Granulocytes: effector cells or immunomodulators in the immune response to helminth infection?

Granulocytes are effector cells in defence against helminth infections. We review the current evidence for the role of granulocytes in protective immunity against different helminth infections and note that for each parasite species the role of granulocytes as effector cells can vary. Emerging evidence also points to granulocytes as immunomodulatory cells able to produce many cytokines, chemokines and modulatory factors which can bias the immune response in a particular direction. Thus, the role of granulocytes in an immunomodulatory context is discussed including the most recent data that points to an important role for basophils under this guise.

Enhanced 5-fluorouracil nucleotide formation after methotrexate administration: explanation for drug synergism.

Exposure of L1210 leukemia cells first to 0.1 to 100 micromolar methotrexate and then to 10 micromolar 5-fluorouracil produces a synergistic effect on the number of cells killed in culture. Methotrexate dose-related increases occur in the concentrations of intracellular 5-fluorouracil ribonucleotides and 5-fluoro-2'-deoxyuridylate and in the incorporation of 5-fluorouracil into RNA. These increases are correlated with increased concentrations of intracellular phosphoribosylpyrophosphate. It is proposed that the enhanced formation of ribonucleotides of 5-fluorouracil and the subsequent incorporation of these compounds into RNA in methotrexate-treated cells may account for synergism between these agents.

Mechanism of synergistic cell killing when methotrexate precedes cytosine arabinoside: study of L1210 and human leukemic cells.

Synergistic killing of L1210 cells occurs when methotrexate (MTX) is administered just before 1-beta-D-arabinofuranosylcytosine (Ara-C). This pehnomenon is dependent upon both the dose and time of exposure to MTX. Such increased killing of cells can be explained by the enhanced intracellular accumulation of Ara-C in cells exposed to MTX. This enhancement of Ara-C entry into cells was only observed when the dose of MTX was high enough (1, 10, and 100 muM) to result in free intracellular nondihydrofolate reductase-bound MTX. At the highest doses of MTX (10 and 100 muM) Ara-C triphosphate was increased eightfold and deoxycytidine triphosphate was decreased by 50%. Therefore, the maximum synergistic cell kill when MTX precedes Ara-C may be the consequence of greater inhibition of DNA polymerase by th;e increased Ara-C triphosphate in the presence of the decreasing natural substrate of this enzyme, deoxycytidine triphosphate. Enhanced Ara-C accumulation after administration of MTX was also observed in human acute myelogenous leukemia cells.

Schedule-dependent cytotoxicity of methotrexate and 5-fluorouracil in human colon and breast tumor cell lines.

The effect of sequential methotrexate and 5-fluorouracil on the clonal growth of the human colon adenocarcinoma cell, HCT-8, and the hormone-dependent human breast carcinoma cell, 47-DN, was examined. In both cell lines, when 5-fluorouracil was given during the last 6 h of a 24 h methotrexate exposure period, there was marked synergistic inhibition of clonal growth. Shorter intervals or the reverse sequence of drugs were either additive or antagonistic. These results indicate the importance of the drug sequence and time interval between drug administration for optimal cytotoxicity in these human cell lines. This information suggests that the administration of methotrexate 18 h before 5-fluorouracil may have potential application in the design of clinical trials for these malignancies.

Levels of thymidylate synthetase during normal culture growth of L1210 cells.

The binding of 5-fluoro-2'-deoxyuridylate generated from 5-fluoro-2'-deoxyuridine in intact cells was used to measure changes in the level of thymidylate synthetase during the course of population growth of murine leukemia L 1210 cells. By the use of elutriation techniques and flow cytometric analysis, the amount and activity of thymidylate synthetase associated with the various phases of the cell cycle were determined for the L 1210 cells during unperturbed in vitro culture growth. Fluctuations of thymidylate synthetase levels were associated with the cell cycle; there was a positive correlation (P less than 0.001) between the percentage of the total cell population in S phase and the concentration of thymidylate synthetase, although there was an increase in the level of this enzyme in association with an increase in G2-M cells, this did not achieve statistical significance. A negative correlation between G1 cells and the concentration of thymidylate synthetase was also observed. The maximum amount of thymidylate synthetase was nearly 900 fmol/10(6) cells and occurred in cell populations during logarithmic growth when the percentage of the population in S phase and G2-M phase was greater than 50 and 20%, respectively. In late culture growth (plateau) when only 25% of the cell population was in S phase and nearly 75% of the population was in G1 phase, the level of enzyme was reduced to 200 fmol/10(6) cells.

