to bind all enzyme sites is required in order to compete with substrate for sites made available by enzyme-drug dissociation. We have examined the role of the polyglutamyl metabolites of MTX-Glu1 containing two to five glutamyl (MTX-Glu2-5) groups in gamma peptide linkage in maintaining an intracellular pool of free drug and in forming slowly dissociable complexes with DHFR. During 24-h incubations of ZR-75-B human breast cancer cells with 2 microM MTX-Glu1, we observed the progressive formation of derivatives with two to five glutamyl groups, which rapidly replaced the parent compound on enzyme binding sites and represented 85% of both unbound and bound intracellular drug at the end of incubation. When cells were then placed in drug-free medium, the rates of disappearance of drug and metabolites from the intracellular bound and free fractions decreased with increasing glutamyl chain length. Over 90% of both bound and free MTX-Glu1 left the cells within 1 h, greater than 90% of MTX-glu2 left within 6 h, and greater than 90% of MTX-Glu3 left the bound and free fractions within 24 h. In contrast, free MTX-Glu4 fell by only 63% and bound by only 23% after 24 h, while free MTX-Glu5 increased by 52% after 6 h in drug-free medium and bound MTX-Glu5 increased threefold after 24 h, as it replaced the other forms of drug bound to DHFR. These results suggested a rapid dissociation of MTX-GLu1 and -Glu2 from the enzyme, and a slower dissociation of the longer chain length derivatives. This conclusion was confirmed by examining the rates at which [3H]MTX-Glu1 through -Glu5 could be replaced on enzyme binding sites by a fivefold or greater excess of unlabeled MTX-Glu1. Bound [3H]MTX-Glu1 and -Glu2 had dissociation t 1/2 of 12 and 30 min, respectively, while -Glu3, -Glu4, and -Glu5 had t 1/2 of 102, 108, and 120 min. These experiments demonstrated that the longer chain polyglutamates have prolonged intracellular retention and can be dissociated less readily than MTX-Glu2 from DHFR, properties likely to make them more efficient DHFR inhibitors than the parent drug and of potential importance in extending the duration of drug action in tumor cells.

Binding Sites↗
PubMed1983

Microangiopathic hemolytic anemia, renal failure, and noncardiogenic pulmonary edema: a chemotherapy-induced syndrome.

Following gastrectomy for locally advanced adenocarcinomas, three patients developed microangiopathic hemolytic anemia and renal failure shortly after completing courses of adjuvant chemotherapy with mitomycin and 5-FU. These complications progressed despite cessation of chemotherapy, and all three patients died of noncardiogenic pulmonary edema precipitated in two cases by blood transfusions. At autopsy, two patients had no residual carcinoma and all had a diffuse microangiopathy involving mainly the kidneys and lungs. There was intimal hyperplasia of many arterioles sometimes associated with complete occlusion of the lumen, prominent nuclear atypia in many capillary cells, and numerous capillary fibrin thrombi. Direct immunofluorescence studies revealed extensive fibrinogen-fibrin deposits in the vascular lesions. Chemotherapy-induced microangiopathic hemolytic anemia and renal failure may predispose patients to fatal episodes of noncardiogenic pulmonary edema that can be triggered by blood transfusions.

Adenocarcinoma↗
PubMed1982

Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.

To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 muM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates. These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

Breast Neoplasms↗
PubMed1980

ACTH-secreting medullary carcinoma of the thyroid: monitoring of clinical course with calcitonin and cortisol assays and immunohistochemical studies.

The clinical course of a patient with Cushing's syndrome secondary to metastatic medullary carcinoma of the thyroid was documented with serial calcitonin and cortisol assays and tumor immunohistochemistry studies. Cortisol levels were originally markedly elevated but returned to normal after total thyroidectomy. These levels rose again when the patient developed liver metastases but normalized during chemotherapy, never to rise again despite the appearance of cervical lymph node metastasis. Calcitonin levels remained elevated throughout the course. The original tumor was composed of two cell lines: one containing both calcitonin and ACTH and another containing only calcitonin-reacting cells. However, the cervical metastasis showed a marked decrease in both cell lines with fever than 1% of cells reacting to ACTH, and only 25% to calcitonin. The almost total disappearance of ACTH-reacting cells may have been therapeutically induced or may represent a consequence of tumor progression.

Adrenocorticotropic Hormone↗