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W Plunkett

Publications and source records attributed to W Plunkett.

At least 73 records · Page 4Linked to original sources
PubMed1995

Metabolism and actions of 2-chloro-9-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)-adenine in human lymphoblastoid cells.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (Cl-F-ara-A) is a new deoxyadenosine analogue that is resistant to phosphorolytic cleavage and deamination. Studies with a variety of cell lines demonstrated that Cl-F-ara-A is a potent cytotoxic agent; in cell-free systems, its triphosphate (Cl-F-ara-ATP) inhibited DNA polymerase alpha and ribonucleotide reductase. To further characterize its mechanism of cytotoxicity, the present study investigated the cellular metabolism of Cl-F-ara-A and the actions of its nucleotide metabolites in human T-lymphoblast leukemia CCRF-CEM cells. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, with the monophosphate as its major metabolite. After washing cells into drug-free medium, the elimination of each Cl-F-ara-A nucleotide was nonlinear with a prolonged terminal phase. Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA; a much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. At low Cl-F-ara-ATP:dATP values, incorporation was mainly in phosphodiester linkages at internal sites, whereas at higher Cl-F-ara-ATP:dATP values, Cl-F-ara-AMP was principally detected at terminal sites. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA. These results suggest that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.

Adenine Nucleotides↗
PubMed1995

Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells.

The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2'-difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

Adenosine Triphosphate↗
PubMed1995

Functional compartmentation of dCTP pools. Preferential utilization of salvaged deoxycytidine for DNA repair in human lymphoblasts.

The utilization of dCTP derived from de novo synthesis through ribonucleotide reductase in exponentially growing CCRF-CEM cells was compared with the metabolic fate of dCTP produced by the salvage pathway. Exogenous dCyd was not effectively incorporated into replicating DNA; instead, dCTP derived from ribonucleotide reductase (labeled by [5-3H]Cyd) was the main precursor for that purpose, apparently because of functional compartmentation of the dCTP pool in these cells. Studies of the metabolic route of incorporation of exogenous [5-3H]dCyd into DNA of growing CCRF-CEM cells demonstrated that it was mainly incorporated through the DNA repair pathway. Incorporation of [5-3H]dCyd into DNA of synchronized cell populations was maximal in G1 cells, whereas [3H]dThd incorporation occurred predominantly in S phase cells. When cellular DNA was density labeled by incubation with BrdUrd, repaired DNA, which was less dense than replicated DNA, was preferentially labeled by [5-3H]dCyd. In contrast, replicated DNA was labeled by both [3H]dThd and [5-3H]Cyd. The DNA-damaging agents methylmethanesulfonate, ultraviolet irradiation, and gamma-irradiation inhibited [3H]dThd incorporation, whereas they stimulated the accumulation of [5-3H]dCyd in DNA. Based on these results, we propose that the dCTP pool is functionally compartmentalized in growing CCRF-CEM cells. dCTP derived from the salvage pathway is utilized predominantly for DNA repair, whereas the de novo pathway supplies dCTP for DNA replication.

Bromodeoxyuridine↗
PubMed1995

Fludarabine- and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event.

The nucleoside analogs fludarabine and gemcitabine inhibit cellular DNA synthesis by two different mechanisms: (1) direct termination of DNA strand elongation after the triphosphate of each drug is incorporation into DNA; and (2) indirect inhibition of DNA synthesis by decreasing cellular dNTPs through inhibition of ribonucleotide reductase. The present study demonstrated that incorporation of the analogs into DNA is critical for the cytotoxic action of these drugs in human T lymphoblastoid CEM cells. S phase cells, which actively incorporated the analogs into DNA, were most sensitive to the cytotoxic action of these compounds. A relatively short-term (5-24 h) cessation of cellular DNA synthesis without analog incorporation was not sufficient to cause cell death. The drug-treated cells died through apoptosis characterized by generation of internucleosomal DNA fragmentation and apoptotic morphology. Induction of high molecular mass (50-500 kb) DNA fragmentation was also observed in cells undergoing apoptosis; this type of DNA degradation was strongly correlated with the analog-induced cell death process. Inhibition of the analog incorporation into DNA by aphidicolin blocked both types of DNA fragmentation and apoptotic morphology, indicating the essential role of analog incorporation into DNA in drug-induced cell death.