Altered 5-azacytidine metabolism following 3-deazauridine treatment of L5178Y and human myeloblasts.

The effect of 3-deazauridine pretreatment on 5-azacytidine metabolism was studied in suspension cultures of L5178Y murine leukemia. A 3-hr exposure to 2 microM 3-deazauridine followed by a 1-hr exposure to 5 microM [14C]-5-azacytidine resulted in a 2-fold increase in total intracellular 5-azacytidine accumulation compared to untreated controls. Under the same conditions, incorporation of 5-azacytidine into the acid precipitable fraction of L5178Y cells was increased 3-fold. Incorporation of 5-azacytidine into RNA increased 85% following 3-deazauridine pretreatment, but 5-azacytidine incorporation into DNA did not change significantly. In cells pretreated with 3-deazauridine, there was an 80% reduction of intracellular cytidine triphosphate, the natural feedback inhibitor of uridine-cytidine kinase, the rate-limiting enzyme in the phosphorylation of 5-azacytidine. Intracellular levels of 5-azacytidine triphosphate, the presumed lethal metabolite of 5-azacytidine, increased from 28.8 pmol/10(6) cells in control cells to 56.4 pmol/10(6) cells following 3-deazauridine treatment. The sequence of 3-deazauridine followed by 5-azacytidine demonstrated synergistic cell killing when measured by an in vitro soft-agar cloning assay. Similar biochemical alterations were also seen in human leukemic myeloblasts. It appears that 3-deazauridine-induced alterations in 5-azacytidine metabolism may account for the enhanced cytotoxicity of this drug sequence.

Biochemical and cytokinetic modulation of L1210 and HL-60 cells by hydroxyurea and effect on 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity.

The modulating effect of hydroxyurea (HU) on 1-beta-D-arabinofuranosylcytosine (ara-C) metabolism and cytotoxicity was evaluated in L1210 cells and the human promyelocytic leukemic cells HL-60. A dose- and time-dependent HU exposure was observed which resulted in maximum deoxycytidine 5'-triphosphate reduction, intracellular ara-C accumulation, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation, and cytotoxicity as determined by soft agar cloning. For the L1210 cells, a 5-hr pretreatment of 5 mM HU was optimum. The best result obtained with the HL-60 cells was after a 24-hr exposure of 1 mM HU. There was also a maximum incorporation of ara-C into DNA in both cells following these optimal pretreatment conditions. Cytofluorometric analysis demonstrated that this HU treatment resulted in a maximum accumulation of L1210 and HL-60 cells in the pre-S phase of the cell cycle and that after removal of the HU there was a rapid progression of the cell population through S phase. Because cytotoxicity of ara-C is considered to be predominantly from the inhibition of DNA polymerase and/or the incorporation into DNA, the cytokinetic and biochemical modulatory effects of HU must both be contributing factors to consider in achieving maximal cell kill from this drug sequence.

Modulation of 5-fluorouracil metabolism and cytotoxicity by antimetabolite pretreatment in human colorectal adenocarcinoma HCT-8.

The modulation of 5-fluorouracil (FUra) metabolism by methotrexate (MTX) pretreatment in monolayer cultures of human colorectal adenocarcinoma. HCT-8, was examined and correlated to clonal growth of this cell line. There was a gradual and nearly linear total intracellular accumulation and incorporation into RNA of FUra for 30 hr in control cells. A 12-hr 10 microM MTX pretreatment before adding 100 microM FUra resulted in approximately a 3-fold increase in total FUra accumulation, 59% of which was fluorouridine triphosphate. Soluble fluorodeoxyuridine monophosphate was increased 5-fold following MTX pretreatment; however, [3H]deoxyuridine incorporation into the acid-precipitable fraction of cells pretreated with MTX was no more than that observed when FUra was given alone. There was also an increase in 5-phosphoribosyl 1-pyrophosphate pools following MTX which was associated with the enhanced FUra metabolism. The maximum synergistic inhibition of clonal growth occurred when FUra was given during the last 6 hr of a 24-hr MTX exposure period. Other antimetabolites associated with elevations of 5-phosphoribosyl 1-pyrophosphate also resulted in an enhanced total intracellular accumulation of FUra.

