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Biomedical subjects

W Plunkett

Publications and source records attributed to W Plunkett.

At least 55 records · Page 3Linked to original sources
PubMed1996

Inhibition of the 3' --> 5' exonuclease of human DNA polymerase epsilon by fludarabine-terminated DNA.

Incorporation of the anticancer drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate; F-ara-AMP) into the 3'-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3' --> 5'-exonuclease activity of DNA polymerase epsilon from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase epsilon was unable to effectively remove F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase epsilon has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase epsilon, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase epsilon to the reaction. Furthermore, the addition of DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3' --> 5' exonuclease of human DNA polymerase epsilon can remove 3'-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."

Base Sequence↗
PubMed1996

Deoxynucleotide pool depletion and sustained inhibition of ribonucleotide reductase and DNA synthesis after treatment of human lymphoblastoid cells with 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine.

The action of the new adenine nucleoside analogue 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (Cl-F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro assay systems with purified DNA polymerases. [3H]Thymidine incorporation into DNA in human lymphoblastoid CEM cells was inhibited by Cl-F-ara-A in a concentration-dependent manner that was not reversed 72 h after removal of Cl-F-ara-A from the medium. Deoxynucleotide pools were depressed after incubation of Cl-F-ara-A for 3 h and only partially recovered following washing the cells into drug-free medium. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the dATP, dGTP, and dTTP pools. This was in concordance with the results of in situ assays of ribonucleotide reductase, which demonstrated profound inhibition of CDP reduction in cells incubated with Cl-F-ara-A; reduction of ADP, GDP, and UDP were affected to lesser extents. Reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and inhibition of the enzyme was saturated when cellular Cl-F-ara-ATP reached 25 microM. In vitro DNA primer extension assays indicated that Cl-F-ara-ATP competed with dATP for incorporation into A sites of the extending DNA strand catalyzed by both human DNA polymerases alpha and epsilon. The incorporation of Cl-F-ara-AMP into DNA inhibited DNA strand elongation; the most pronounced effect was observed at Cl-F-ara-ATP:dATP values >1. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular concentration ratio of dATP to Cl-F-ara-ATP, which favors analogue incorporation into DNA, an action that has been strongly correlated with loss of viability.

Adenine Nucleotides↗
PubMed1996

Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.

Therapeutic agents used in the treatment of chronic lymphocytic leukemia (CLL) are capable of inducing apoptosis in some (but not all) patient isolates. It is not yet clear whether cells that are resistant to one agent will also be resistant to others, and the mechanisms contributing to differential apoptosis sensitivity are not known. Here we report that glucocorticoid hormone and a clinically relevant chemotherapy combination (fludarabine plus mitoxantrone) fail to induce apoptosis in four of 24 CLL patient isolates. Apoptosis resistance was associated with elevated BCL-2 and BAX expression. Interestingly, incubation in vitro led to down-regulation of BCL-2 expression in both apoptosis-sensitive and apoptosis-resistant cells, whereas parallel down-regulation of BAX occurred only in the resistant samples. Evaluation of nuclear endonuclease content indicated that all of the apoptosis-sensitive samples contained appreciable levels of activity, whereas the endonuclease was not detected in the four populations of resistant cells. Our results indicate that nuclear endonuclease activity represents an excellent prognostic indicator of CLL apoptosis sensitivity that may be controlled by differential BCL-2 family polypeptide expression and signals from the in vivo microenvironment.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions.