Antimetabolites, Antineoplastic↗
PubMed1995

Preclinical characteristics of gemcitabine.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) is a nucleoside analogue of deoxycytidine in which two fluorine atoms have been inserted into the deoxyribofuranosyl ring. Once inside the cell gemcitabine is rapidly phosphorylated by deoxycytidine kinase, the rate-limiting enzyme for the formation of the active metabolites gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP). Gemcitabine diphosphate inhibits ribonucleotide reductase, which is responsible for producing the deoxynucleotides required for DNA synthesis and repair. The subsequent decrease in cellular deoxynucleotides (particularly dCTP) favours gemcitabine triphosphate in its competition with dCTP for incorporation into DNA. Reduction in cellular dCTP is an important self-potentiating mechanism resulting in increased gemcitabine nucleotide incorporation into DNA. Other self-potentiating mechanisms of gemcitabine include increased formation of active gemcitabine di- and triphosphates, and decreased elimination of gemcitabine nucleotides. After gemcitabine nucleotide is incorporated on the end of the elongating DNA strand, one more deoxynucleotide is added, and thereafter the DNA polymerases are unable to proceed. This action, termed "masked chain termination", appears to lock the drug into DNA because proof-reading exonucleases are unable to remove gemcitabine nucleotide from this penultimate position. Incorporation of gemcitabine triphosphate into DNA is strongly correlated with the inhibition of further DNA synthesis. Compared with ara-C, gemcitabine serves as a better transport substrate, is phosphorylated more efficiently, and is eliminated more slowly. These differences, together with self-potentiation, masked chain termination and the inhibition of ribonucleotide reductase, which are not seen with ara-C, may explain why gemcitabine is, and ara-C is not, active in solid tumours. This unique combination of metabolic properties and mechanistic characteristics suggests that gemcitabine is likely to be synergistic with other drugs that damage DNA, and also with other modalities such as radiation.

Animals↗
PubMed1995

Gemcitabine: a modulator of intracellular nucleotide and deoxynucleotide metabolism.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) is a deoxycytidine (dCyd) analog that extensively modulates intracellular CTP and dCTP metabolism. In Chinese hamster ovary (CHO) cells, a 4-hour exposure to gemcitabine (100 mumol/L) reduced cellular CTP and dCTP concentrations to 5.9% and 50%, respectively. Intracellular UTP concentrations increased, indicating a metabolic block at CTP synthetase. Pool-sizes of ATP and GTP remained unaffected. In contrast, a CHO mutant deficient in deoxycytidine kinase, and thus unable to accumulate dFdCTP, maintained its CTP pools under identical conditions, suggesting that the CTP pool depletion was dependent on dFdC phosphorylation. Neither 100 mumol/L arabinosylcytosine nor 5 mmol/L hydroxyurea affected CTP levels, indicating that inhibition of DNA synthesis by analog incorporation or by depletion of dNTP pools were not the causes of the CTP pool perturbation. Metabolic studies demonstrated that incorporation of [3H]uridine into the UTP pool was not impaired by dFdC treatment, whereas the specific activity of the CTP pools decreased as a function of increasing gemcitabine concentration and time of exposure. Comparable results were obtained using 3-deazauridine, a known inhibitor of CTP synthetase. We conclude that high cellular concentrations of dFdCTP deplete cellular CTP concentrations by inhibition of the dCTP pool and also may be a limiting factor for RNA synthesis.

Animals↗
PubMed1995

Induction of apoptosis by gemcitabine.