Modulation of 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity in L1210 cells by fluoropyrimidine pretreatment.

The effect of pretreatment with the fluoropyrimidines 5-fluorouracil (FUra), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) on the intracellular metabolism and in vitro cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was explored in L1210 cells. A 4-hr exposure to 100 microM FUra, FUrd, or FdUrd produced greater than 3-fold increments in the intracellular accumulation of [3H]deoxycytidine, and 2-fold increments in the intracellular accumulation of [3H]ara-C were produced over a 1-hr exposure. Intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate levels in cells exposed to these 3 agents were also increased over 2-fold. L1210 cells exposed sequentially to 1 microM FUra, FUrd, or FdUrd followed by 5 microM ara-C for 1 hr resulted in synergistic cell killing measured by soft-agar cloning. Although intracellular 2'-deoxycytidine 5'-triphosphate levels were reduced in cells treated for 4 hr with FUra and FUrd, this was not associated directly with the maximum accumulation of ara-C. FdUrd treatment was as effective as FUra and FUrd at enhancing ara-C accumulation and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation and producing synergistic cytotoxicity; however, the 2'-deoxycytidine 5'-triphosphate pools were minimally affected. 5'-Deoxy-5-fluorouridine did not increase intracellular ara-C accumulation, reduce intracellular 2'-deoxycytidine 5'-triphosphate levels, or enhance ara-C cytotoxicity. This sequential drug interaction is similar to that which we have observed when methotrexate precedes ara-C and provides a rational basis from which further in vivo studies can be designed.

Biochemical alterations during unperturbed suspension growth of L1210 cells.

The following parameters were evaluated at several points throughout unperturbed suspension culture growth of L1210 cells: cell volume; DNA histograms; the mean content of cellular DNA, RNA, and protein; ribonucleoside and deoxyribonucleoside triphosphate pools; phosphoribosyl pyrophosphate; and the incorporation of glycine into purine bases. The cell volume, the incorporation of glycine into purine bases, and the intracellular pools of phosphoribosyl pyrophosphate and dexoyribunucleotides began to decrease significantly during the midportion of logarithmic cell growth. However, there was no significant change in the DNA content per cell during culture growth. The RNA, protein content, and ribonucleotides all demonstrated a biphasic pattern with the highest values obtained during the midportion of logarithmic growth followed by rapid decline as the culture approached plateau growth. These intracellular fluctuations in de novo synthesis and precursor pools were correlated with the variable intracellular accumulation of three fluoropyrimidines (5-fluorouracil, 5-fluorouridine, and 5-fluorodeoxyuridine) and their active metabolites (5-fluorouridine triphosphate and 5-fluorodeoxyuridylate). These studies were performed to demonstrate that multiple biochemical alterations occur during logarithmically growing suspension cell cultures and could result in misleading conclusions of experiments with antimetabolites unless these factors are considered in the context of the performed studies.

Tamoxifen and 5-fluorouracil in breast cancer: modulation of cellular RNA.

Biochemical studies were undertaken with the human breast carcinoma cell line, 47-DN, to explore the mechanisms underlying cytotoxic synergy between tamoxifen (TAM) and the fluoropyrimidines, 5-fluorouracil (FUra) and 5-fluorouridine (FUrd). The influence of TAM pretreatment was measured on intracellular FUra accumulation, FUra nucleotide formation, and incorporation of fluoropyrimidines into cellular RNA. Unlike other modulators of FUra metabolism and toxicity. TAM decreased intracellular FUra accumulation and total RNA incorporation by 20 to 60%. Cells treated with TAM contained 10 to 20% less cellular RNA and showed reduced transcription and altered RNA turnover, independent of fluoropyrimidine treatment. Newly synthesized RNA from control and TAM-treated cells was fractionated by sucrose gradient centrifugation. The specific incorporation of FUrd (2 hr) was compared to that of FUra (6 hr) and labeled uridine incorporation into controls. Compared to its effect on uridine incorporation, TAM produced nearly twice as much FUra incorporation and 3 times as much FUra incorporation into 32 to 45S RNA. Since accumulation of this high-molecular-weight RNA has been associated with fluoropyrimidine toxicity and impaired ribosomal RNA processing, it is believed that TAM enriched the RNA-mediated toxicity of FUra and FUrd in this breast carcinoma cell line.