The effectiveness of arabinosylcytosine (ara-C) for the treatment of acute myelogenous leukemia (AML) depends on the formation of its active metabolite, the triphosphate of ara-C (ara-CTP). Using biochemical modulation strategies to increase the accumulation of ara-CTP in leukemia blasts, a clinical protocol was designed combining 2-chlorodeoxyadenosine (CdA), an inhibitor of ribonucleotide reductase, and ara-C for adults with AML. The protocol stipulated an infusion of 1 g/m2 of ara-C over 2 hours on day 1. A continuous infusion of CdA (12 mg/m2/d) begun 24 hours later and continued for 5 days. Identical doses of ara-C were administered on days 3, 4, 5, and 6. Pharmacokinetic and pharmacodynamic interactions between CdA and ara-C during therapy were investigated. To complement these studies, molecular actions of the triphosphate of ara-C and CdA on DNA extension by human DNA polymerase alpha in an in vitro model system was conducted. In the circulating leukemia blasts of 7 of the 9 patients studied, ara-CTP pharmacokinetics showed a median 40% increase in the rate of ara-CTP accumulation after 24 hours of CdA infusion. The ex vivo effect of CdA on accumulation of ara-CTP in AML blasts was similar to that during therapy except that the enhancement was less. The DNA synthetic capacity of the circulating blasts was inhibited to a greater extent by administration of CdA and ara-C in combination than by either one alone. Additionally the lowered level of DNA synthesis was maintained until the next infusion of ara-C. Endogenous levels of deoxynucleotides increased 24 hours after ara-C infusion. Administration of CdA in general lowered the concentrations of all dNTPs. DNA pol alpha incorporated CdATP and ara-CTP with high affinity in a DNA primer extending over an oligonucleotide template of defined sequence. Human DNA polymerase alpha extended DNA primers terminated by CdA monophosphate (CdAMP) at its 3'-end by incorporating ara-C monophosphate (ara-CMP). The tandem incorporation of CdAMP and ara-CMP resulted in nearly complete inhibition of DNA primer extension. The insertion of two analogs in sequence, inhibition of ribonucleotide reductase, and the metabolic potentiation of ara-CTP by CdA infusion may be responsible for sustained inhibition of DNA synthesis in the circulating leukemia blasts during therapy with this combination regimen.

Adenosine Triphosphate↗
PubMed1996

Natural killer cell activity in chronic lymphocytic leukemia patients treated with fludarabine.

Fludarabine, the 5'-monophosphate of 9-beta-D-arabinofuranosyl-2- fluoroadenine (FaraAMP), is effective in the treatment of chronic lymphocytic leukemia (CLL) and has been demonstrated to increase natural killer (NK) cell lytic activity (NKa) in humans and mice. To determine the effect of FaraAMP on NK cells in CLL, we analyzed NKa toward K562 targets after in vitro incubation with FaraAMP and after in vivo exposure to fludarabine. Pretreatment analysis of peripheral blood from 12 CLL patients (9 untreated) revealed: median number of NK cells 500/microliter (range 290-1160); median NKa lytic unit30/10(6) cells (range 5-80). These results were similar to those from healthy adult donors. After exposure to 3, 30 or 300 microM FaraAMP, the median maximum stimulation index (NKa FaraAMP/NKa) was 1.2 (range 0.9-1.5), within the range observed in normal adults. FaraA also stimulated NKa in vitro toward autologous CLL cells in two of five patients as measured by a dye-exclusion assay. In three patients following three or more treatment courses of fludarabine (30 mg/m2 per day for 5 days) the NK cell number and NKa were maintained near pretreatment values. Phenotypic analysis of the peripheral mononuclear cells in 34 consecutive CLL patients revealed a marked reduction in CD5/CD20 and CD4 cell numbers after three courses of fludarabine with less effect on CD8 and CD56 cells. These results indicate that fludarabine spares NK cells and may stimulate NKa in some CLL patients.

Adult↗
PubMed1996

The effect of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 1-beta-D-arabinofuranosylcytosine on the cell cycle phase distribution, topoisomerase II level, mitoxantrone cytotoxicity, and DNA strand break production in K562 human leukemia cells.