Inhibition of cellular DNA synthesis is the major action of gemcitabine. In cells, this drug is converted to its triphosphate (dFdCTP), which is incorporated into DNA and terminates DNA strand elongation. After incorporation of gemcitabine nucleotide into the DNA strand, one more deoxynucleotide is incorporated, and thereafter the DNA polymerases are unable to proceed ("masked chain termination"). Gemcitabine also inhibits DNA synthesis indirectly by decreasing cellular dNTP pools via inhibition of ribonucleotide reductase. Incubation of human leukemia cells (CEM) with gemcitabine leads to apoptotic cell death. Two types of DNA fragmentation were observed in the gemcitabine-treated cells: (1) large-sized double-stranded DNA fragments range from 5 kb to 500 kb with the majority of the fragments located at 50 kb, and (2) nucleosomal-sized DNA fragments. Both types of drug-induced DNA fragmentation were detected in exponentially growing cells and were much more prominent in cells synchronized at S phase. The gemcitabine-induced DNA fragmentation in either synchronized or nonsynchronized cells was inhibited by the DNA synthesis inhibitor, aphidicolin. Thus, incorporation of gemcitabine into DNA is essential to induce DNA fragmentation. The intracellular calcium chelator BAPTA-AM inhibited the drug-induced nucleosomal DNA fragmentation but did not prevent the large-sized DNA fragmentation, suggesting that the nucleosomal DNA fragmentation is a calcium-dependent event, whereas the large-sized DNA fragmentation is independent of calcium. Furthermore, BAPTA-AM did not prevent the morphologic appearance of apoptotic bodies in cells incubated with gemcitabine, indicating that degradation of DNA to nucleosomal fragments is not an essential element of the apoptotic process. Phorbol 12-myristate 13-acetate also inhibited drug-induced nucleosomal DNA fragmentation, but prevented neither large-sized DNA fragmentation nor formation of apoptotic bodies. In contrast, aphidicolin inhibited both types of DNA fragmentation and blocked the formation of apoptotic bodies in the presence of gemcitabine. These data suggest that the generation of large-sized DNA fragments caused by incorporated gemcitabine monophosphate in DNA is critical in gemcitabine-induced apoptosis, whereas nucleosomal DNA fragmentation is not a requirement in this cell death process.

Aphidicolin↗
PubMed1995

Gemcitabine: metabolism, mechanisms of action, and self-potentiation.

Gemcitabine (dFdC) is a new anticancer nucleoside that is an analog of deoxycytidine. It is a pro-drug and, once transported into the cell, must be phosphorylated by deoxycytidine kinase to an active form. Both gemcitabine diphosphate (dFdCTP) and gemcitabine triphosphate (dFdCTP) inhibit processes required for DNA synthesis. Incorporation of dFdCTP into DNA is most likely the major mechanism by which gemcitabine causes cell death. After incorporation of gemcitabine nucleotide on the end of the elongating DNA strand, one more deoxynucleotide is added and thereafter, the DNA polymerases are unable to proceed. This action ("masked termination") apparently locks the drug into DNA as the proofreading enzymes are unable to remove gemcitabine from this position. Furthermore, the unique actions that gemcitabine metabolites exert on cellular regulatory processes serve to enhance the overall inhibitory activities on cell growth. This interaction is termed "self-potentiation" and is evidenced in very few other anticancer drugs.

Animals↗
PubMed1995

Difluorodeoxyguanosine: cytotoxicity, metabolism, and actions on DNA synthesis in human leukemia cells.

The success of gemcitabine (2',2'-difluorodeoxycytidine; dFdC) resulted in new interest in its purine congeners. Based on the structure-activity relationship studies of catabolism and anabolism, 2',2'-difluorodeoxyguanosine (dFdG) emerged as a lead candidate among the difluoropurine analogs. The cytotoxicity, metabolism, and actions of dFdG on DNA synthesis were studied in the human leukemia lymphoblastoid line CCRF-CEM. The IC50 values of dFdG after a 72-hour continuous incubation were 0.01, 0.03, and 0.28 mumol/L for CCRF-CEM, K562, and HL-60 cells, respectively. A cell line deficient in dCyd kinase was equally sensitive to dFdG, suggesting that, in contrast to dFdC, dFdG may be activated by other deoxynucleoside kinase(s). Consistent with these data, coincubation with dGuo spared the dFdG-mediated toxicity; however, up to 500 mumol/L dCyd failed to reverse the toxicity of dFdG. These observations indicated that dGuo kinase, which phosphorylates arabinosylguanine, also appears to play a major role in activating dFdG. CCRF-CEM cells incubated with varying concentrations of [3H]dFdG accumulated dFdGTP in a dose-dependent manner; a 3-hour incubation with 1 mmol/L dFdG resulted in more than 600 mumol/L intracellular dFdGTP. This is in contrast to the gemcitabine triphosphate accumulation, which is saturated at 10 to 20 mumol/L of exogenous dFdC. dFdG metabolites affected ribonucleotide reductase, resulting in a lowering of the dCTP pool; this is in agreement with the effect of dFdC on dNTP pools in leukemia cell lines. The major effect of dFdG on macromolecular synthesis was inhibition of DNA synthesis. DNA primer extension over a defined template revealed that dFdGTP was a good substrate for DNA polymerase alpha and incorporated opposite C sites of the template. Unlike arabinosyl analogs, but similar to gemcitabine triphosphate, dFdGTP incorporation caused DNA polymerase to pause after one normal deoxynucleotide was incorporated beyond the analog. The unique activation requirements of dFdG, its novel mode of inhibition of DNA synthesis, and its potent toxicity to human leukemia cells make it a promising new antimetabolite.

