RESUMO
Iron is a metal essential for cellular metabolism. However, excess iron available for reactions contributes to the formation of dangerous reactive oxygen species, such as the hydroxyl radical, via the Fenton reaction. Therefore, intracellular iron levels are tightly constrained by a control system of proteins. This paper contains a mathematical model, in the form of a system of five ordinary differential equations, of the core of this control system, including the labile iron pool as well as proteins that regulate uptake, storage, and export and are connected through negative feedback loops. The model is validated using data from an overexpression experiment with cultured human breast epithelial cells. The parameters in the mathematical model are not known for this particular cell culture system, so the analysis of the model was done for a generic choice of parameters. Through a mixture of analytical arguments and extensive simulations it is shown that for any choice of parameters the model reaches a unique stable steady state, thereby ruling out oscillatory behavior. It is shown furthermore that the model parameters are identifiable through suitable experiments.
Assuntos
Mama/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Modelos Biológicos , Mama/citologia , Células Cultivadas , Células Epiteliais/metabolismo , Retroalimentação Fisiológica/fisiologia , Feminino , HumanosRESUMO
We investigated whether phorbol-12-acetate-13-myristate (PMA)-activated human polymorphonuclear leukocytes (PMNs) induce base modifications in target cell DNA in vivo. Human PMNs produced 9.4 +/- 0.8 (SD) nmol of H2O2/10(6) cells during 50 min of exposure to 2 micrograms/ml PMA and 13.7 +/- 2.8 nmol/10(6) cells during exposure to PMA plus 5 mM NaN3. Neither nonstimulated PMNs, nor PMA alone, nor NaN3 alone induced base modifications in chromatin-associated DNA of human Ad293 cells above control levels, when assayed by gas chromatography/mass spectrometry with selected-ion monitoring. However, a 60-min exposure to 1.7 +/- 0.4 x 10(6) PMNs/ml in the presence of 2 micrograms/ml PMA induced a 2-3-fold increase in the level of all modified bases detected by gas chromatography/mass spectrometry with selected-ion monitoring. The guanine-derived products 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and the adenine-derived product 4,6-diamino-5-formamidopyrimidine were induced to the highest levels among those bases detected. These data demonstrate that exposure to activated PMNs causes DNA base modifications in target cells in vivo typical of those induced by hydroxyl radical attack. The induction of potentially promutagenic modified bases may contribute to the mutagenicity of activated PMNs.
Assuntos
Cromatina/metabolismo , DNA/metabolismo , Neutrófilos/fisiologia , Linhagem Celular , Humanos , Peróxido de Hidrogênio/toxicidade , Hidróxidos , Radical Hidroxila , Neutrófilos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive HCT 116 cells. Eight NQO1 cDNA clones were isolated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from HCT 116-R30A, are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC-resistant sublines showed 75-90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of cDNA fragment of nucleotide 298-617 revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO1 activity. Transcriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.
