The Role of Fludarabme-Induced Apoptosis and Cell Cycle Synchronization in Enhanced Murine Tumor Radiation Response in Vivot Vincent Grã©Goire,2Nguyent

Home , Cell synchronization

[CANCERRESEARCH54, 6201-6209, December 1, 1994] The Role of Fludarabme-induced Apoptosis and Cell Cycle Synchronization in Enhanced Murine Tumor Radiation Response in Vivot Vincent GrÃ©goire,2NguyenT. Van, L. Clifton Stephens, William A. Brock, Luka Milas, William Plunkett, and Walter N. Hitteiman@

Departments of Clinical Investigation (V. G., W. P., W. N. H.J, Hematology (N. T. V.1, Veterinary Medicine and Surgery (L. C. SI, and Experimental Radiotherapy (L U., W. A. B.]. The University ofTexas M. D. Anderson Cancer Center. Houston, Texas 77030

ABSTRACT INTRODUCTION

We have previously reported that fludarabine, an adenine nucleoside The efficacy of radiotherapy for local tumor control has been analogue, significantly enhances radiation-Induced tumor regrowth delay postulated to be determined by a number of factors, including intrinsic and local cure in several mouse tumors. Although fludarabine potentiated cellular radioresistance (1â€”6).One important component of cellular tumor regrowth delay at various times from -36 h to +6 h In a SA-NH radioresistance is the capability of the cell to repair radiation-induced mouse sarcoma model, the greatest enhancement was observed when DNA or chromosome damage (7â€”i1).Thus, one potential strategy for fludarabine was administered 24 h before Irradiation. The purpose of this improving radiotherapy outcome is to combine radiation with repair study was to understand the basis for in vivo enhancement of radiation inhibitors that would selectively poison those tumor cells with the efficacy by fludarabme. To examine the effect of fludarabine on DNA highest capability for repair. synthesis and cell cycle progression, tumor-bearing mice were given flu Since cellular DNA synthesis and DNA repair share some common darabine by an Lp route and then bromodeoxyuridlne at various times up enzymatic pathways (12), DNA synthesis inhibitors have been inves to 36 h, followed 0.5 h later by tumor harvest. Two-parameter flow cytometry analysis of the tumor cells using an antl-bromodeoxyuridine tigated for their potential as DNA repair inhibitors when combined antibody demonstrated that an 800-mg/kg fludarabine dose stops DNA with radiation (13â€”17).However, some of the repair inhibitors shown synthesis within 3 h with recovery starting at 12 h. By 24 h after fludara to be active in vitro do not appear to be useful in the in vivo setting due bine treatment, a synchronized wave of cycling tumor cells appeared in to limiting biochemical and pharmacological considerations. For cx G2-Mphase. The degree ofDNA synthesis shutdown and the timing of the ample, although ara-A4 was previously found to be an effective repair relnitiation of DNA synthesis and cell cycle progression were all fludara inhibitor in vitro and enhanced the cytotoxicity of radiation, its trans bine dose dependent. Interestingly, DNA synthesis reinitlated only at the lation to the in vivo setting was limited by rapid deamination and G1-S boundary; cells in the S phase at the time of fludarabine adminis subsequent loss of activity. On the other hand, F-ara-A, a fluorinated tratlon appeared to disappear from the tumor popalatlon. To confirm analogue of ara-A, is relatively resistant to deamination (18). More these observations more directly, we pretreated tumor-bearing mice i.p. over, its active triphosphate form, F-ara-ATP, has been shown to be a with chlorodeoxyuridlne to mark the cells in the S phase, gave them potent inhibitor of DNA primase (19), DNA polymerase a and â‚¬(20, fludarabine 0.5 h later, and then gave them lododeoxyurldlne 0.5 h before tumor harvest Flow cytometry analysis using antibodies specific for 21), DNA ligase (22), and ribonucleotide reductase (21), and can chlorodeoxyuridlne- and Iododeoxyuridlned-labeled cells confirmed that cause DNA chain termination. In vitro F-nsa-A is a potent inhibitor of cells in the S phase at the time of fludarabine administration never DNA repair after UV radiation5 and chromosome break repair after relnitiated DNA synthesis and disappeared from the tumor population. ionizing radiation (23). In vivo the monophosphate form (fludarabine) Immunohlstological analysis of tumor sections obtained after fludarabine has already shown potent clinical activity in the treatment of chronic administration demonstrated that prelabeled S-phase cells took on an lymphocytic leukemia and lymphoma (24). apoptotic appearance and gradually disappeared from the tumors. An in Based on these background observations, we chose to explore the situ DNA end labeling assay demonstrated DNA fragmentation In these ability of fludarabine to enhance radiation activity in vivo. Using morphologically apoptotic cells. These results suggest that the mechanism several mouse solid tumor models, fludarabine was found to potently of fludarabine enhancement of radiation response involves induced S enhance radiation-induced regrowth delay and local tumor cure after phase cell loss through an apoptotic pathway and subsequent synchronl zation ofthe remaining cells to a more radiosensitive cell cycle phase at the single and fractionated radiation schedules (dose modification factors time of irradiation. range, 1.24â€”2.14;Refs. 25 and 26). Moreover, except for mucosal tissue, the sensitizing effect on tumors appeared to be more pro