Animals↗
PubMed1995

A 3-day schedule of fludarabine in previously treated chronic lymphocytic leukemia.

This study was undertaken to determine the efficacy and toxicity of a shorter schedule of fludarabine administration (30 mg/m2 i.v. daily for 3 days every 4 weeks) in patients with previously treated chronic lymphocytic leukemia (CLL). Eighty patients with previously treated advanced (Rai III-IV) (54%) or progressive Rai stage 0-II (46%) were treated. The results of this trial were retrospectively compared with those of previous fludarabine trials using different schedules of administration. The complete response (CR), nodular CR and partial response rates were 10, 15 and 21%. The overall response rate (46%) was slightly lower than prior regimens using a 5-day schedule of fludarabine; however, this difference was not significant. Further evaluation by dual-parameter flow cytometry and immunoglobulin gene rearrangement analysis revealed that minimal residual disease was more common in a 3-day schedule. The overall incidence of infections per treatment course (14%) was significantly lower than that observed on the 5-day or weekly regimens (P < 0.001). The incidence of minor, atypical and viral infections were similar. There was no difference in survival in the various trials. In conclusion, a 3-day schedule of fludarabine in previously treated CLL patients was associated with a lower infection rate and similar survival to a 5-day schedule. These data support the use of a 3-day schedule of fludarabine as a single agent and in combination with other active agents.

Adult↗
PubMed1995

Arabinosyl-2-fluoroadenine augments cisplatin cytotoxicity and inhibits cisplatin-DNA cross-link repair.

Cytotoxicity was increased significantly when arabinosyl-2-fluoroadenine (F-ara-A) was administered in simultaneous combination with cisplatin (CDDP) to human colon tumor cell lines relatively sensitive (LoVo) or resistant (CP2.0) to CDDP. Because the mechanism of action of F-ara-A indicates that it may be an effective inhibitor of DNA repair, we hypothesized that F-ara-A induces cytotoxic augmentation by suppressing cellular repair in CDDP-damaged DNA lesions. To test this, we compared the repair of CDDP-induced DNA interstrand cross-links in the total genome and in ERCC1 gene-specific sequences of LoVo and CP2.0 cells for treatments with CDDP and CDDP plus F-ara-A. We determined the DNA repair by measuring the rate of removal of the cross-links, using two methods, i.e., an ethidium bromide fluorescence binding assay, which detects the DNA lesion in the total genome, and a method combining denaturation/renaturation neutral agarose gel electrophoresis and Southern hybridization to detect gene-specific lesions. When F-ara-A (15 microM) was coadministered with CDDP (15 micrograms/ml for LoVo cells and 30 micrograms/ml for CP2.0 cells) for 4 hr, the initial cross-link index for the total genome was increased 67% (4.5 versus 2.7 with CDDP alone) in LoVo cells and 93% (2.9 versus 1.5 with CDDP alone) in resistant CP2.0 cells. At 10 hr after the treatment, only 5% of the cross-links had been removed in combination-treated LoVo cells, compared with 40% in CDDP-treated LoVo cells; in CP2.0 cells, F-ara-A inhibited the removal of cross-links from 95% to 45%. Similar results were obtained for ERCC1 gene-specific DNA sequences. These data suggest that F-ara-A enhances the accumulation of CDDP-induced cross-links in LoVo and CP2.0 cells by suppressing the repair of such lesions, thereby enhancing the cytotoxicity of CDDP in combination treatment.

Antineoplastic Agents↗
PubMed1995

Modulation of the cellular metabolism of cytarabine and fludarabine by granulocyte-colony-stimulating factor during therapy of acute myelogenous leukemia.

