Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent.

The therapy of Pneumocystis carinii (PC) pneumonia is often unsuccessful, particularly in patients with acquired immune deficiency syndrome (AIDS). Because of difficulties in growing the organism in vitro or obtaining purified organisms, current treatment choices have been made with little information on the metabolic effects of therapeutic agents on PC. This report quantitates the effects of the commonly used antifolates as well as the classic antineoplastic antifolate methotrexate and a lipid-soluble analogue, trimetrexate, on the target enzyme, dihydrofolate reductase (DHFR), in the PC organisms. Trimethoprim and pyrimethamine were found to be weak inhibitors (ID50 = 39,600 and 2,800 nM, respectively), while methotrexate and trimetrexate were potent reductase inhibitors (ID50 = 1.4 and 26.1 nM, respectively). transport studies with radiolabeled compounds showed that compounds with the classic folate structure (methotrexate and leucovorin) were not taken up by the intact PC organisms. In contrast, trimetrexate exhibited rapid uptake. These results suggest a major therapeutic advantage may be gained by combining a potent, readily transported PC DHFR inhibitor such as trimetrexate with the reduced folate leucovorin to achieve a highly potent antiprotozoan effect while preventing toxicity to mammalian cells.

Mineralogy and composition of lunar fines and selected rocks.

Mineralogical descriptions and both wet chemical analyses and microprobe analyses are given of the glasses and crystalline components of the lunar fines and of the minerals in microgabbros (samples 10050 and 10047). The principal minerals described are various clinopyroxenes, plagioclase, olivine, low cristobalite, low tridymite, ilmenite, iron-nickel, iron, schreibersite, cohenite, troilite, and a new CaFe pyroxenoid. Descriptions are given of small craters produced by hypervelocity particle impact on glass and iron-nickel fragments in the fines. The rounding of grains in the fines and of surface rocks is attributed to mechanical ahrasion and not to cratering.

Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate.

Trimetrexate, a highly lipid-soluble quinazoline antifolate now undergoing trials as an anticancer agent, was found to be a potent inhibitor of the dihydrofolate reductase (DHFR) isolated from Toxoplasma gondii. The concentration required for 50% inhibition of protozoal DHFR was 1.4 nM. As an inhibitor of this enzyme, trimetrexate was almost 600-fold (amount of antifolate required to inhibit catalytic reaction by 50%) and 750-fold (inhibition constant) more potent than pyrimethamine, the DHFR inhibitor currently used to treat toxoplasma infection. When the protozoan was incubated with 1 microM trimetrexate, the drug rapidly reached high intracellular concentrations. Since toxoplasma organisms lack a transmembrane transport system for physiologic folates, host toxicity can be prevented by co-administration of the reduced folate, leucovorin, without reversing the antiprotozoal effect. The effectiveness of trimetrexate against toxoplasma was demonstrated both in vitro and vivo. Proliferation of toxoplasma in murine macrophages in vitro was completely inhibited by exposure of these cells to 10(-7) M trimetrexate for 18 h. When used alone, trimetrexate was able to extend the survival of T. gondii-infected mice.

Purification and properties of cytidine deaminase from normal and leukemic granulocytes.

Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70 degrees C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10(-5) M) than for ara-C (8.8 x 10(-5) M) or 5-azaC (4.3 x 10(-4) M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2'-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10(-8) M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52+/-1.86 x 10(3)/mg protein) than chronic myelocytic leukemia (CML) cells (1.40+/-0.70 x 10(3) U/mg protein) or acute myelocytic leukemia (AML) cells (0.19+/-0.17 x 10(3) U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells.

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.

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.

Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines.