Assuntos
Neoplasias do Colo/enzimologia , DNA Complementar/genética , DNA de Neoplasias/genética , Mitomicina/metabolismo , Quinona Redutases/metabolismo , Sequência de Bases , Western Blotting , Neoplasias do Colo/genética , Resistência a Medicamentos , Escherichia coli/enzimologia , Humanos , Indofenol/análogos & derivados , Indofenol/metabolismo , Dados de Sequência Molecular , Quinona Redutases/análise , Quinona Redutases/genética , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
We investigated the mechanism of antitumor activity of the water-soluble derivative of menadione, menadione sodium bisulfite (vitamin K3), versus murine leukemia L1210. Vitamin K3, in concentrations greater than 27 microM, caused time- and concentration-dependent depletion of the acid-soluble thiol (GSH) pool. Maximal GSH depletion to 15% of control occurred at 45 microM vitamin K3. Vitamin K3-mediated GSH depletion and vitamin K3-mediated growth inhibition were abrogated by coincubation with 1 mM cysteine or 1 mM reduced glutathione but not by 1 mM ascorbic acid or 180 microM alpha-tocopherol. Low concentrations of vitamin K3 (9-27 microM) elevated both the GSH pool and the total glutathione pool, the latter to a greater degree. Vitamin K3 also caused an increased rate of superoxide anion generation by L1210, maximal at 45 microM vitamin K3 (300% of control), and a concentration-dependent depletion of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) and total nicotinamide adenine dinucleotide phosphate (NADP) pools. Forty-fifty % depletion of the NADPH pool occurred after exposure to 27 microM vitamin K3 and 100% occurred at 36 microM vitamin K3; 27 microM vitamin K3 is a nontoxic concentration of vitamin K3. Loss of NADPH and total NADP was prevented by coincubation with 1 mM cysteine but not by coincubation with ascorbic acid or alpha-tocopherol. We conclude that tumor cell growth inhibition by vitamin K3 is modulated by acid-soluble thiols and may be caused by GSH pool and/or NADPH depletion. Toleration of partial NADPH depletion by L1210 cells may indicate that a threshold level of NADPH loss of greater than 50% is necessary for toxicity. NADPH depletion may be a toxic effect common to quinone drugs. Equitoxic concentrations of vitamin K3, phylloquinone, lapachol, dichlorolapachol, and doxorubicin caused L1210 NADPH pools to deplete to 30 +/- 10 (SD), 60 +/- 10, 60 +/- 11, and 80 +/- 12% of control, respectively. In contrast, GSH depletion may not be a common mechanism of toxicity. Of these quinones, only vitamin K3 caused significant GSH depletion when studied in equitoxic concentrations.
Assuntos
Glutationa/metabolismo , Leucemia L1210/metabolismo , Vitamina K/análogos & derivados , Animais , Cisteína/farmacologia , Relação Dose-Resposta a Droga , Cinética , Leucemia L1210/patologia , Camundongos , NADP/metabolismo , Oxirredução , Superóxidos/metabolismo , Vitamina K/farmacologia , Vitamina K 3RESUMO
We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd). Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar. Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium. The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM. For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM. Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10). In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively. Incorporation of [3H]dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo. Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance [3H]dCyd incorporation in either HL-60 or K-562 cells. Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning. These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself.
Assuntos
Desoxirribonucleosídeos/farmacologia , Desoxirribonucleotídeos/metabolismo , Leucemia/patologia , Adenosina Desaminase/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , DNA/biossíntese , Desoxiadenosinas/farmacologia , Desoxicitidina/farmacologia , Desoxiguanosina/farmacologia , Humanos , Leucemia/metabolismoRESUMO
We investigated the expression of the genes for several antioxidant and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is greater than 500-fold resistant to doxorubicin by clonogenic assay. Enzyme activity determinations in the cytoplasmic compartment of MCF-7/Dox revealed a 25-fold increase in glutathione peroxidase level compared to the parent line (mean +/- SD, 10 +/- 2.8 versus 0.4 +/- 0.24 nmol/min/mg; P less than 0.005). The activity of the other major hydrogen peroxide-detoxifying enzyme, catalase, was diminished in MCF-7/Dox (2.0 +/- 0.4 versus 4.8 +/- 1.4 mumol/min/mg; P less than 0.025 compared to MCF-7). Superoxide dismutase activity did not differ between the two cell lines. The specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was 4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 +/- 45 versus 509 +/- 123 nmol/min/mg; P less than 0.005). Daunorubicinol-producing carbonyl reductase activity was equal in the two lines. Northern blot analysis demonstrated a 0.9-kilobase band of glutathione peroxidase mRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase superoxide dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When normalized to 28S RNA, no difference in the mRNA levels of catalase and superoxide dismutase in MCF-7/Dox and MCF-7 could be determined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous to the putative carbonyl reductase cDNA was also easily detectable in both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobiotic-detoxifying enzymes was decreased 2- to 4-fold in the doxorubicin-resistant cells. Southern blot analysis of PstI- and MspI-restricted genomic DNA revealed no evidence for amplification or rearrangement of the glutathione peroxidase gene. These results indicate that, in addition to the previously described overexpression of anionic glutathione S-transferase in MCF-7/Dox cells, an augmented glutathione peroxidase mRNA level is the major alteration in antioxidant and xenobiotic-detoxifying enzyme expression that could contribute to doxorubicin insensitivity in these multidrug-resistant breast cancer cells.