Previous in vitro investigations demonstrated that human leukemia cells, when incubated with hematopoietic growth factors such as granulocyte-colony-stimulating factor (G-CSF), augment the accumulation of the triphosphate 1-beta-D-arabinofuranosylcytosine (ara-C cytarabine). To test whether G-CSF infusion prior to ara-C infusion would biologically modulate the accumulation of ara-9-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) and other ara nucleotides in the leukemia blasts during therapy, protocols were designed to infuse G-CSF prior to fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate) and ara-C to increase the accumulation of the active triphosphates [9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) and ara-CTP] in acute myelogenous leukemia (AML) blasts during therapy. To complement these in vivo studies, ex vivo accumulation of ara-CTP was also investigated before and after G-CSF infusion. Patients (n = 5) treated on the fludarabine/ara-C/G-CSF regimen received a 30 mg/m2 dose of fludarabine followed by a 2 g/m2 dose of ara-C infused i.v. for 4 h. Beginning at 24 h, and every day, patients received a 6-h infusion of 400 microgram/m2 G-CSF. At 48 h, the fludarabine and ara-C couplet was repeated. Comparison of F-ara-ATP pharmacokinetics in circulating AML cells of patients on the fludarabine/ara- C/G-CSF regimen demonstrated that the area under concentration time curve (AUC) of F-ara-ATP increased significantly (median, 1.4-fold; range, 0.9-1.5; P = 0.045) after G-CSF infusion. This was due to an increased rate of F-ara-ATP accumulation by AML cells. The AUC of ara-CTP, on the other hand, was not affected (median, 1.0-fold; range, 1.0-1.2; P = 0.571) after G-CSF infusion. Because fludarabine potentiates the accumulation of ara-CTP, the effect of G-CSF on ara-CTP metabolism may not be evident in the AML blasts of patients on the fludarabine/ara-C/G-CSF regimen. To determine the effect of G-CSF when ara-C was infused alone, four additional patients were treated on a pilot protocol in which ara-C (2 g/m2) was infused on days 1 and 3 and G-CSF on day 2. The AUC of ara-CTP accumulation in these patients decreased by a median of 48% after G-CSF infusion. Consistent with these in vivo investigations, ex vivo ara-CTP accumulation was decreased in the AML blasts after G-CSF infusion. Based on these data it could be concluded that (a) infusion of G-CSF before fludarabine augmented the rate of F-ara-ATP synthesis in circulating AML blasts during therapy, suggesting that G-CSF may benefit fludarabine therapy by biological modulation; (b) G-CSF did not increase ara-CTP accumulation, rather it may have caused it to decrease; and (c) these data imply that when G-CSF and ara-C are used in combination, administration of fludarabine prior to ara-C may maintain the ara-CTP AUC.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

High molecular weight DNA fragmentation: a critical event in nucleoside analogue-induced apoptosis in leukemia cells.

Cleavage of DNA into internucleosomal fragments is one of the characteristics of apoptosis. However, searches for in vivo evidence of nucleosomal DNA fragmentation in leukemia cells freshly obtained from patients during chemotherapy frequently failed to reveal nucleosomal multimers (DNA ladders). It is not clear whether this type of DNA cleavage is an essential event in drug-induced apoptosis and thus a denominator of cell killing, or whether the internucleosomal DNA fragments are merely the by-products of the apoptotic process. Here, we report our investigation into the role of DNA fragmentation in apoptotic cell death induced by anticancer nucleoside analogues, both in cell culture and in leukemia patients undergoing chemotherapy. Using a 5'-end DNA-labeling technique and pulsed field gel electrophoresis, we detected fragmentation of DNA in two distinct size classes, internucleosomal and high molecular weight (predominantly 50 kb) DNA fragments, in a human leukemia cell line exposed to the nucleoside analogues fludarabine and gemcitabine. We further demonstrated that the two types of DNA fragmentation were separate events, distinguishable by their requirements for Ca2+ and responses to phorbol ester treatment. The drug-treated cells underwent morphological changes of apoptosis even after internucleosomal DNA fragmentation was selectively inhibited by intracellular Ca2+ chelation, or by treatment with phorbol ester. In contrast, neither apoptotic morphology nor internucleosomal DNA fragmentation was observed when the high molecular weight DNA fragmentation was blocked by inhibition of nucleoside analogue incorporation into DNA. These results suggest that cleavage of DNA into large fragments may be an initial event that is critical for drug-induced apoptosis, whereas activation of a Ca2+-dependent endonuclease to cleave DNA at internucleosomal sites is not an absolute requirement for the execution of the apoptotic cell death program. Further studies of leukemic lymphocytes obtained from 9 patients with chronic lymphocytic leukemia during therapy with fludarabine revealed high molecular weight DNA fragmentation, which was correlated with a decrease of peripheral lymphocytes in 6 patients, whereas only 1 of the 15 patients evaluated for nucleosomal DNA fragments showed the DNA ladders. These results indicate that high molecular weight DNA fragmentation occurs in vivo, and may be correlated with the cytotoxic action of the anticancer drugs. Further study of the association of high molecular weight DNA fragmentation with clinical response to chemotherapy is warranted.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

The role of fludarabine-induced apoptosis and cell cycle synchronization in enhanced murine tumor radiation response in vivo.