Thymidylate synthase (TS) (EC 2.1.1.45) is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. Using the TS 106 monoclonal antibody to human TS, we have compared the immunological quantitation of TS by Western immunoblot and immunofluorescent techniques to the conventional biochemical 5-fluorodeoxyuridine monophosphate binding assay in a panel of 5-fluorouracil (5-FU)-sensitive and -resistant human cancer cell lines. Densitometric quantitation of TS 106-labeled Western immunoblot analysis of cell lysates from two 5-FU-resistant colon carcinoma cell lines, NCI H630R1 and NCI H630R10, revealed 12.8- and 16-fold increases in TS, respectively, compared to the parent 5-FU-sensitive NCI H630 colon cell line. By biochemical analysis, the TS level was 15- and 23-fold higher, respectively, in these resistant cell lines. Similarly, immunoblot analysis of cell lysates from two 5-FU-resistant breast cancer cell lines, MCF-Ad5 and MCF-Ad10, detected a 2.3- and 6.3-fold increase in TS, respectively, compared to the parent MCF-7 cell line. By biochemical assay the TS activity was 1.8- and 7.0-fold higher in these resistant breast cell lines. Western immunoblotting analysis revealed a 35-fold range of TS protein concentrations among the 10 cell lines examined, compared to a 38-fold range demonstrated by the biochemical assay. Direct comparison of Western blotting and the biochemical assay revealed a highly significant correlation (r2 = 0.93) between the two assays. Moreover, using the monoclonal antibody TS 106, the Western blotting technique was capable of detecting TS protein levels as low as 0.3 fmol in cellular lysates. Quantitation of TS in intact cells by immunofluorescent TS labeling and flow cytometric analysis was performed using three of the cell lines, NCI H630, NCI H630R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630R10 line compared to the parent NCI H630 line and a 3.5-fold increase in TS compared to the 5-FU-resistant MCF-Ad10 breast cell line. The 5-FU-resistant MCF-Ad10 breast cell line, in turn, displayed a 7.7-fold increase in TS, compared to the 5-FU-sensitive NCI H630 cell lines. TS immunofluorescent analysis was capable of measuring TS within individual cells. The development of these immunological assays using an anti-TS monoclonal antibody will facilitate the quantitation of TS in cell lines and tissue samples.

The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer.

PURPOSE: We assessed the prognostic importance of the level of thymidylate synthase (TS) expression in patients with primary rectal cancer and whether, for Dukes' B and C cancer patients, the benefit of chemotherapy was associated with TS expression. PATIENTS AND METHODS: The level of TS expression in the primary rectal cancers of 294 of 801 patients enrolled on protocol R-01 of the National Surgical Adjuvant Breast and Bowel Project (NSABP) was immunohistochemically assessed with the monoclonal antibody TS 106. RESULTS: Forty-nine percent of patients whose tumors had low TS levels (n = 91) were disease free at 5 years compared with 27% of patients with high levels of TS (n = 203; P < .01). Moreover, 60% of patients with low TS levels were alive after 5 years compared with 40% of patients with high TS levels (P < .01). The level of TS protein was significantly associated with Dukes' stage (P < .01); patients with a more advanced Dukes' stage had a significantly higher level of TS. The level of TS expression remained prognostic for both disease-free survival (P < .01) and survival (P < .05) independent of Dukes' stage and other pathologic characteristics evaluated. Thirty-eight percent and 54% of patients with high TS levels (n = 71) were disease free and alive, respectively, after 5 years when treated with chemotherapy, compared with 17% and 31%, respectively, of similar patients when treated with surgery alone (n = 64) (P < .01). No difference was noted in disease-free survival (P = .46) or survival (P = .43) in patients with low TS levels. CONCLUSION: The expression of TS is an important independent prognosticator of disease-free survival and survival in patients with rectal cancer. Adjuvant fluorouracil (5-FU)-based chemotherapy demonstrated significant improvement in disease-free and overall survival for patients with high TS levels. Prospective studies measuring TS levels will be needed to understand further the role of TS as a prognosticator of survival and chemotherapeutic benefit.

Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer.

The efficacy and toxicity of leucovorin 500 mg/m2 administered intravenously (IV) over 30 minutes daily for five days followed in one hour by fluorouracil (5-FU) 375 mg/m2 administered IV daily for five days, each given every 3 weeks, was assessed in 54 previously treated patients with metastatic breast cancer. An overall objective response rate of 24% was achieved (95% confidence interval, 13% to 38%), with an additional 56% of patients maintaining stable disease. Eleven of 12 patients who responded had received previous 5-FU therapy. Toxicity of this regimen included grade 3 diarrhea in 13%, grade 3 or 4 mucositis in 33%, grade 3 or 4 granulocytopenia in 65%, and grade 3 or 4 thrombocytopenia in 19%. Delay of treatment was required for hematologic toxicity in 44 patients. Thirty-eight patients required dose reductions due to toxicity. Biochemical evaluation of tumor biopsy specimens obtained from 17 patients used as their own controls with and without leucovorin was performed. These studies reveal an increased stabilization of the 5-fluorodeoxyuridylate (FdUMP)-thymidylate synthase (TS) folate ternary complex with the addition of leucovorin. There was a 71% +/- 14% occupancy or inhibition of the enzyme with the use of both 5-FU and leucovorin, v 30% +/- 13% for 5-FU alone (P2 less than .037). The percent TS bound in responding patients was substantially higher than in those patients with progressive disease. Finally, the mean total tumor TS pre-therapy in seven patients was 31 fmol/mg compared with a mean of 81 fmol/mg in these same seven patients 24 hours after therapy. This 2.6-fold increase suggests that there is an induction of the enzyme, TS, with 5-FU treatment.