Received 6/13/94; accepted 10/3/94. nounced than on normal tissues, suggesting the therapeutic index The costs of publication of this article were defrayed in part by the payment of page would be >1. The degree of radiotherapeutic enhancement was found charges. This article must therefore be hereby marked advertisement in accordance with to be dependent on the timing of fludarabine administration relative to 18 U.S.C. Section 1734 solely to indicate this fact. 1 This investigation was supported by Grants CA-06294, CA-27931, and CA-28596 radiation, dose of fludarabine, tumor type, and schedule of radiation. and Core Grant CA-16672 (for flow cytometry) awarded by the National Cancer Institute. Based on the in vitro experiments, the working hypothesis was that Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current fludarabine acts as a repair inhibitor; thus its greatest enhancing regulations and standards of the United States Department of Agriculture and Department activity was expected when the drug was given proximal to the time of Health and Human Services, NIH. of irradiation so that the F-ara-ATP intracellular concentrations were 2 Present address: St-Luc University Hospital, Radiation Oncology Department, 10 Avenue Hippocrate, 1200 Brussels, Belgium. highest during the repair interval. However, when the optimal sched 3 To whom requests for reprints should be addressed, at Department of Clinical ule for fludarabine administration was explored in a SA-NH mouse Investigation, Box 019, University of Texas M. D. Anderson Cancer Center, 1515 sarcoma (i.e., from â€”36h to +6 h of irradiation), the most pro Holcombe Boulevard, Houston, TX 77030.

4 The abbreviations used are: ara-A, 9-@3-n-arabinofuranosyladenine; F-ara-A, 9-$-o- nounced radiosensitization occurred when fludarabine was given 24 h arabinofuranosyl-2-fluoroadenine; fludarabine, F-ara-AMP, 9-@-D-arabinofuranosyl-2- before irradiation. fluoroadenine-5'-monophosphate; F-ara-ATP, 9-f3-o-arabinofuranosyl-2-fluoroadenine 5'-triphosphate; BrdUrd, bromodeoxyuridine; CIdUrd, chlorodeoxyuridine; IdUrd, Previous studies of fludarabine activity in murine leukemia models iododeoxyuridine; PBS, calcium- and magnesium-free phosphate-buffered saline; PBTB, suggested that it was unlikely that intracellular levels of F-ara-ATP calcium- and magnesium-free phosphate-buffered saline with 0.5% Tween 20 and 0.5% would still be high 24 h after drug administration (27). The fmding of bovine serum albumin; P1, propidium iodide. â€˜WalterN.Hittelman, unpublished data. maximum radiosensitization at this time point suggested that 6Luka Milas, unpublished data. additional mechanisms might contribute to the interaction between 6201