We have previously reported that fludarabine, an adenine nucleoside analogue, significantly enhances radiation-induced tumor regrowth delay and local cure in several mouse tumors. Although fludarabine potentiated tumor regrowth delay at various times from -36 h to +6 h in a SA-NH mouse sarcoma model, the greatest enhancement was observed when fludarabine was administered 24 h before irradiation. The purpose of this study was to understand the basis for in vivo enhancement of radiation efficacy by fludarabine. To examine the effect of fludarabine on DNA synthesis and cell cycle progression, tumor-bearing mice were given fludarabine by an i.p. route and then bromodeoxyuridine at various times up to 36 h, followed 0.5 h later by tumor harvest. Two-parameter flow cytometry analysis of the tumor cells using an anti-bromodeoxyuridine antibody demonstrated that an 800-mg/kg fludarabine dose stops DNA synthesis within 3 h with recovery starting at 12 h. By 24 h after fludarabine treatment, a synchronized wave of cycling tumor cells appeared in G2-M phase. The degree of DNA synthesis shutdown and the timing of the reinitiation of DNA synthesis and cell cycle progression were all fludarabine dose dependent. Interestingly, DNA synthesis reinitiated only at the G1-S boundary; cells in the S phase at the time of fludarabine administration appeared to disappear from the tumor population. To confirm these observations more directly, we pretreated tumor-bearing mice i.p. with chlorodeoxyuridine to mark the cells in the S phase, gave them fludarabine 0.5 h later, and then gave them iododeoxyuridine 0.5 h before tumor harvest. Flow cytometry analysis using antibodies specific for chlorodeoxyuridine- and iododeoxyuridined-labeled cells confirmed that cells in the S phase at the time of fludarabine administration never reinitiated DNA synthesis and disappeared from the tumor population. Immunohistological analysis of tumor sections obtained after fludarabine administration demonstrated that prelabeled S-phase cells took on an apoptotic appearance and gradually disappeared from the tumors. An in situ DNA end labeling assay demonstrated DNA fragmentation in these morphologically apoptotic cells. These results suggest that the mechanism of fludarabine enhancement of radiation response involves induced S-phase cell loss through an apoptotic pathway and subsequent synchronization of the remaining cells to a more radiosensitive cell cycle phase at the time of irradiation.

Animals↗
PubMed1994

Fludarabine improves the therapeutic ratio of radiotherapy in mouse tumors after single-dose irradiation.

PURPOSE: Fludarabine, an adenine nucleoside analogue, and an effective inhibitor of chromosome repair, was previously shown to synergistically enhance radiation-induced regrowth delay in three murine tumors. The purpose of this study was to assess whether fludarabine can increase the therapeutic ratio of radiotherapy in murine tumors, that is, to increase local tumor control without significantly modifying the radiation-induced normal tissue response. METHODS AND MATERIALS: Mice bearing 8-mm tumors in the right thigh (SA-NH sarcoma and MCA-K mammary carcinoma) were given 800 mg/kg fludarabine IP 3 h or 24 h before single doses of photon irradiation. Local tumor control was assessed by the TCD50 assay 100 days after treatment. Acute normal tissue toxicity was assessed in the skin (degree of epilation 30 days after irradiation) and in the jejunum (crypt regeneration assay), and late normal tissue toxicity was assessed by a leg contracture assay 120 days after treatment. RESULTS: In both tumors and with both drug schedules, fludarabine enhanced radiation-induced local tumor control (dose modification factors (DMF) of 1.24 (95% confidence limits 1.19-1.31) and 1.26 (95% confidence limits 1.20-1.32) for SA-NH, and 1.38 (95% confidence limits 1.25-1.50) and 1.35 (95% confidence limits 1.22-1.16) for MCA-K tumors). When given 3 h before radiation, fludarabine offered a slight protection from skin toxicity (DMF = 0.83, 95% confidence limits 0.77-0.86) but enhanced jejunum toxicity (DMF = 1.53). When fludarabine was given 24 h before irradiation, the reverse trend was observed (DMF = 1.11 (95% confidence limits 1.07-1.16) and 0.89, respectively). No enhancement of leg contracture was observed for either fludarabine schedule. CONCLUSION: The data presented here demonstrate that fludarabine can potentiate local tumor control induced by single-dose irradiation. While jejunum sensitization limited the relative effectiveness when fludarabine was administered 3 h before irradiation, a therapeutic ratio greater than one was always achieved when fludarabine was given 24 h before irradiation.