Assuntos
Oxirredutases do Álcool/genética , Neoplasias da Mama/genética , Doxorrubicina/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/genética , Quinona Redutases/genética , RNA Mensageiro/análise , Neoplasias da Mama/enzimologia , Resistência a Medicamentos , Glutationa Peroxidase/metabolismo , Humanos , Quinona Redutases/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
The frequency of oxidative base damage along the human p53 and PGK1 genes was determined at nucleotide resolution by cleaving DNA at oxidized bases with endonuclease III and formamidopyrimidine DNA glycosylase and then using the ligation-mediated PCR technique to map induced break frequency. Damage was induced either in vivo by exposing cultured human male fibroblasts to H2O2 or in vitro by exposing purified genomic DNA to H2O2 plus ascorbate in the presence of Cu(II), Fe(III), or Cr(VI) metal ions. All four base damage patterns from either in vivo or in vitro treatments were nearly identical in both regions of the genome. The frequency of base damage varied along the DNA, with guanine being the most commonly damaged base. In the Fe(III)-mediated in vitro reactions, single-stranded breaks were almost completely suppressed by addition of sucrose, which facilitated mapping of base damage. The in vitro base damage pattern generated by Cr(VI), ascorbate, and H2O2 was similar to that of the other metal ions, with the exception of several unique positions; these were heavily damaged only in the presence of Cr(VI). Isolated nuclei suffered little oxidative base damage in the presence of ascorbate and H2O2, and we conclude that during H2O2 in vivo treatment of cells, metal ions (or metal-like ligands) are freed from the cytoplasm to migrate into the nucleus and supply the redox cycling ligands necessary for oxidative base damage. These data simplify the complexity of H2O2-induced oxidative damage and mutagenesis studies by demonstrating the commonality of damage catalyzed by different transition metal ions and by showing that the pattern of H2O2-mediated oxidative base damage is determined almost entirely by the primary DNA sequence, with chromatin structure having a limited effect. Our data suggest a model for base damage in which DNA-metal ion binding domains can equally accommodate a variety of different metal ions and thus are a key factor in determining the local probability of DNA damage.
Assuntos
Dano ao DNA , Peróxido de Hidrogênio/farmacologia , Ácido Ascórbico/farmacologia , Cromo/farmacologia , Cobre/farmacologia , DNA/efeitos dos fármacos , DNA/genética , Fibroblastos/efeitos dos fármacos , Humanos , Ferro/farmacologia , MasculinoRESUMO
We studied the ability of the purine deoxynucleoside deoxyguanosine (dGuo) to enhance 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA of HL-60 cultured human leukemia cells. The effects of dGuo on ara-C incorporation into DNA were compared to those of thymidine (dThd), a pyrimidine deoxynucleoside known to augment ara-C effects both in vitro and in vivo. Both deoxynucleosides doubled the cells in the S phase of the cell cycle within the exposure periods (up to 48 hr) and concentrations (10 to 1000 microM) tested. Both deoxynucleosides enhanced ara-C incorporation into DNA equally. However, dThd and dGuo differed in the schedule required to achieve this effect. Simultaneous exposure of cells to ara-C and dThd increased ara-C incorporation into DNA approximately 3.5-fold. Preincubation of cells with dThd for 16 hr prior to the addition of ara-C further enhanced ara-C incorporation into DNA (to approximately 5-fold) in direct proportion to the dThd-induced increase in cells in S phase. Preincubation was essential for dGuo, since 16-hr preincubation of cells with concentrations as low as 30 microM caused augmentation of ara-C incorporation into DNA; but simultaneous exposure of cells to dGuo and ara-C caused no augmentation of ara-C incorporation into DNA. The augmentation of ara-C incorporation into DNA caused by preincubation of the cells with dGuo results from a number of factors, including the cytokinetic effect of increasing the percentage of cells in S phase and the reduction of intracellular dCTP pools. Maximal dGuo enhancement of ara-C incorporation into DNA (approximately 5-fold) required greater than 100 microM dGuo, 16-hr preincubation with dGuo, and final incubation of cells with ara-C after removal of dGuo. We explain this further augmentation of ara-C incorporation into DNA caused by the removal of dGuo prior to adding ara-C by our observed inhibition of ara-C phosphorylation by dGuo concentrations greater than 100 microM.