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. APOPTOSIS AND CELL CYCLE SYNCHRONIZA11ON INDUCED BY F-ara-AMP radiation and fludarabine. The purpose of the present study was to resuspended in 5 g.@g/mlP1 (Aldrich Chemical Co., Milwaukee, WI) in 2 ml explore other potential mechanisms by which fludarabine might po PBTB. The nuclear suspension was stored at 4Â°C.RNasewas added 30 mm tentiate radiotherapy in vivo. One hypothesis is that fludarabine pre before flow cytometry analysis to a final concentration of 10 @g/ml. treatment induces tumor cell cycle redistribution and subsequent For double-label experiments (DNA labeled with both CdUrd and IdUrd), partial cell synchronization to a more radiosensitive phase of the cell 2 X 106 nuclei were first exposed to adUrd-s@cffic BR3 antibody as cycle. To test this hypothesis, we performed comprehensive cell cycle described above. After being washed twice in PBTB, nuclei were resuspended in 150 @tlofphycoerythrin-conjugatedgoat anti-mouse IgO (M3004â€”4;Caltag kinetic studies with flow cytometry and immunohistochemical anal Laboratories, Inc.) diluted 1:100 v/v in PBTB and incubated for 1 h at room ysis in the SA-NH murine tumor model using halogenated thymidine temperature. After being washed in PBTB, nuclei were resuspended in 20 g.d analogues to differentially label S-phase cells. The present study of IdUrd-specific flourescein-conjugated 844 antibody (7583; Becton Dickin shows that fludarabine induced inhibition of DNA synthesis in the son, San Jose, CA) diluted 1:2 v/v in PBTB, and incubated for 1 h at room tumor, a subsequent loss of S-phase cells from the tumor through an temperature. Nuclei were then washed and stained with P1 as described above. apoptotic mechanism, and a cell cycle redistribution such that a partial RNase was added 30 min before flow cytometry. synchronization of tumor cells in the radiosensitive G2-M phase Flow CytOmetry Analysis. Flow cytometry was performed using a FAC occurred at the time of radiation. Thus, multiple mechanisms can Scan (Becton Dickinson) equipped with an air-cooled argon laser delivering 15 contribute to the radiosensitization of tumors by fludarabine. mW at 488 nm. Red fluorescence (P1measuring DNA content) was collected through a long-pass filter (630 nm LP) and amplified in linear mode to better reflect DNA ploidy. Green (fluorescein isothiocyanate) and orange (phyco MATERIALS AND METHODS erythrin) fluorescences were collected through band-pass filters of 530 nm Â±30 nm and 575 mu Â±30 mu, respectively, and amplified in a logarithmic Tumor Model. The SA-NH murine tumor model system is a nonimmuno mode to better record the wide range of incorporation of halogenated genic mouse sarcoma of spontaneous origin that is syngeneic to C3Hf/Kam thymidine analogues into DNA of different cells. mice (28). Tumor cells of the third isotransplant generations, stored in liquid For each sample, 10,000â€”15,000eventswere collected in list mode. Cell nitrogen, were injected after thawing into the mouse flank to generate new debris and doublets were removed from the data acquisition using a doublet tumor sources. These tumors were excised and single-cell suspensions ob discriminator (Becton Dickinson). Compensation between red and green fluo tamed by trypsin and DNase digestion of nonnecrotic tissue as described rescences was not necessary due to their minimal spectral overlap. However, previously (29). Cell viability was evaluated by trypan blue dye exclusion compensationwasusedforredchannelsoverlappingintoorangechannelsand, under phase contrast microscopy and was usually more than 95%. For these to a lesser extent, for orange channels emitting into the green channels; under experiments, tumors were then generated by i.m. injection of 5 X 10@tumor the conditions of these experiments, only moderate compensation levels were cells in 10 pi Hsu's medium into the right thigh of C3Hf/KAM mice and required. Typically, for voltages of 520, 410, and 455 V on green (FL1), allowed to grow to 8 mm in diameter. The volume doubling time for SA-NH orange (FL2), and red (FL3) photomultipliers, respectively, compensations of tumors, calculated during growth from a mean diameter of 8â€”14mm,was 17.5%for FL2-%FL3 and 2.5% for FL1-%FL2 were applied. Dataacquiredon 2.5 Â±0.1 days. an HP computer were converted to a PC format (HP Reader, Verity Software) Fludarabine Treatment. Fludarabine was generously supplied by Berlex and analyzed using PCLYSIS II software (Becton Dickinson). Labeled cells Laboratories (Richmond, CA). Before each experiment, the drug was recon were separated from unlabeled cells by visual inspection on a scattergram of stituted in calcium- and magnesium-free PBS, the pH adjusted to 6.8â€”7.0,and relative DNA content versus relative BrdUrd or IdUrd content. A single line the solution filtered through a 0.45-pm Millipore filter and stored at 4Â°C.The drawn just above the bulk of dots representing the different G1 and G2-M concentrations of fludarabine in solution were adjusted to allow injection of populations was used to divide the two regions. 