Antimetabolites and topoisomerase (topo) II-reactive drugs are frequently combined in the therapy of acute leukemia. The two types of agents are thought to be synergistic in their actions against malignant blasts but the mechanism for this synergism is incompletely described. This study sought to determine whether the combination of two rather than one anti-metabolite with the topo II-reactive intercalator mitoxantrone would be greater than the effect of the single antimetabolite ara-C on mitoxantrone's cytotoxic actions. We also aimed to determine a mechanism for synergism should it occur. The model system used was K562 human leukemia cells. The second anti-metabolite selected was F-ara-A, the active form of fludarabine. The resultant combination (F-ara-A, ara-C, and a topo II reactive drug) is one currently being tested against acute myelogenous leukemia in clinical trials. F-ara-A itself had little effect on the cytotoxicity or the topo II-mediated DNA cleaving actions of mitoxantrone, while ara-C potentiated these actions as it does those of other topo II-reactive drugs. Surprisingly F-ara-A enhanced the actions of ara-C on mitoxantrone-associated cytotoxicity by at least an order of magnitude. The effect of the addition of F-ara-A to ara-C on mitoxantrone-induced DNA cleavage was considerably smaller, but present. Antimetabolite treatment did not increase the amount of topo II within cells measured directly by immunoblotting or indirectly by quantifying the maximum number of topo II-DNA complexes stabilized by mitoxantrone. Rather, the anti-metabolites altered the distribution of the cells in the cell cycle. Antimetabolite treatment caused a large increase in S-phase cells, a phase in which cells are more sensitive to topo II-reactive drugs than the associated topo II-mediated DNA cleavage would predict. Therefore, it is likely that this shift in the distribution of the cells within the cell cycle accounts for both the enhanced cytotoxicity of mitoxantrone in antimetabolite pretreated cells and the discrepancy between the magnitude of antimetabolite action on topo II-mediated DNA cleavage.

Antineoplastic Agents↗
PubMed1996

Bcl-2 expression in chronic lymphocytic leukemia and its correlation with the induction of apoptosis and clinical outcome.

Transcriptional deregulation of the Bcl-2 gene has been demonstrated to extend cell viability via an inhibition of apoptotic cell death. Chronic lymphocytic leukemia (CLL) cells are inherently susceptible to apoptosis during short-term culture. Because increased expression of the Bcl-2 gene has been reported in CLL, we sought to correlate Bcl-2 protein expression with the in vitro propensity towards apoptosis and also clinical outcome. Immunoblot analysis of Bcl-2 protein revealed interpatient variability with nine of 42 (21%) cases demonstrating similar or greater expression than a t(14;18) containing lymphoma cell line and 18 of 42 (43%) cases demonstrating a level of expression similar to or less than that seen in normal peripheral blood lymphocytes. Bcl-2 expression did not correlate with clinical features, or with apoptosis, as measured by an in vitro DNA fragmentation assay. However, analysis of survival in the 33 untreated patients revealed significant differences based on the level of Bcl-2 expression, with higher expression being an adverse feature (P<0.02). This data suggests that Bcl-2 is important in the pathogenesis and progression of CLL and that quantitation of Bcl-2 protein may provide useful prognostic information.

Apoptosis↗
PubMed1996

Clinical and laboratory studies of 2-chlorodeoxyadenosine +/- cytosine arabinoside for relapsed or refractory acute myelogenous leukemia in adults.