5'-Deoxy-5-fluorouridine selective toxicity for human tumor cells compared to human bone marrow.

Studies were completed to establish the comparative cytotoxicity of 5'-deoxy-5-fluorouridine (5'-dFUrd) and other fluoropyrimidines in human bone marrow stem cells and several cultured human tumor cell lines (i.e., 47-DN and MCF-7 breast carcinomas, MG-63 osteosarcoma, HCT-8 colon tumor, Colo-357 pancreatic tumor, and HL-60 promyelocytic leukemia). In vitro clonogenic assays were used to measure cytotoxicity following a 3-hr drug exposure. 5'-dFUrd was less potent than was 5-fluorouracil or 5-fluoro-2'-deoxyuridine in all cells examined, exhibiting its best activity against the 47-DN [concentration that prevented 50% clonal growth compared to untreated control (LD50) = 32 microM] and MCF-7 (LD50 = 35 microM) breast carcinomas and MG-63 osteosarcoma (LD50 = 41 microM). Intermediate activity was observed against HCT-8 (LD50 = 200 microM) and Colo-357 (LD50 = 150 microM) gastrointestinal tumors. 5'-dFUrd had very poor activity against the HL-60 leukemia (LD50 = 470 microM). The suppression of the clonal growth of human bone marrow stem cells required the greatest amount of 5'-dFUrd (LD50 = 580 microM). With use of these studies, a therapeutic ratio (concentration that prevented 25% clonal growth compared to untreated control of bone marrow divided by LD50 of tumor) was calculated for each drug in each tumor. 5'-dFUrd had values ranging from 1.2 to 7.5 for the solid tumors and 0.5 in HL-60 cells. This was in marked contrast to 5-fluorouracil, or 5-fluoro-2'-deoxyuridine, which failed in all cases to have ratios greater than or equal to one. The results indicate that 5'-dFUrd can exhibit a cytotoxic selectivity for human tumor cells compared to human bone marrow stem cells that does not exist for 5-fluorouracil or 5-fluoro-2'-deoxyuridine. This suggests that 5'-dFUrd may be of greater therapeutic benefit in the treatment of certain human cancers than the fluoropyrimidines used currently.

Differential effect of N-(phosphonacetyl)-L-aspartate on 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity in human leukemia and normal bone marrow progenitors.

The effect of N-[phosphonacetyl]-L-aspartate (PALA) on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in the human promyelocytic leukemic cell line, HL-60, and in normal human bone marrow. HL-60 cells exposed to 0.1 mM PALA for 12 hr accumulated 58.7 pmol ara-C per 10(6) cells after a 45-min exposure to 1 microM ara-C, compared to 27.8 pmol ara-C per 10(6) cells in untreated control cells. This PALA concentration and exposure interval was associated with a greater than 2-fold increase in both the 45-min generation and 4-hr retention of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate compared to untreated control HL-60 cells. Exposure of HL-60 cells to PALA followed by ara-C produced greater than additive effects on the inhibition of DNA synthesis, the inhibition of cell growth, and clonogenicity. In contrast, exposure of normal human bone marrow to the same PALA-ara-C schedule was not associated with a synergistic inhibition of colony-forming units in soft agar. If these perturbations also occur in vivo, an improvement in the therapeutic index of ara-C in patients with acute leukemia might result.

Potentiation of 1-beta-D-arabinofuranosylcytosine metabolism and cytotoxicity by 2,3-dihydro-1H-imidazolo[1,2-b]pyrazole in the human promyelocytic leukemic cell, HL-60.