0.01 ml/g mouse body weight. Fludarabine was injected i.p. at room temper Determination of Apoptosis. For histological examinations, tumors were ature. Mice were sacrificed by neck dislocation at designated times after removed and fixed in neutral-buffered 10% formalin (Fisher Scientific, PiUs fludarabine administration. burgh, PA) and embedded in paraffin. Tumor sections were stained with Tumor Labeling. To label tumor cells in the S phase in vivo.we injected hematoxylin and eosin and scored for the presence of apoptotic bodies as SA-NH tumor bearing mice i.p. before and/or after fludarabine administration with described previously (32). Briefly, apoptotic bodies were counted under the halogenated thymidine analogues dissolved in PBS at a dose of 60 mg/kg. For light microscope at a magnification of X 400. Five nonnecrotic fields were single-labeling experiments, either BrdUrd (Sigma chemical Co., St. Louis, MO) analyzed per tumor section, and in each field the number of apoptotic bodies or CIdUrd(Sigma)was used.For double-labelingexperiments,CldUrdand IdUrd per 100 nuclei scored was expressed as a percentage. The following criteria (Sigma)were used.The pH had to be progressivelyincreasedto 8.5 to completely were used to distinguish apoptotic bodies: (a) isolated distribution of apoptotic dissolve IdUrd. Solutionsof 6 mg/mIwere prepared,filteredthrough a 0.45-sm bodies; (b) shrunken cells usually with empty space between neighboring cells; Millipore filter, and stored at 4Â°Cuntil use. (c) eosinophilic cytoplasm; (d) condensation of nuclei into dense particles; (e) Isolation and Staining of Nuclei for Flow Cytometry. For flow cytom fragmentation of the nuclei into several bodies; and (e) absence of inflamma etry, the tumors were removed, cut in small pieces, and fixed in cold 70% tory reaction surrounding the apoptotic cells. At least, criteria 1 to 4 were ethanol (AAPER Alcohol and Chemical Co., Shelbyville, KY) at 4Â°Cforat required to score a cell as apoptotic. least 24 h. Isolation and staining of nuclei were done as described previously The presence of apoptotic bodies on tumor sections was also determined by (30, 31). Briefly, small pieces of tumor were removed from ethanol, minced an in situ end-labeling technique (33, 34). Briefly, [email protected] were finely, and incubated in a spinner flask with 10 ml 0.04% pepsin (E. M. dewaxed in xylene (Fisher Scientific) for 5 mm three times each, and progres Science, Cherry Hill, NJ) w/v in 0.1 N HO (Mallinckrodt, Inc., Paris, KY) for sively hydrated by immersing the slides for 3 miii each in 100%, 90%, 70%, 1 h in a 37Â°Cwaterbath to obtain a nuclear suspension. The solution was then and 30% ethanol solutions. Endogenous peroxidase was inactivated by im passed through an 18-gauge needle, filtered through a 35-@tmnylonmesh, and mersing the slides for 20 mlii in 0.75% H2O2 (Fisher Scientific) v/v in 100% centrifuged. To denature the DNA before antibody incubation, the resulting methanol (E. M. Science). After the slides were washed in PBS, sections were pellet was resuspended in 2 ml 2 N HO (Mallinckrodt, Inc.) in a 37Â°C water digested with 0.1% pepsin (Fisher Scientific) w/v in 0.1 N HC1in a coplin jar bath for 20 mm. The nuclear suspension was then neutralized with 0.1 M for 5 mm at 37Â°Candthen extensively washed in PBS. Sections were then sodium tetraborate (Sigma) and washed twice in PBS with PBTB. incubated in a moist chamber at 37Â°Cfor1 h with an end-labeling cocktail For single-label experiments (DNA labeled with either BrdUrd or CldUrd including the following: terminal deoxynucleotidyl transferase (USB 70033), alone), 2 x 106 nuclei were resuspended in 150 @lofthe BrdUrd/CldUrd 0.5 enzyme unit4al; biotinylated dUTP (Sigma), 0.06 mM; enzyme 5 X buffer specific BR3 antibody (MD5300; Caltag Laboratories, Inc., San Francisco, (Curtin Matheson Scientific), 10 @l;double-distilledwater up to 50 s.d.The CA) diluted 1:500 v/v in PBTB and incubated for 1 h at room temperature. reaction was terminated by immersing the slides in a buffer containing 300 mM Nuclei were washed twice in PBTB and resuspended in 150 ,xl of flourescein sodium chloride (Sigma) and 30 mM sodium citrate (Sigma) in double-distilled conjugated goat anti-mouse IgG (Sigma) diluted 1:50 v/v in PBTB and water. After the slides were washed in PBS, sections were incubated with incubated for 1 h at room temperature. After being washed, nuclei were horseradish peroxidase-conjugated avidin (VectaStain ABC kit; Vector 6202