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.

Presence of 2,4-diamino-N10-methylpteroic acid after high-dose methotrexate.

Assay of plasma methotrexate has been established as important to its safe use. We have investigated the specificity of 2 assay procedures for methotrexate: the competitive dihydrofolate reductase binding assay (CRBA) and the radioimmunoassay (RIA). The RIA of plasma methotrexate resulted in consistently higher values than the CRBA, with greater differences at later measurement times. A compound that strongly cross-reacts in the RIA, but not the CRBA, has been identified in plasma and urine of patients on high-dose methotrexate therapy, and appears to be the carboxypeptidase cleavage product (2,4-diamino-N10-methylpteroic acid) on the basis of chromatographic and ultraviolet spectral properties. Although this compound is present as a minor contaminant in commercial methotrexate preparations, quantitative assessment of urinary excretion suggests that in man a major portion of the compound is derived from methotrexate metabolism.

Treatment of Pneumocystis carinii pneumonia with trimetrexate in acquired immunodeficiency syndrome (AIDS).

In vitro studies have shown that trimetrexate, a lipid-soluble analogue of methotrexate, is 1500 times more potent than trimethoprim as an inhibitor of dihydrofolate reductase from Pneumocystis carinii. Furthermore, trimetrexate is readily taken up by P carinii, while performed folates such as leucovorin are not. These observations suggest that the combination of trimetrexate plus leucovorin, which can specifically protect mammalian host tissues from the toxic effects of the antifolate, may be useful in the treatment of pneumocystis pneumonia. This concept was tested in a clinical study of 49 patients with acquired immunodeficiency syndrome (AIDS) and P carinii pneumonia who were treated for 21 days with trimetrexate and leucovorin. Patients were divided into three groups: 16 patients who were unable to tolerate or had failed both pentamidine isethionate and trimethoprim-sulfamethoxazole therapy were treated with trimetrexate plus leucovorin (Group I); 16 patients who were unable to tolerate sulfonamide therapy were treated with trimetrexate with leucovorin as initial therapy (Group II); and 17 patients in whom trimetrexate with leucovorin plus sulfadiazine was used as initial therapy (Group III). Response and survival rates were 69% and 69% in Group I; 63% and 88%, respectively, in Group II; and 71% and 76%, respectively, in Group III. Toxicity was minimal. The results indicate that trimetrexate with leucovorin is safe and effective for initial therapy in AIDS patients with P carinii pneumonia and in those intolerant or unresponsive to standard therapies.

Quantitation of thymidylate synthase in human tumors using an ultrasensitive enzyme-linked immunoassay.

Thymidylate synthase (TS; EC 2.1.1.45) is an important therapeutic target for fluoropyrimidine cytotoxic drugs that are widely used for the treatment of solid tumors. Using the monoclonal antibody TS 106, we have developed an ultrasensitive enzyme-linked immunoassay (ELISA) for the detection and quantitation of TS. Using a chemiluminescent ELISA technique, TS was detectable in serially diluted lysates from NCI H630 and HCT 116 human colon carcinoma cell lines. The ELISA assay was reliably able to detect activity down to a level of 30 attamol of TS protein above background (P2 = 0.016). The usable range of detection was from 0.03 to 500 fmol of enzyme. There was a close correlation between the optical density signal and the total TS enzyme between both cell lines (r2 = 0.96). The ELISA was used to measure TS in cytosolic extracts from human tumor samples, and it was able to quantitate TS levels using as little as 1-mg tumor biopsy samples. The mean total TS measured by ELISA in seven tumor samples from patients with breast cancer and sarcomas was 131 fmol/mg cytosolic protein (range 60-240) compared with a mean TS of 85 fmol/mg cytosolic protein (range 35-163) using the fluorodeoxyuridine monophosphate binding assay. While the TS levels were uniformly higher when measured by ELISA, there was close proportional agreement between both assays (r2 = 0.84). Thus, the chemiluminescent TS ELISA would appear to be an extremely sensitive and specific assay that may be used to quantitate TS in tumor tissue specimens.

Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil.