Previous studies in pediatric patients with acute myelogenous leukemia (AML) have suggested that 2-chlorodeoxyadenosine (2CdA) is an effective therapeutic agent. Santana et al (J Clin Oncol 1992; 10: 364-370) reported a CR rate of 8/17 (95% Cl 23-72%) in children with relapsed AML and a median first CR of 21 months. The activity of 2CdA in adults with relapsed or refractory leukemia was therefore investigated in a phase I study. In the phase II study, based on biochemical modulation rationale, 2CdA was combined with Ara-C for adults with relapsed AML to test the effectiveness of this combination therapy. In the phase I study 27 patients (25 AML and two MDS) with a median first CR duration of 21 weeks, received 2CdA at doses ranging from 5 to 13 mg/m2/day by continuous infusion (CI) for 7 days. In vitro and ex vivo pharmacologic studies performed to determine the effect of pretreatment with 2CdA on Ara-CTP accumulation in leukemic blasts demonstrated a 50-65% increase in the rate of Ara-CTP accumulation. Based on this biochemical modulation, 2CdA (12 mg/m2/day x 5 days by CI) was combined with Ara-C (1 g/m2/day over 2 h) in a phase II study. Seventeen patients (15 AML, two MDS) with relapsed AML (median 1st CR of 19 weeks) were treated. In the phase I study two patients died before the day 14 marrow (ED). Marrow hypoplasia developed in 16 of the remaining 25. Leukemic regrowth occurred in nine after a median hypoplastic period of 2 weeks (range 1-3 weeks). The other seven patients died with aplastic marrows, median duration of hypoplasia was 2 weeks, range 1-4 weeks. None achieved CR and the median survival was 10.5 weeks. Toxicity generally was mild except for three late occurring cases of grade III or IV renal dysfunction and two cases of tumor lysis syndrome. The MTD was 10.8 mg/m2/day x 7 days. In the phase II study two patients, both with AML, achieved CR (95% CI 1-33%). In both cases leukemia relapsed after 10 weeks and 17 weeks. There was one ED. Most (11/16) cleared their marrow although leukemic infiltrate regrew in six cases. Toxicity was generally mild, with two episodes of grade 2 GI bleeding, one episode of severe renal dysfunction and one case of grade 2 CNS toxicity. We conclude that as a single agent 2CdA at the MTD is a cytoreductive agent but is not sufficient to achieve CR in adults with relapsed AML. While combination of Ara-C with 2CdA increases the Ara-CTP uptake in these heavily treated patients this regimen does not appear to be an improvement over existing modalities.

Adult↗
PubMed1996

Gemcitabine: preclinical pharmacology and mechanisms of action.

Gemcitabine is a nucleoside analog which exhibits metabolic characteristics that distinguish it from related compounds and may explain its activity in solid tumors. The active nucleotide forms are effectively accumulated to high concentrations in cells. This is due to both efficient phosphorylation and relatively slow elimination. The diphosphate is a potent inhibitor of ribonucleotide reductase, an action that reduces deoxynucleotide pools. Decreased cellular concentrations of deoxycytidine triphosphate permit more rapid phosphorylation of gemcitabine and decreases the metabolic clearance of gemcitabine nucleotides by deoxycytidine monophosphate deaminase. Most importantly, the ratio of the cellular concentrations of gemcitabine triphosphate to deoxycytidine triphosphate increases, favoring analog incorporation into DNA, which is strongly associated with loss of viability.

Animals↗
PubMed1996

Improvement in the therapeutic ratio of radiotherapy for a murine sarcoma by indomethacin plus fludarabine.

Fludarabine, an effective repair inhibitor of radiation-induced chromosome breaks, and indomethacin, an inhibitor of prostaglandin synthesis, were shown previously to improve the therapeutic ratio of radiotherapy for murine tumors. The purpose of this study was to determine whether the combination of these two radiosensitizers with different mechanisms of action could further increase the therapeutic ratio of radiotherapy in an FSA mouse sarcoma after single and fractionated irradiation. The effect of the combined treatment on tumors was assessed by the local tumor control assay (TCD50) in mice bearing an FSA sarcoma in the leg. The effect of the combination on normal tissues was assessed by skin desquamation, hair loss and leg contracture in the legs of non-tumor-bearing mice. For the TCD50 assay, after single irradiation, the radiation dose modification factor (DMF) reached 1.2 for both indomethacin (35 micrograms/ml in the drinking water for 10 days) and fludarabine (800 mg/kg intraperitoneally 3 h prior to irradiation). For both drugs combined, the DMF increased to 1.7. No significant increase in normal tissue toxicity was observed with any of the combinations. After fractionated irradiation (16 fractions over 4 days), the DMFs for local tumor control reached 1.3 for indomethacin and 1.8 for fludarabine darabine (400 mg/kg every day for 4 days). The combination of both drugs produced a DMF of 2.0. None of the combinations altered the effects of radiation on skin desquamation, hair loss or leg contracture significantly. The present study suggests that the therapeutic ratio of radiotherapy for a murine sarcoma can be improved by the combination of indomethacin and fludarabine, two agents differing in their mechanisms of radiopotentiation.