The effect of IMPY (2,3-dihydro-1H-imidazolo[1,2-b]pyrazole) on the metabolism and cytotoxicity of subsequently administered 1-beta-D-arabinofuranosylcytosine (ara-C) was examined in the human promyelocytic leukemic cell line HL-60. Cells exposed to 3 mM IMPY for 12 hr followed by a 1-hr exposure to 1 microM [3H]ara-C accumulated 27.5 +/- 4.8 (S.D.) pmol ara-C/10(6) cells compared to 14.0 +/- 3.5 pmol/10(6) cells in untreated controls. Cells experienced greater than a 2-fold increment in 1-beta-D-arabinofuranosylcytosine 5'-triphosphate generation and retention following this same IMPY exposure and nearly a 4-fold increment in incorporation of ara-C into HL-60 nucleic acids. These alterations in ara-C metabolism were associated with a 36% reduction in the intracellular concentration of deoxycytidine 5'-triphosphate and reductions in deoxyadenosine 5'-triphosphate and deoxyguanosine 5'-triphosphate concentrations to undetectable levels. Coincubation of cells with IMPY along with other pyrimidine antagonists such as thymidine, N-(phosphonacetyl-L-aspartate), deoxyadenosine, and deoxyguanosine, produced up to 4-fold increments in ara-C intracellular accumulation. Pretreatment of HL-60 cells with 3 mM IMPY followed by a continuous exposure to 10 nM ara-C produced synergistic inhibitory effects on both suspension culture growth and soft agar clonogenicity. In contrast, exposure of normal human bone marrow progenitor cells (CFU-GM) to the same schedule of IMPY and ara-C produced subadditive or antagonistic effects on the growth of these cells in soft agar. These findings may have implications for the design of in vivo regimens using IMPY and ara-C.

Dose- and schedule-dependent activation and drug synergism between thymidine and 5-aza-2'-deoxycytidine in a human promyelocytic leukemia cell line.

The ability of thymidine (dThd) to enhance the metabolism and cytotoxicity of subsequent administered 5-aza-2'-deoxycytidine (5-aza-dCyd) was studied in L1210 cells and in the human promyelocytic leukemic cell line, HL-60. Exposure of L1210 cells to 0.1 mM dThd for 5 h resulted in an increase in the total intracellular and acid-precipitable accumulation of 5-aza-dCyd. Higher dThd concentrations and longer exposure intervals resulted in smaller increments in 5-aza-dCyd accumulation. In contrast, in HL-60 cells, a 24-hr exposure in 1 mM dThd resulted in the greatest intracellular accumulation of 5-aza-dCyd, 3.3 times more accumulation than in control cells. There was also a 4-fold increase in the acid-precipitable accumulation and nearly a 3-fold increase in DNA incorporation of 5-aza-dCyd in HL-60 cells exposed to the same dThd schedule. High-pressure liquid chromatographic analysis demonstrated a greater than 3-fold increase in the intracellular amounts of 5-aza-dCyd metabolites eluting in the triphosphate region in these human cells under identical conditions. Shorter dThd incubation exposure intervals (6 hr) and lower dThd concentration (0.1 mM) produced smaller increments in these studies. Both growth and clonogenic assays of HL-60 cells demonstrated a dose- and schedule sequence-dependent synergism between dThd and 5-aza-dCyd.

Optimal schedule of methotrexate and 5-fluorouracil in human breast cancer.