Laboratories, Burlingame, CA) for 1 h at 37Â°Cina moisture chamber. Staining was developed using 3,3'-diaminobenzidine (Sigma), and sections were I- counterstained with methyl green. z Immunoblstochemlcal Detection of Cells with CldUrd-substituted DNA. To detect cells labeled in vivo with CldUrd on embedded tumor 0 1@1h sections, 4-pin paraffin sections were incubated overnight in an oven at 60Â°C, C.) dewaxcd in xylene (Fisher Scientific) baths for 5 mm each three times, and progressively hydrated by immersing the slides for 3 mm in 100%, 90%, 70%, @0 and 30% ethanol solutions. Endogenous peroxidase was inactivated by im m mersing the slides for 20 min in 0.75% H202 (Fisher Scientific) v/v in 100% methanol (E. M. Science). After the slides were washed in PBS, sections were digestedwith0.1%pepsin(CurtisMathesonScientific)w/vin doubledistilled 40 10203040 water for 1 h in a moist 37Â°Cchamber.Slides were then washed with PBS and RELATIVE DNA CONTENT immersed in 2 N H@ (Mallinckrodt, Inc.) for 50 mm at room temperature to denature the DNA. Acid neutralization was done with 0.1 Msodium tetraborate Fig. 1. Fludarabine-induced cell cycle redistribution in SA-NH tumors. Mice bearing 8-mm tumors were given i.p. injections of fludarabine (800 mg/kg). At various times after (Sigma) for 2 mm, and sections were washed in PBS. The slides were the drug administration, S-phase cells were pulse-labeled in vivowith BrdUrd and 30 miii incubated for 20 mm at 37Â°Cwith normal horse serum (Vector Laboratories, later the tumors were harvested. Samples were then processed for flow cytometry analysis. Inc.) diluted 1:66 v/v in PBS to block nonspecific binding. Sections were then The relative DNA content (linear scale, abscissa) versus relative BrdUrd content (log incubated with BR3 antibody (MD5300; Caltag) diluted 1:500 v/v in PBTB for scale, ordinate) are presented for various time periods after fludarabine administration. The times indicated represent the time elapsed between fludarabuneadministration and 1 h in a moist 37Â°Cchamber.Sections were then washed in PBS and incubated tumor harvest. The 0-h time (top left) was obtained from an untreated tumor. SA-NH with a biotinylated antimouse antibody (Vectastain ABC kit; Vector Labora tumors have a diploid as well as two aneuploid populations. The diploid population has tories, Inc.) diluted 1:66 v/v in PBS for 1 h in a moist 37Â°Cchamber.After a G1 relative DNA content slightly below 10; the aneuploid G@populationshave relative being washed with PBS, sections were incubated with horseradish peroxidase DNAcontentat 20 and40, respectively. conjugated avidin (Vectastain ABC kit; Vector Laboratories) for 1 h in a moist 37Â°Cchamber.Staining was developed using 3,3'-diaminobenzidine (Sigma) and sections were counterstained in hematoxylin. and 19.2 Â±0.6% at 12, 15, 18, 24, and 36 h, respectively (Fig. 1). Interestingly, DNA synthesis appeared to reinitiate only from cells at RESUL@S the G1-S boundary, whereas cells already in the S phase at the time of fludarabine administration did not reinitiate DNA synthesis. As a CellCycleRedistributionafterFludarabineAdministration.In result, a cell synchronization effect was generated whereby cells were vitro studies have shown that F-ara-A is a potent inhibitor of chro accumulated in early S phase at 15 h (relative DNA content at 10 or mosome repair after ionizing radiation (23). Thus, it could be postu 20), late S or G2-M phase at 24 h (relative DNA content around 20 or lated that the greatest potentiation of radiation effect would be around 40), and early-mid S phase (relative DNA content between 10 achieved if fludarabine were administered proximal in time to radia and 15, or between 20 and 30) in the subsequent cell cycle, 36 h after tion such that high intracellular levels of F-ara-ATP would be present fludarabine treatment. No major differences in fludarabine cell cycle during the period of repair. However, when fludarabine was given at redistribution was observed for the diploid and aneuploid tumor various times before or after radiation in a mouse tumor model, while subpopulations. Of major interest, the time of greatest radiosensitiza all schedules used showed some improvement of radiation effect, the tion occurred 24 h after fludarabine treatment, which corresponded to greatest enhancement of radiation-induced regrowth delay occurred the time of greatest accumulation of cells in G2-M phase, a relatively when fludarabine was given 24 h before radiation (25). Since this radiosensitive cell cycle phase. observation is difficult to explain by repair inhibition, another radio Dose-Effect Relationship for Fludarabine-induced Cell Cycle sensitization mechanism must be implicated. One possibility was that Redistribution. We previously reported that the degree of enhance fludarabine caused a cell cycle redistribution such that a synchronized ment of radiation-induced tumor growth delay was dependent on the cohort of cells in a more radiosensitive phase of the cell cycle dose of fludarabine administered (25). For fludarabine doses of 800 occurred at the time of irradiation 24 h after fludarabine was given. mg/kg and 400 mg/kg, the maximum radiation enhancement was To determine the effect of fludarabine on tumor cell cycle progres observed when fludarabine was administered 24 h before irradiation. sion, mice bearing 8-mm SA-NH tumors were injected with 800 However, this effect was markedly decreased with lower fludarabine mg/kg fludarabine, and tumors were harvested for flow cytometry at doses (i.e., 200 mg/kg and 100 mg/kg). It was therefore of interest to 0-36 h after injection. To pulse label the S-phase cells and thus to determine whether the degree of DNA synthesis inhibition and cell determine the effect of fludarabine on DNA synthesis, BrdUrd was cycle redistribution was also dependent on the dose of fludarabine administered to the mice 30 mm before tumor harvest. Using two administered. To address this question, mice bearing 8-mm SA-NH parameter flow cytometry (i.e., DNA content and BrdUrd uptake in tumors were treated with 400, 200, or 100 mg/kg fludarabine and then S-phase cells), both cell cycle distribution and DNA synthesis capac tumors were harvested at various times after treatment. In these ity could be evaluated in the same analysis. Single parameter DNA studies, mice were treated with IdUrd 30 mm before tumor harvest to content analysis indicated that the SA-NH tumor has a diploid assess DNA synthetic activity using two-parameter flow cytometry. population as well as two aneuploid populations with DNA indices of AsshowninFig.2,boththedegreeanddurationofDNAsynthesis 1.2 and 2.2 (data not shown). A two-parameter plot of relative DNA inhibition was dependent on the dose of fludarabine administered. At content (in abscissa) and relative BrdUrd uptake (in ordinate) in 400 mg/kg, DNA synthesis was substantially inhibited at 3 h, was untreated tumors (Fig. 1) indicates that 22.6 Â±0.2%of the cells were reinitiated at the Ga-S boundaries by 6 h, and was restored to control actively synthesizing DNA in the S phase. By 3 h after fludarabine levels by 12 h. DNA synthesis was also substantially inhibited 3 h administration, DNA synthesis (i.e., BrdUrd uptake) was completely after 200 mg/kg fludarabine, but reinitiation apparently took place shut off and remained inhibited for several hours. before 6 h. After 100 mg/kg fludarabine, DNA synthesis was only Reinitiation of DNA synthesis began 12 h after fludarabine admin partially inhibited at 3 h, and DNA synthesis could be reinitiated to a istration, and the fraction of BrdUrd-labeled cells progressively greater degree throughout the S phase. As a consequence of the increased to 0.8 Â±0.3%, 19.6 Â±0.1%, 33.0 Â±1.9%, 21.3 Â±5.3%, decreased effects on the degree and duration of DNA synthesis 6203