Animals↗
PubMed1994

Potentiation of radiation-induced regrowth delay in murine tumors by fludarabine.

Fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine-5'-monophosphate), an adenine nucleoside analogue, has previously been shown to inhibit the repair of radiation-induced chromosome damage. Thus fludarabine may have therapeutic utility in combination with photon irradiation. The purpose of this study was to determine whether fludarabine could enhance radiation-induced murine tumor regrowth delay and to determine the most effective dose and schedule of the combination. A significant (P < 0.05) absolute regrowth delay enhancement was observed in three murine tumor models (SA-NH, a sarcoma; and MCA-K and MCA-4, mammary carcinomas) when fludarabine (800 mg/kg) was given 1 h prior to 25 Gy gamma-irradiation. While fludarabine enhanced radiation-induced tumor regrowth delay when given between -36 h and +6 h of radiation (SA-NH tumor), the greatest enhancement was observed when fludarabine was given at -24 h prior to irradiation (radiation dose modification factor of 1.82 at -24 h compared to 1.57 at -3 h prior to radiation). The degree of fludarabine enhancement (at -3 or -24 h) was dose dependent at doses above 200 mg/kg. When fludarabine and radiation were administered on a fractionated schedule (fludarabine given 3 h prior to radiation each day for 4 days), the dose modification factor increased to 2.14 (1.63 if the effect of fludarabine alone is subtracted). These results suggest that fludarabine enhances radiation-induced tumor regrowth delay in a more than additive fashion after both single and fractionated treatments, and the degree of enhancement is dependent on the sequence and timing of administration, the fludarabine dose, and the tumor type. Thus, fludarabine may have clinical potential as a radiation enhancer in the treatment of solid tumors.

Animals↗
PubMed1994

Quantification of topoisomerase-DNA complexes in leukemia cells from patients undergoing therapy with a topoisomerase-directed agent.

Several clinically important drugs utilized in cancer chemotherapy inhibit type I (Topotecan) or type II (amsacrine, etoposide) DNA topoisomerases by stabilizing the formation of DNA-topoisomerase complexes (topoisomerase-DNA cross-links). In various cell lines, the magnitude of drug-induced DNA-protein cross-link production correlates with the magnitude of cytotoxicity induced by the drugs. We developed a simple filter-binding assay that can measure drug-induced DNA-protein cross-links in leukemia cells obtained directly from patients because the assays most widely used for assessment of drug-induced DNA-protein cross-links in cells [sodium dodecyl sulfate (SDS)/KCl precipitation and alkaline elution] are not readily applicable for use on patient material. HL-60 human leukemia cells or freshly isolated patients' leukemia cells were incubated with Topotecan, etoposide, or amsacrine; lysed with SDS; and applied to nitrocellulose filters in a low-salt buffer. DNA is retained on the filter only if it is covalently bound to protein. The amount of DNA retained on the filter is quantified by hybridization to the alu sequence of DNA, which is distributed ubiquitously in the human genome. Using radiolabeled cells, we compared the filter-binding assay directly with the SDS/KCl precipitation assay in the detection of etoposide- or amsacrine-induced DNA-protein cross-links in HL-60 cells and amsacrine-resistant HL-60/AMSA cells. Both the SDS/KCl precipitation assay and the filter-binding assay detected etoposide-induced DNA-protein cross-links in HL-60 and HL-60/AMSA cells and detected a greater frequency of amsacrine-induced DNA-protein cross-links in HL-60 cells than in HL-60/AMSA cells. The filter-binding assay detected DNA-protein cross-links in freshly isolated leukemia cells exposed to Topotecan in vitro. The ratios of DNA retention for Topotecan-treated versus untreated cells from leukemia patients ranged from 1.8 to 11.5. The heterogeneity of this detected cross-linking was as might be expected if the assay were predictive of the antileukemic action of Topotecan, which is variable. This new filter-binding technique may be useful for predicting the sensitivity of individual patients' tumors to drugs that inhibit type I or type II DNA topoisomerases.

Amsacrine↗