Animals↗
PubMed1996

Pharmacology of purine nucleoside analogues.

Three new purine nucleoside analogues, pentostatin, cladribine and fludarabine, have demonstrated remarkable clinical activity in a variety of hematologic malignancies. Although they share structural similarities, their plasma pharmacology, metabolism, and mechanisms of action differ qualitatively and quantitatively. The plasma pharmacokinetics and the cellular pharmacodynamics are reviewed to provide a basis for dose schedules and rationales for combinations with other anticancer drugs.

Animals↗
PubMed1996

Clinical experience with fludarabine in hemato-oncology.

Fludarabine monophosphate (Fludara) is a purine analogue which entered clinical trials in 1982. Although inactive in solid tumors, Fludara has marked activity in indolent lymphoproliferative disorders. The exact mechanism of action of Fludara is uncertain. Fludara has been established as the most active single agent in chronic lymphocytic leukemia (CLL) in single arm and comparative clinical trials. The activity has been demonstrated in both previously treated and initially treated patients. Marked activity has been noted in patients with low grade lymphoma, in particular, those with a follicular morphology and in Waldenstrom's macroglobulinemia. Combinations of fludarabine with alkylating agents, anthracyclines, and anthraquinones have led to clinically useful combination approaches. The ability of fludarabine to modulate the levels of the triphosphate form of cytosine arabinoside (ara-C) in acute leukemia cells has led to the development of combinations of fludarabine and ara-C. These combinations have demonstrated marked activity in treatment of relapsed and previously untreated patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). The ability to modulate the activity of pyrimidines and to inhibit repair of DNA damage caused by alkylating agents, anthracyclines, and other DNA active drugs suggest that the future of fludarabine will be in combination approaches to modulate the activity of other agents. These activities may extend its role to use in solid tumors.

Antineoplastic Agents↗
PubMed1996

Fludarabine-mediated inhibition of nucleotide excision repair induces apoptosis in quiescent human lymphocytes.

Incorporation of fludarabine, 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A), into replicating DNA inhibits further chain elongation and is the critical event in F-ara-A-mediated cytotoxicity. We have used the normal cellular process of nucleotide excision repair to create an opportunity for F-ara-A incorporation into the DNA of noncycling cells. Irradiation of quiescent lymphocytes with UV light (254 nm, 0. 5-30 J/m2) in the presence of [3H]F-ara-A produced a dose-dependent increase in F-ara-A monophosphate incorporation into DNA that reflected a 60-70% inhibition of DNA repair at 2 h. Lymphocytes pretreated with 3 micrometer F-ara-A for 2 h before irradiation with 0.5 or 2.0 J/m2 were incubated for 24 h in the presence or absence of F-ara-A. Morphological features of apoptosis and DNA cleavage into high molecular weight fragments were increased in cells treated with UV plus F-ara-A compared to those treated with UV or F-ara-A alone. FACScan analysis confirmed the morphological and biochemical results. A 2-h pulse of F-ara-A produced intracellular F-ara-ATP levels of 40 micrometer, and removal of F-ara-A from the media resulted in a monophasic elimination (r2 = 0.88) in F-ara-ATP levels with a half-life of 5.6 h. Lymphocytes undergoing apoptosis demonstrated a G0 DNA content, indicating that entry into the cell cycle was not required. This study demonstrates that F-ara-AMP is incorporated into DNA during UV-induced repair in quiescent lymphocytes and that this is associated with the inhibition of ongoing DNA repair and an increased incidence of apoptosis. Combinational therapies involving fludarabine with agents and modalities that initiate DNA repair may have clinical relevance in the treatment of human malignancies.

Antineoplastic Agents↗
PubMed1996

Influence of fludarabine on pharmacokinetics and pharmacodynamics of cytarabine: implications for a continuous infusion schedule.