Assuntos
Citarabina/metabolismo , Desoxiguanosina/farmacologia , Linhagem Celular , Células Clonais , Citarabina/toxicidade , Replicação do DNA/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Leucemia Linfoide/metabolismo , Fosforilação , Timidina/metabolismo , Timidina/farmacologia , TrítioRESUMO
We compared the relative susceptibility of HL-60, a human acute progranulocytic leukemia cell line, and the normal human myeloid progenitor cell, the colony-forming unit in culture, to growth inhibition by thymidine. Normal human myeloid colony formation was more sensitive to thymidine than was HL-60 colony formation, the former being inhibited by greater than or equal to 0.5 mM thymidine and the latter by greater than or equal to 5.0 mM. Colony inhibition by thymidine was irreversible in both cases after a seven-day incubation of the colony-forming unit in culture in liquid medium enriched with thymidine and subsequent replating in thymidine-free soft agar. Thymidine-induced inhibition of HL-60 cloning could be partially prevented by deoxycytidine, whereas normal myeloid cloning could not, suggesting that high-concentration thymidine may affect processes necessary for cloning in addition to deoxyribonucleotide synthesis. HL-60 growth in liquid suspension culture could be suppressed transiently by 1.0 mM thymidine, suppressed totally by greater than or equal to 5.0 mM thymidine, and rescued completely by concomitant addition of deoxycytidine. The development of resistance to 1.0 mM thymidine could not be accounted for by reduction in thymidine pool size or by reduction in thymidine kinase activity. Replating of HL-60 cells growing in the presence of 1.0 mM thymidine in liquid medium in thymidine-free soft agar produced significant colony inhibition, also suggesting that thymidine affects more than just deoxyribonucleotide synthesis or that the clonogenic HL-60 cell presents a distinct subpopulation with more sensitive deoxyribonucleotide-synthetic control mechanisms.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mieloide/patologia , Timidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Desoxicitidina/farmacologia , Humanos , Fatores de TempoRESUMO
Twenty-seven patients with advanced cancer were entered in a phase I study of bolus i.v. 5-fluorouracil at a dose of 370 mg/m2/day for 5 days combined with a continuous i.v. infusion of (6S)-folinic acid for 5.5 days, starting 24 h in advance of the first 5-fluorouracil dose. The dose of (6S)-folinic acid was escalated in cohorts of patients from 250 mg/m2/day to a maximum of 1000 mg/m2/day. The pharmacokinetics of (6S)-folinic acid were studied in the 3 patients given 250 mg/m2/day and in 6 patients given 1000 mg/m2/day. The mean steady-state plasma concentrations of (6S)-folinic acid and its principal metabolite (6S)-5-methyltetrahydrofolate at the 250 mg/m2/day dose were 2.7 and 5.1 microM, respectively. Both concentrations were comparable to the concentrations produced when (6S)-folinic acid was administered as half of a (6R,S)-folinic acid mixture (E. M. Newman et al., Cancer Res., 49:5755-5760, 1989). At the 1000 mg/m2/day dose of (6S)-folinic acid, the concentration of (6S)-folinic acid was 15.3 microM, more than the 4-fold increase predicted by linear pharmacokinetics, while the concentration of (6S)-5-methyltetrahydrofolate was only 16.5 microM. The change in the ratio of the parent compound to its metabolite was accounted for by a decrease in the nonrenal clearance of (6S)-folinic acid, probably indicating saturation of its metabolism. The toxicities observed in this phase I trial, including stomatitis, diarrhea, neutropenia, and anemia, did not differ in nature or severity from those produced by 5-fluorouracil and (6R,S)-folinic acid when administered on the same schedule. Finally, the degree of toxicity did not appear to depend on the dose of (6S)-folinic acid over the range of doses tested.