3h 6h 12h 24h later, immediately following a 30-mm pulse injection with IdUrd to label tumor cells capable of synthesizing DNA at the time of tumor 400 harvest. As shown in Fig. 4, a similar set of events occurred as was Imp/kg ... observed at 800 mg/kg. (a) By 24 h after fludarabine admmlstratlon, â€˜U the fraction of cells labeled with CldUrd (i.e., the cells in the S phase at the initiation of the experiment) decreased to 5% of the tumor 0 200 @ C-) I kg population. (b) DNA synthesis appeared to reimtiate preferentially in the portion of the tumor population that was not in the S phase at the @0 time of fludarabine administration. Interestingly, at these lower flu 8' darabine doses, DNA synthesis did reinitiate from a portion of the -a 100 @ mgI kg cells that were in the S phase at the time of fludarabine administration (i.e., 0.5% and 4% for doses of 400 and 200 mg/kg, respectively). It 10 20 30 40 10203040 10203040 10203040 should be noted, however, that these values for the fraction of S phase RELATIVEDNACONTENT tumor which could reinitiate DNA synthesis after fludarabine admin Fig. 2. Dose-dependent fludarabine-induced cell cycle redistribution in SA-NH tumors. istration might be an underestimate because by 24 h after fludarabine, Mice bearing 8-mm tumors were given i.p. injections of 400, 200, or 100 mg/kg some surviving S-phase tumor cells might have recovered and tiudarabine, and, at various times after drug administration, tumor cells were pulse labeled progressed to the G@phase of the next cell cycle (Fig. 2). with IdUrd to monitor DNA synthesis reinitiation. Tumors were harvested 30 mm later. Samples were then processed for flow cytometry analysis. Scattergrams of relative DNA Hudarabine-induced Apoptosis. The preceding data suggested content (linear scale) versus relative IdUrd content (log scale) are presented for the three that S-phase cells at the time of fludarabine administration not only doses of fludarabine. The times indicated represent the time elapsed between fludarabine administration and tumor harvest. were unable to reinitiate DNA synthesis, but were removed from the tumor population in a time- and dose-dependent fashion. We therefore investigated the mechanism of tumor cell loss after fludarabine treat inhibition seen at progressively lower fludarabine doses, the extent of cell cycle synchronization also depended on dose. At 400 mg/kg, moderate synchronization at G2-M and early G1 phases 24 h after fludarabine administration was induced. At 200 mg/kg, cells were already into early S phase of the next cell cycle by this time, and at 100 mg/kg fludarabine little cell cycle redistribution occurred. Thus the significant radiosensitization observed at 24 h after fludarabine treatment was associated with a high degree of G2-M (and early G@at 400 mg/kg) synchronization induced by fludarabine at this time point. Fludarabine-induced S-Phase Cell Loss. The studies described above demonstrated that fludarabine could effectively shut down DNA replication in S-phase tumor cells and that DNA synthesis Log CIdUrd CONTENT reinitiated preferentially from cells at the Ge-S boundary, at least for Fig. 3. Fludarabine-induced S-phase tumor cell loss in SA-NH tumors. Mice bearing doses above 200 mg/kg. To better examine the kinetics of this phe 8-mm tumors were given i.p. injections of CIdUrd to label the S-phase cells. One-half h nomenon we injected mice bearing 8-mm SA-NH tumors with CIdUrd later, an 800-mg/kg fludarabunedose was given i.p., and, at 2.5 or 23.5 h after fludarabine to label the S-phase cells of the tumors. The animals were treated 0.5 administration, tumor cells were pulse labeled again with IdUrd to monitor DNA synthesis reinitiation. Tumors were harvested 30 mm later. Samples were then processed for flow h later with 800 mg/kg fludarabine, and tumors were harvested 3 or 24 cytometry analysis. Scattergrams of relative CldUrd content (log scale) versus IdUrd h later immediately after a 30-mm pulse label of IdUrd to label the content (log scale) are presented. The times indicated represent the time elapsed between fiudarabine administration and tumor harvest. The CIdUrd-positive cells are cells that cells synthesizing DNA at the time of tumor harvest. The use of were in the S phase at the time of fludarabine administration. The IdUrd-positive cells are antibodies that preferentially recognize CldUrd- or IdUrd-containing cells that reunitiatedDNA synthesis after fludarabine administration. DNA in combination with flow cytometry made it possible to better determine from which cell cycle phase DNA synthesis reinitiated after 400 mg/kg 200 mg/kg F-Ara-ATP drug levels decreased. I- As shown in Fig. 3, DNA synthesis was completely shut down in the tumor cells by 3 h after 800 mg/kg fludarabine: neither cells in the Iâ€” S phase (i.e., CldUrd-positive cells) nor cells not in the S phase (i.e., CldUrd-negative cells) incorporated IdUrd. When tumors were har vested 24 h after fludarabine treatment, DNA synthesis was apparent but only in a fraction of the cells that were not in the S phase at the time of fludarabine treatment (i.e., CldUrd-negative/IdUrd-positive cells). In contrast, the cells in the S phase at the time of fludarabine administration (CldUrd positive) did not incorporate IdUrd, and thus these cells appeared unable to reinitiate DNA synthesis. Moreover, LogCidUrdCONTENT comparison of Figs. 1 and 3 suggests a preferential loss of tumor cells Fig. 4. Dose-dependent fludarabine-induced S-phase cell loss in SA-NH tumors. Mice that were in the S phase at the time of fludarabine administration: the bearing 8-mm tumors were given i.p. injections of CidUrd to label the S-phase cells. fraction of CIdUrd-prelabeled cells decreased from 22.6% at the One-half h later, a 400- or 200-mg/kg fludarabunedose was given i.p., and, at 233 h after initiation of the experiment to 17.6% and 2.6% at 3 and 24 h after fludarabine administration, tumor cells were pulse labeled again with IdUrd. Tumors were harvested 30 mm later. Samples were then processed for flow cytometry analysis. fludarabine administration, respectively. Scattergrams of relative OdUrd content versus relative IdUrd content are presented. The To examine the fludarabine dose-response effect for this phenom CldUrd-negative and IdUrd-positive cells are cells that were not in the S phase at the time of fludarabune administration and that reinitiated DNA synthesis. The CIdUrd-positive enon, tumor-bearing mice were preinjected with CldUrd, treated with cells are cells that were in the S phase at the time of fludarabine administration. The either 400 or 200 mg/kg fludarabine, and tumors were harvested 24 h IdUrd-positivecells are cells that reunitiatedDNAsynthesisafter fludarabuneadministration. 6204