We have shown previously that methotrexate pretreatment of murine leukemia and human colon carcinoma cell cultures results in augmented intracellular accumulation of 5-fluorouracil metabolites. Both of these drugs are commonly used for the treatment of women with breast cancer; thus, sequencing of methotrexate before 5-fluorouracil was evaluated in vitro using a human mammary carcinoma cell line, 47-DN. Intracellular 5-fluorouracil accumulation was maximally increased 4-fold in cultures pretreated with 10 microM methotrexate for 24 hr. This enhancement of 5-fluorouracil metabolism was associated with increased intracellular levels of 5-phosphoribosyl 1-pyrophosphate, resulting from the antipurine effect of methotrexate. Brief exposure to exogenous hypoxanthine at physiological concentrations reversed the biochemical synergism between methotrexate and 5-fluorouracil. Other antimetabolites associated with elevations of 5-phosphoribosyl 1-pyrophosphate enhanced intracellular accumulation of 5-fluorouracil up to 2.5-fold. In cloning assays, 18 hr of methotrexate pretreatment followed by 5-fluorouracil resulted in optimal synergistic cytotoxicity, which could be prevented if high concentrations of leucovorin were given between methotrexate and 5-fluorouracil administration. Since these results indicated that optimal breast tumor toxicity in vitro was achieved by 18- to 24-hr sequencing of methotrexate and 5-fluorouracil, clinical toxicity study was carried out to assess whether this drug schedule could be tolerated. Seven patients with advanced cancer were treated with 21 courses of sequential therapy. No toxicity occurred with 38% of treatment courses; mild to moderate leukopenia and mucositis occurred with 29 and 38% of courses respectively. Toxicity was related to treatment interval and not cumulative drug dose or elevated serum methotrexate levels. These clinical results suggest that Phase II studies evaluating 24-hr-sequenced methotrexate and 5-fluorouracil in breast cancer are warranted.

Effect of N-(phosphonacetyl)-L-aspartate on 5-azacytidine metabolism in P388 and L1210 cells.

The effect of N-phosphonacetyl-L-aspartate (PALA) pretreatment on the metabolism and cytotoxicity of 5-azacytidine (5-aza-Cyd) was studied in two murine leukemic cell lines. Exposure of P388 and L1210 cells to 3 mM PALA for 3 hr before adding 5-aza-Cyd at 75 microM was accompanied by a two-fold increment in acid-soluble and 3-fold increment in acid-insoluble incorporation of 5-aza-Cyd in both cell lines. RNA incorporation of 5-aza-Cyd increased from 97.5 +/- 3.4 pmol 5-aza-Cyd per microgram D-ribose in control cells to 299.2 +/- 4.2 pmol 5-aza-Cyd per microgram D-ribose in PALA-treated cells; a smaller increment in DNA incorporation of 5-aza-Cyd was also noted. Sequential treatment of cells with PALA and 5-aza-Cyd was associated with a 40% reduction in protein synthesis compared to only a 2 and 8% reduction, respectively, produced by the drugs given alone. Sequential administration of PALA and 5-aza-Cyd resulted in greater than additive cytotoxicity as measured by both growth inhibition and in vitro soft-agar cloning assays. Exposure of both cell lines to 3 mM PALA for 3 hr produced 50 and 65% reductions in intracellular levels of cytidine triphosphate and uridine triphosphate; intracellular accumulation of 5-azacytidine triphosphate, the lethal metabolite of 5-aza-Cyd, increased from 43.4 +/- 2.1 pmol/10(6) cells to 92.4 +/- 3.3 pmol/10(6) cells in PALA-treated cells. PALA was able to augment the metabolism and cytotoxicity of 5-aza-Cyd in a uridine-cytidine kinase-mutant 5-aza-Cyd-resistant L5178Y subline. This sequential drug combination has a rational biochemical basis and may offer significant advantages over either drug when administered alone, especially in cells which are resistant to 5-aza-Cyd.

Tamoxifen and 5-fluorouracil in breast cancer: cytotoxic synergism in vitro.

The cytokinetic and cytotoxic interactions involved in combining tamoxifen, methotrexate, and 5-fluorouracil were studied in two hormone-dependent human breast cancer cell lines, 47-DN and MCF-7. These cells had measurable cytosol and nuclear estrogen receptor and cytosol progesterone receptor. Growth of the MCF-7 cells in medium containing gelding serum was stimulated maximally by addition of 10 pM estradiol. Both MCF-7 and 47-DN cells showed dose-dependent in vitro growth inhibition on exposure to tamoxifen, and toxicity from tamoxifen at concentrations up to 10 microM could be prevented by 1 nM estradiol. After exposure of 47-DN cells to 10 microM tamoxifen, cytosol progesterone and nuclear estrogen receptor levels were still detectable at 30 and 60% of control values. With this same concentration of tamoxifen, 47-DN cells in S phase declined 50% in association with a buildup of G0-1 cells. By clonogenic assay, tamoxifen enhanced 47-DN and MCF-7 cytotoxicity to 5-fluorouracil and 5-fluorouridine, but not to methotrexate alone. When given either concurrently or using a pretreatment-synchronizing schedule, tamoxifen enhanced markedly the growth inhibition of sequentially combined methotrexate and 5-fluorouracil. Isobologram analysis was used to prove that the cytotoxic interaction between tamoxifen and 5-fluorouracil was synergistic.