Assuntos
Fluoruracila/uso terapêutico , Leucovorina/uso terapêutico , Neoplasias/metabolismo , Adulto , Idoso , Esquema de Medicação , Avaliação de Medicamentos , Sinergismo Farmacológico , Feminino , Fluoruracila/farmacocinética , Humanos , Infusões Intravenosas , Leucovorina/farmacocinética , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológicoRESUMO
We have established a variant of the human breast cancer cell line MCF7, designated MCF7/H2O2, which is 5-fold resistant to H2O2 by clonogenic assay. The specific activity of the H2O2 disposal enzyme catalase was elevated 3-fold in MCF7/H2O2; activities of other antioxidant enzymes, including glutathione peroxidase and superoxide dismutase, were not increased. The steady-state level of catalase mRNA was only slightly elevated (approx. 1.6-fold) in MCF7/H2O2 cells; however, degradation of catalase mRNA was markedly retarded in MCF-7/H2O2 compared to MCF-7 (82% of catalase mRNA remained 24 h after inhibition of RNA synthesis by actinomycin D in MCF-7/H2O2 vs. 32% in MCF7). The degradation rates of superoxide dismutase mRNA and 28 S ribosomal RNA were not reduced in MCF-7/H2O2; however, the rate of degradation of another mRNA species, beta-actin, was also significantly decreased. These data suggest that resistance to H2O2 in MCF7/H2O2 cells is mediated by elevated catalase activity which can be explained by stabilization of certain mRNA species, including catalase mRNA.
Assuntos
Neoplasias da Mama/enzimologia , Catalase/genética , Peróxido de Hidrogênio/farmacologia , RNA Mensageiro/metabolismo , Actinas/genética , Catalase/metabolismo , Dactinomicina/farmacologia , Resistência a Medicamentos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Meia-Vida , Humanos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: The use of leucovorin (LV) to modulate fluorouracil (FU)-mediated inhibition of thymidylate synthase has been shown both in vitro and in vivo to improve the antitumor activity of this drug. Based on our previous demonstration that this combination was active in heavily pretreated patients with prior FU exposure, we performed a phase II study of FU and high-dose intravenous calcium LV in patients with advanced breast cancer who had been exposed to no more than one prior chemotherapy regimen. PATIENTS AND METHODS: Fifty-one female patients with metastatic breast cancer were entered onto this trial. Patients with metastatic disease limited to soft tissue, lymph nodes, skin, and pulmonary nodules were allowed no prior chemotherapy for advanced disease. Those with metastases in the liver or a lymphangitic pattern on chest x-ray were allowed either a single prior regimen for advanced disease or no therapy for metastatic disease if less than 1 year had elapsed since the completion of adjuvant chemotherapy. FU was given daily for 5 days at 400 mg/m2/d with calcium LV, 500 mg/m2/d, beginning 24 hours before and continuing 12 hours after the first and last FU doses, respectively. RESULTS: The overall objective response rate among 45 eligible patients was 36% (95% confidence interval, 22% to 51%). Fourteen of 31 patients in the soft tissue category responded (45%), and two of 14 in the visceral category experienced an objective response (14%). The median response duration was 5 months. Toxicities were moderate leukopenia and mucositis. CONCLUSIONS: FU plus LV is an active first-line regimen with antitumor efficacy comparable to that of the anthracyclines, which warrants further exploration in combination with other agents active in advanced breast cancer. FU plus LV in this schedule is also an excellent alternative for patients with medical contraindications to more intensive combination chemotherapy regimens.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/patologia , Esquema de Medicação , Feminino , Fluoruracila/administração & dosagem , Humanos , Infusões Intravenosas , Leucovorina/administração & dosagem , Pessoa de Meia-Idade , Metástase Neoplásica , Resultado do TratamentoRESUMO