Fig. 5. Fludarabine-induced apoptosis in SA-NH tumors. Ught micrographs of 4-pin tumor sections stained with hematoxylin and eosin. Mice bearing 8-mm tumors were given i.p. injections of an 800-mg/kg fludarabine dose (bottom). Tumors were harvested 12 h after fludarabine administra tion, fixed in neutral-buffered 10% formalin, and paraffin embedded. Apoptotic nuclei (arrows) are clearly identified from morphological criteria in cludingemptyspacebetweentheneighboringcells, I condensation of nuclei into dense particle, and frag .@â€˜I mentation into several bodies. Untreated tumor (0 h) is shown (top). X 1000.

â€¢@Ibâ€¢ .JÃ¸@ c . ,. ment. To this end, mice bearing 8-mm SA-NH tumors were treated termination associated with the effect of fludarabine on DNA with 800 mg/kg fludarabine and tumors were harvested at various synthesis. times after drug administration and prepared for histological analysis. A time course of fludarabine-induced apoptosis is shown in Fig. 7. As shown in Fig. 5, hematoxylin- and eosin-stained tissue sections The pretreatment background level of apoptotic cells in this tumor from tumors harvested 12 h after fludarabine treatment contained cells was 0.7%. After fludarabine administration (800 mg/kg), the fre exhibiting the typical morphology of apoptotic bodies. To determine quency of apoptotic bodies increased by 3 h, peaked 7 h after drug whether these apoptotic-appearing cells were undergoing DNA frag administration, and then progressively declined. Interestingly, a sec mentation, adjacent tissue sections were incubated with terminal de ond smaller apoptotic peak (5.7%) was observed at 27 h, suggesting oxynucleotidyl transferase in the presence of biotinylated dUTP to that fludarabine might set off a cyclical process of apoptosis induc label DNA ends and the incorporated precursors were visualized by tion. It was also of interest that the frequencies of apoptotic figures immunocytochemical means utilizing horseradish peroxidase-conju (1.3%) observed at 36 and 48 h were still slightly above the gated avidin. As illustrated in Fig. 6, nearly all of the morphologically background level. apparent apoptotic figures became highly stained by this in situ Because flow cytometry had suggested a preferential loss of cells in end-labeling procedure. The findings that no end labeling was the S phase at the time of treatment, it was of interest to determine observed in nonapoptotic cells and that the peak of apoptotic directly whether these S-phase cells were induced by fludarabine to figures occurred well after the shut-off of DNA synthesis suggest undergo apoptosis. To this end, mice bearing SA-NH tumors were that this assay is identifying DNA fragmentation associated with injected with CldUrd 30 mm before fludarabine administration (800 cells undergoing apoptosis, rather than premature DNA chain mg/kg). At various times (0, 6, 12, and 24 h) after fludarabine 6205

APOFIOSIS AND CELL CYCLE SYNCHRONIZATIONINDUCED BY F-ara-AMP

might also induce apoptosis in a subpopulation of cells not in the S @ phase at the time of fludarabine administration.