Arabinosylcytosine (ara-C) is a cytotoxic agent with major activity against acute leukemias. To exert this effect, it must first be phosphorylated to its active 5'-triphosphate, ara-CTP, which is incorporated into DNA. Our previous studies demonstrated that preincubation with arabinosyl-2-fluoroadenine (F-ara-A) increased the rate of ara-CTP accumulation in leukemia cells when incubated with 10 microM ara-C. Such concentrations of ara-C are readily obtained during intermittent bolus infusions of ara-C, and clinical trials were conducted using fludarabine in combination with 2-h infusions of intermediate-dose ara-C. During continuous infusion of ara-C, however, serum ara-C levels are <10 microM. Because the effectiveness of ara-C depends on the levels of intracellular ara-CTP and its incorporation into DNA, we sought to investigate the influence of fludarabine on pharmacodynamics of ara-C at concentrations of ara-C achieved during continuous infusion. Using the K562 human leukemic cell line, we established that incubation with 30 microM F-ara-A was able to modulate intracellular dNTP pools and achieve maximum enhancement of ara-CTP levels at all concentrations of ara-C tested (0.3-10.0 microM). The relative enhancement of ara-CTP concentrations ranged from 2.2- to 2.8-fold. Combination of F-ara-A with 1.0 and 3.0 microM ara-C also increased the incorporation of ara-CTP into DNA. To model the influence of F-ara-A on continuous infusion ara-C, cells were incubated with 1 microM ara-C alone or in combination with F-ara-A. The F-ara-A-incubated cells accumulated effective intracellular concentrations of F-ara-ATP, which resulted in greatly increased intracellular ara-CTP levels. These studies demonstrate the capacity of clinically attainable concentrations of F-ara-ATP to enhance the formation of ara-CTP at concentrations of ara-C that are achieved during a continuous infusion schedule. Given the important role intracellular ara-CTP concentrations and ara-CMP incorporation into DNA have on the ultimate cytotoxic capacity of ara-C against acute myelogenous leukemia blasts, these studies suggest a promising pharmacological model for improving the efficacy of the continuous infusion ara-C regimen.

Antimetabolites, Antineoplastic↗
PubMed1995

Biochemical pharmacology of penclomedine (NSC-338720).

Penclomedine (PEN) is a synthetic pyridine derivative that has been selected for clinical development based on its activity against human and mouse breast tumors implanted in mice. Its mechanism of action was unclear, and we were interested in determining its mechanism of cytotoxicity in vitro and in vivo. We found chromosome breaks, gaps, and exchanges in P388 ascites cells from BD2F1 mice treated with 200 mg/kg PEN. Maximal observed damage occurred 24 hr after drug administration. Alkaline elution indicated only limited DNA strand breaks and interstrand cross-linking. In vitro, PEN (75 micrograms/mL) inhibited RNA and DNA syntheses almost completely. In addition, incubation of [14C]PEN with rat liver S-9 fraction in the presence of calf thymus DNA resulted in the stable transfer of radioactivity to DNA. Addition of butylated hydroxytoluene, a free radical scavenger, to the incubation mixture inhibited the binding of drug to DNA, implicating free radicals as the ultimate reactive species. These data suggest that PEN can be metabolized to free radical, DNA-reactive products, and that its cytotoxicity is due to chromosomal damage produced by monofunctional alkylation. As an alternate mechanism, the ability of PEN to inhibit cellular dihydroorotate dehydrogenase was explored. Although PEN is an inhibitor of this enzyme in cells in vivo, in vitro, and in isolated cell sonicates, HPLC analyses of ribonucleotide triphosphate pools in P388 cells showed that all triphosphates had increased, especially UTP. Addition of uridine to the cell culture failed to prevent PEN-mediated cytotoxicity, suggesting that inhibition of de novo pyrimidine biosynthesis was not likely to be an important mechanism of action of this drug. These data suggest that PEN is activated in cells to a free radical that binds DNA.

Animals↗