PURPOSE: To evaluate the antitumor activity of combination cisplatin (CDDP) and alpha-interferon (alpha-IFN) in advanced, measurable metastatic melanoma. PATIENTS AND METHODS: Adult patients with metastatic melanoma were required to have bidimensionally measurable lesions and a Karnofsky performance status > or = 60%. Serum creatinine < or = to 1.5 mg/dL, creatinine clearance > or = 60 mL/min, adequate organ and bone marrow function, and radiologic proof of the absence of brain metastases were required. CDDP 40 mg/m2 intravenously (IV) on day 1 and day 8, and alpha-IFN 3 million units/m2 subcutaneously on days 1 to 5 and 8 to 12 were administered every 3 to 4 weeks. RESULTS: Forty-two patients were entered onto this phase II trial and were assessable for response and toxicity. Three patients achieved complete responses (CRs) that lasted 31+, 5, and 8+ months. Seven patients had partial responses (PRs) and a median response duration of 4.4 months. The overall objective response rate was 24% (95% confidence interval, 12% to 39%). Toxicities were mild. Only 11% of the courses required dose reduction of alpha-IFN, and three of 128 courses required CDDP dose reduction for reversible nephrotoxicity. CONCLUSION: The combination of moderate-dose CDDP and alpha-IFN as administered in this schedule is well tolerated and possesses encouraging activity in metastatic melanoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Melanoma/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cisplatino/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Interferon-alfa/administração & dosagem , Masculino , Melanoma/secundário , Pessoa de Meia-IdadeRESUMO
We investigated whether human circulating myeloid progenitor cells (CFU(c)) exhibit diurnal variation. Peripheral venous blood samples were drawn every 3 h from each of the nine healthy male volunteers. Blood mononuclear cells were harvested by the Ficoll-Hypaque method. CFU(c) content of blood was determined by the soft agar cloning assay. Human placental conditioned medium (HPCM) was used as colony stimulating factor. We found a 24 h cyclic variation of colonies/10(5) mononuclear cells plated with a maximum at 9 a.m. (P < 0.01). Analysis of the colonies by in situ staining indicated that this variation could be attributed almost entirely to colonies composed of eosinophilic cells (representative of CFU(eos)). Neutrophil/macrophage colonies (representative of CFU(G,M)) exhibited a trend toward a 9 a.m. maximum, but this variation was not statistically significant. We conclude that time of blood collection should be standardized in experiments measuring human peripheral blood CFU(c).
Assuntos
Ritmo Circadiano/fisiologia , Macrófagos/fisiologia , Células Progenitoras Mieloides/fisiologia , Neutrófilos/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Macrófagos/citologia , Masculino , Células Progenitoras Mieloides/citologia , Neutrófilos/citologiaRESUMO
Interleukin-2 (IL-2) and gamma interferon (gamma-IFN) may be synergistic in inducing cell-mediated antitumor cytotoxicity. In order to determine the dose-limiting toxicities and define a maximum tolerated dose of these two agents in combination, we performed a Phase I clinical trial of intravenous IL-2 plus intramuscular gamma-IFN. Patients received both agents on a thrice-weekly schedule consisting of 4 weeks of treatment followed by 2 weeks of rest. Twenty-five patients were treated and received gamma-IFN doses between 0.05-0.25 mg/m2 (1-4 x 10(6) U/m2) with IL-2 doses from 0.33 mg/m2 to 2.33 mg/m2 (6-42 x 10(6) IU/m2). Two patients had partial responses of melanoma and adenocarcinoma of the lung lasting greater than 11 and 8 months, respectively. The toxicities of the combination were those expected from each agent, with no unusual effects, no irreversible organ toxicities, and no patient deaths. The doses recommended for outpatient administration on this schedule are IL-2, 2.0 mg/m2 plus gamma-IFN, 0.25 mg/m2, a dose combination that is unassociated with significant organ toxicity.