Sequential infusions of methotrexate and 5-fluorouracil in advanced cancer: pharmacology, toxicity, and response.

Preclinical studies have suggested that synergistic antitumor toxicity occurs when methotrexate (MTX) is administered prior to 5-fluorouracil (FUra). A protocol of sequenced, overlapping infusions of MTX and FUra was designed to achieve 5 microM MTX serum levels lasting 36 h and 1 to 5 microM FUra levels lasting 24 h, with leucovorin started at the end of the MTX infusion. Thirty-nine patients with metastatic neoplasms received a total of 127 treatment courses; two-thirds of the patients had received prior treatment with radiation therapy or chemotherapy; most of the latter treatment regimens included MTX or FUra. In three patients, the duration of FUra infusion was prolonged up to 72 h to determine the toxic limits of therapy. Blood samples were collected during treatment courses to estimate the half-lives and total-body clearances of MTX and FUra. The initial serum half-lives and total-body clearances of both MTX and FUra appeared within the range of reported normal values. The terminal half-life of MTX appeared less than previously reported values, and there appeared to be a substantial delay in achieving a FUra steady-state concentration; these two differences may have resulted from either the prolonged intervals of drug infusion or from metabolic interaction between the two drugs. During the 127 courses of treatment, nearly one-half of the patients experienced mild toxicity occurring after at least one treatment, but this toxicity was predominantly Grade I mucositis and/or diarrhea. Of the three patients who received extended intervals of FUra infusion, none was able to tolerate more than 48 h of FUra without developing mucositis. Thirty-four patients were evaluable for response; no one experienced a complete response, but 11 (32%) patients had either a partial or minimal response. Adenocarcinomas as a group, arising from the lung, gut, breast, and unknown site, appeared to respond best. Sequenced MTX-FUra infusion by this schedule is a generally well-tolerated regimen that deserves further clinical assessment.

Uridine and cytidine metabolism following inhibition of de novo pyrimidine synthesis by pyrazofurin.

Pyrazofurin, an inhibitor of orotidylate decarboxylase, imposes an absolute nutritional requirement for exogenous uridine to maintain normal growth of L5178Y, P388, L1210, W256 and S180 cells in vitro. The amount of uridine necessary for cell division when de novo uridine nucleotide synthesis is inhibited by pyrazofurin is: L5178Y, 30.5; P388, 39.7; L1210, 53.3: W256, 70.6; and S180, 886 fmol/cell. Cytidine, which can be deaminated to uridine, will substitute for uridine to maintain normal cell growth in the presence of growth-inhibitory concentrations of pyrazofurin (5 microM). The requirements for cytidine and uridine are identical. If cytidine deamination is prevented by tetrahydrouridine (100 microM), cytidine can no longer support growth in the presence of pyrazofurin. Cytidine and uridine, as expected, are additive in their effect to permit normal growth of pyrazofurin treated cells. Tetrahydrouridine does not alter this additive effect, indicating that when both nucleotides are added to pyrazofurin treated cells each nucleotide replenishes their respective nucleotide pools and cytidine deamination is unnecessary to allow cell growth. Incorporation of [14C]uridine into the acid insoluble cell fraction of L5178Y cells was 25 fmol/cell at 48 h and remained constant during the remaining growth of the pyrazofurin treated cell suspension. The [14C]uridine acid soluble pool of 4 fmol/cell also was maximum at 48 h but declined during the subsequent growth of the suspension culture to approx. 2 fmol/cell at 96 h. This decline in the acid soluble pool is correlated with a 42% decrease in modal cell volume during this phase of cell growth which would maintain a constant specific activity of uridine in this pool. This may explain the decline in the acid soluble pool while the acid insoluble pool remains constant during growth of suspension cultures of L51878Y cells. The block in pyrimidine synthesis de novo induced by pyrazofurin provides a useful and quick method for the evaluation of uridine and cytidine metabolism of tumor cell specimens.