A series of 5-fluorouracil (5-FU)-resistant human colon H630 cancer cell lines were established by continuous exposure of cells to 5-FU. The concentration of 5-FU required to inhibit cell proliferation by 50% (IC50) in the parent colon line (H630) was 5.5 microM. The 5-FU IC50 values for the resistant H630-R1, H630-R10, and H630-R cell lines were 11-, 29-, and 27-fold higher than that for the parent H630 cell line. Using both the radioenzymatic 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) binding and catalytic assays for measurement of thymidylate synthase (TS) enzyme activity, there was significantly increased TS activity in resistant H630-R1 (13- and 23-fold), H630-R10 (37- and 40-fold), and H630-R (24- and 34-fold) lines, for binding and catalytic assays, respectively, compared with the parent H630 line. The level of TS protein, as determined by western immunoblot analysis, was increased markedly in resistant H630-R1 (23-fold), H630-R10 (33-fold), and H630-R (26-fold) cells. Northern analysis revealed elevations in TS mRNA levels in H630-R1 (18-fold), H630-R10 (39-fold), and H630-R (36-fold) cells relative to parent H630 cells. Although no major rearrangements of the TS gene were noted by Southern analysis, there was significant amplification of the TS gene in 5-FU-resistant cells, which was confirmed by DNA slot blot analysis. These studies demonstrate that continuous exposure of human colon cancer cells to 5-FU leads to TS gene amplification and overexpression of TS protein with resultant development of fluoropyrimidine resistance.

Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines.

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.

5-fluorouracil-mediated thymidylate synthase induction in malignant and nonmalignant human cells.

Thymidylate synthase (TS, EC 2.1.1.45) is an important target enzyme for the fluoropyrimidines used in cancer chemotherapy. Studies have documented a 2- to 4-fold induction of TS protein following 5-fluorouracil (5-FU) treatment of malignant cells. We measured the effect that 5-FU exposure had on TS protein expression in nonmalignant human breast (MCF-10 and HBL-100), colorectal (ATCC Co18, Co112, and Co33), and bone marrow cells along with malignant breast (MCF-7) and colon (NCI-H630) cells. Twenty-four hours after plating, cells were treated with 0.01 to 10 microM of 5-FU for a period of 24 hr. TS was quantitated by Western immunoblot using monoclonal antibody TS106. Absolute levels of TS in nonmalignant cells were substantially lower than in the malignant lines, ranging from approximately 40% in HBL-100 cells to less than 10% in the colon lines. An approximately two-fold induction in the level of TS was found for all cell lines examined, and there was a strong dependence on 5-FU exposure concentration in free TS levels of MCF-WT, and total TS levels of H630-WT, normal bone marrow, and MCF-10 cells. The induction of TS following 5-FU exposure is a generally observed phenomenon in both malignant and nonmalignant cells, suggesting that a selective means for inhibiting this induction may be critical for the development of alternative therapeutic strategies using 5-FU and the antifolate TS inhibitors.

Dose-dependent inhibition of aspartate carbamoyltransferase in peripheral blood mononuclear cells in patients receiving N-(phosphonacetyl)-L-aspartate.

Forty-eight patients with adenocarcinoma of the gastrointestinal tract were treated on this trial. The MTD of 5-FU given as a 72 hour infusion with high-dose leucovorin was initially determined to be 2000 mg/m2/d. Patients were treated at PALA dose levels ranging from 250 to 2848 mg/m2. Biochemical assessment of target enzyme activity was performed at each PALA dose level. We conclude that compared to each patient's own baseline, PALA at 250 mg/m2 failed to appreciably inhibit ACTase activity at 24 hours in most patients. More consistent inhibition of ACTase activity was seen with PALA at or above 1266 mg/m2, but toxicity was prohibitive with 2848 mg/m2 PALA. Even with the highest PALA doses, ACTase activity was back to baseline by 96 hours in most patients. PALA at 1266 mg/m2 given 24 hours prior to the start of 72 hour infusional 5-FU plus high-dose leucovorin was associated with acceptable toxicity and did not appear to compromise 5-FU dose-intensity. Finally, because of interpatient variability in the degree of ACTase inhibition following PALA, biochemical monitoring of target enzyme activity may permit more rational adjustment of the PALA dose in individual patients.

Ara-C metabolism: implications for drug resistance and drug interactions.

Clinical studies of resistance to cytosine arabinoside have not produced agreement as to the specific biochemical lesions responsible for altered sensitivity, although experimental and clinical work supports the concept that a decreased ability to generate ara-CTP must be the ultimate effect of this lesion. 3-deazauridine, an inhibitor of CTP synthetase, was found to enhance ara-CTP production in murine tumor cells, and in the present study, was shown to inhibit deamination of ara-C at both the nucleoside and nucleotide level. Enhanced ara-CTP formation was observed in cells lacking cytidine deaminase (L1 210 and HL60), indicating that 3-deazauridine inhibition of deoxycytidylate deaminase may be important in this drug interaction.