Assuntos
Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/terapia , Melanoma/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Avaliação de Medicamentos , Feminino , Humanos , Interferon gama/administração & dosagem , Interleucina-2/administração & dosagem , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologiaRESUMO
Cyclo(His-Pro) is believed to be a metabolite of TRH. A specific antiserum directed against cyclo(His-Pro) was used to detect immunoreactive material in human serum. Gel filtration column chromatography was used to establish that cyclo(His-Pro)-like immunoreactivity was found not only in free form established to be identical to cyclo(His-Pro) by high pressure liquid chromatography analysis on reverse phase and cation exchange columns, but also in a fraction of about 100,000 mol wt. Free cyclo(His-Pro) was released by heating column fractions of 70,000 mol wt that were not immunoreactive before heating. A procedure for estimation of the content of free cyclo(His-Pro) in human serum was developed. Normal levels of cyclo(His-Pro) determined by this procedure were in the range of 11-33 pmol/ml. The level of cyclo(His-Pro) in sera of individuals with hypothyroidism, hyperthyroidism, or alcoholic cirrhosis was in the normal range, while patients with renal failure had approximately 3-fold elevated levels of the peptide. Analysis of human sera drawn at 3-h intervals over a 24-h period suggested a circadian rhythm of cyclo(His-Pro) levels.
Assuntos
Peptídeos Cíclicos/sangue , Piperazinas/sangue , Hormônio Liberador de Tireotropina/metabolismo , Animais , Química Encefálica , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Ritmo Circadiano , Temperatura Alta , Humanos , Hipertireoidismo/sangue , Hipotireoidismo/sangue , Nefropatias/sangue , Cirrose Hepática Alcoólica/sangue , Masculino , Peptídeos Cíclicos/análise , Piperazinas/análise , Radioimunoensaio , Ratos , Valores de ReferênciaRESUMO
The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.
Assuntos
Ácido Ascórbico/farmacologia , Cobre/metabolismo , Cobre/farmacologia , Dano ao DNA , DNA/metabolismo , Peróxido de Hidrogênio/farmacologia , DNA/efeitos dos fármacos , Diálise , Ácido Edético/farmacologia , Eletroforese em Gel de Ágar , Fibroblastos , Glioxal , Humanos , Peróxido de Hidrogênio/metabolismo , Cinética , MasculinoRESUMO
Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydrio-2,4-dioxopyrimidin-5-yl)-methyl]- L-tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.
Assuntos
Reagentes de Ligações Cruzadas , DNA/metabolismo , Proteínas/metabolismo , Animais , Ácido Ascórbico/farmacologia , Linhagem Celular , Cromatina/química , Cobre/farmacologia , Meios de Cultura , Dano ao DNA/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Compostos Ferrosos/farmacologia , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Hibridomas , Peróxido de Hidrogênio/farmacologia , Camundongos , Timina/química , Tirosina/químicaRESUMO
We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.
Assuntos
Citarabina/farmacologia , Desoxiguanosina/farmacologia , Leucemia/metabolismo , Linhagem Celular , Citarabina/metabolismo , Desoxiguanosina/metabolismo , Sinergismo Farmacológico , Guanosina Trifosfato/metabolismo , Humanos , Leucemia/tratamento farmacológico , FosforilaçãoRESUMO
We developed a method to map oxidative-induced DNA damage at the nucleotide level using ligation-mediated polymerase chain reaction (LMPCR) technology. In vivo and in vitro DNA base modification patterns inflicted by reactive oxygen species (ROS) in the human P53 and PGK1 gene were nearly identical in vitro and in vivo. In human male fibroblasts, these patterns are independent of the transition metal used (Cu (II), Fe(II), or Cr(VI). Therefore, local probability of H2O2-mediated DNA base damage is determined primarily by DNA sequence. Moreover, in cells undergoing severe oxidative stress, extranuclear sites contribute metals that enhance nuclear DNA damage. The role of the base excision repair pathway in human cells responsible for the repair of the majority of ROS base damage is also discussed.