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Biomedical subjects

E Cadman

Publications and source records attributed to E Cadman.

At least 55 records · Page 3Linked to original sources
PubMed1982

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.

Breast Neoplasms↗
PubMed1982

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.

Animals↗
PubMed1982

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.

Antimetabolites, Antineoplastic↗
PubMed1981

Use of periodate and methylamine for the quantitation of intracellular 5-fluoro-2'-deoxyuridine-5'-monophosphate by high-performance liquid chromatography.

High-performance liquid chromatography was used for the quantitation of [3H]fluorouracil and metabolites in L1210 cells with or without pre-treatment with methotrexate. Ribonucleotide pools were evaluated on a chemically bonded anion-exchange column. Deoxynucleotide pools were determined following periodate and methylamine treatment which eliminated the ribonucleotides (greater than 99.9% complete) and allowed for a rapid quantitation of the acid-soluble 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) pools. FdUMP levels as low as 10 fmol/10(6) cells were detectable by polystyrene anion-exchange chromatography. Confirmation of these FdUMP values was performed by rechromatography on a reversed-phase ion-pair column. The use of periodate and methylamine as a means for the reliable elimination of the ribonucleotides will allow accurate isolation of deoxynucleotides by high-performance liquid chromatography.

Animals↗
PubMed1981

Preliminary report on the efficacy of sequential methotrexate and 5-fluorouracil in advanced breast cancer.

Recent in vitro and animal tumor studies suggest that sequential methotrexate (MTX) and 5-fluorouracil (5-FU) may be much more tumoricidal than either drug when used alone or in conventional combinations. Seventeen evaluable female patients with advanced carcinoma of the breast were treated with MTX 200 mg/M2I.V. followed one hour later with 5-FU 600 mg/M2I.V. Leucovorin 10 mg/M2 was begun orally 24 hours later and continued every six hours for six doses. Thirteen of these patients had not responded to prior drug treatment. Of the 17 patients, 11 responded to therapy, 9 had an objective response of greater than 50% tumor regression, and 2 had a minimal response of less than 50% tumor regression. The greatest number of responses was seen in skin and soft tissue. Disease-free interval, hormonal status, and prior therapy, with the exception of MTX and 5-FU containing drug regimens, did not appear to affect response rates. Toxicity was minimal and not life threatening. Based on these results it is believed that large trials with this drug sequence are warranted.

Adult↗
PubMed1981

The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells.

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.

Animals↗
PubMed1981

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.

Animals↗
PubMed1981

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.

Animals↗
PubMed1981

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.

Adenocarcinoma↗
PubMed1980

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.

Amides↗
PubMed1980

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.

Adenocarcinoma↗
PubMed1980

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.

3-Deazauridine↗
PubMed1979

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.

Animals↗