a. . DISCUSSION -,, !...:.@ , @. The experiments described here were carried out to better under â€¢0 -@ stand the mechanisms involved in the enhancement of radiation m induced tumor control by fludarabine. Previous experiments have @ :. â€¢ .:@b,,. shown that fludarabine was an effective inhibitor of chromosome . .â€˜â€˜ â€˜- damage repair after ionizing radiation in vitro (23) and therefore might act as a radiation sensitizer in vivo. Subsequent in vivo studies using murine tumor models supported this hypothesis by demonstrat ing that when given proximally in time to a dose of radiation, @ @â€˜â€˜: :.â€˜ fludarabine enhanced radiation-induced tumor growth delay and local @%,a tumor cure beyond that expected by additivity (25, 26). However, @@ ..8 , unexpectedly,themostsignificantradiationenhancementbyfludara @ @:â€˜@Iia#dlII@@ bineoccurredwhenadministered24hbeforeirradiation,especiallyat @ :@ ,: q@, doses of 400 and 800 mg/kg. Thus, an additional mechanism of

@ â€˜@. . drug-radiation interaction must have occurred. The present @ .. . I experimentsweredesignedtoexplorethispossibility. . C@ . @t . f. : Fludarabine is known to be an efficient inhibitor of DNA synthesis , â€˜ as well as other cellular functions associated with DNA metabolism @ @. â€˜ .@ , . , S (e.g., inhibition of ribonucleotide reductase activity), and the degree @ ..- ,,. 4. ._4..k@,,...â€˜ a effecthasbeenshowntoberelatedtotheF-ara-ATPlevels @ . I.. : . :@ achieved intracellularly (35, 36). For example, our previous studies â€œ_4 .sâ€”@.', , hadshownthatF-ara-Ainhibitedchromosomerepairinadose @@@@ â€¢1'@'@ @..â€”I . dependentfashion, with high dosesinhibiting both fast and slow @ . ,-@ . chromosome repair and lower doses only interfering with slow chro I .â€˜ 4 mosome repair (23). Although it is difficult to directly measure the @ â€¢.: , - @- . â€˜ F-ara-ATP levels achieved in tumors in vivo in order to obtain @@@ - â€˜ . . correlationswith tumor control, an indirect measureof F-ara-ATP @ ;,: , â€¢ , inhibitory activity is the degree to which DNA synthesis is inhibited @ i@, ...@â€”,#...@, , â€˜@.,;b . . # in the tumor cells after fludarabine administration. By using the @ ,, incorporation of halogenated pyrimidine as a measure of effect on .@@.,1up@â€”.â€¢@ V @â€¢ â€˜ DNAsynthesisinvivo,thepresentstudyshowedthatfludarabine

@â€” effectively shut down DNA synthesis in vivo in a dose-dependent Fig. 6. Fludarabine-induced DNA fragmentation in SA-NH tumors. Light micrograph fashion. Within 3 h, higher doses of fludarabine (800 and 400 mg/kg) of a 4-sm tumor section processed for an in situ end-labeling assay. Mice bearing 8-mm tumors were given i.p. injections of an 800-mg/kg fludarabine dose. Tumor was harvested were highly effective in blocking DNA synthesis, whereas lower 12 h after fludarabine administration, fixed in neutral-buffered 10% formalin, and paraffin doses (200 and 100 mg/kg) were somewhat less effective. Moreover, embedded. Tissue sections were incubated with terminal deoxynucleotidyl transferase in the presence of dUTP and the incorporated precursors were visualized using immunocy the duration of DNA synthesis inhibition in the tumor was fludarabine tochemistry. The section was counterstained with methyl green. Nearly all of the mor phologically apparent apoptotic figures became highly stained (arrows). Only a few apoptotic cells were not stained by this assay (arrowheads). X 400.

treatment, tumors were harvested and prepared for histological anal (I) 10 ysis, and CldUrd-containing cells were visualized using immunohis 8 tochemical means with an antibody to CldUrd containing DNA. As shown in Fig. 8, in untreated tumors, the fraction of CIdUrd-labeled 6 cells was in the range of 20â€”25%.After fludarabine treatment, there 2 I- was a progressive decline in the number of prelabeled cells per field, z 4 and by 24 h after fludarabine, very few labeled cells were visible in â€˜U U the tumors, confirming the preferential loss of cells that were in the S 2 phase at the initiation of fludarabine treatment. At all times studied, â€˜Ua. well-defined apoptotic bodies were observed in cells that were labeled 0 with the CIdUrd-staining technique; however, the dark staining, the @ small size of some of the apoptotic bodies, and the slight loss of tissue â€”@T II I architecture under the conditions used for immunohistochemical anal 0 4 8 12 16 20 24 28 32 36 40 44 48 ysis made quantitative evaluation of labeled apoptotic bodies difficult. TIME AFTER FLUDARABINE ADMINISTRATION (h)

Nevertheless, these studies strongly suggested that apoptosis played a Fig. 7. Kinetics of fludarabine-induced apoptosis in SA-NH tumor. An 800-mg/kg substantial role in the gradual clearance from the tumor of cells that fludarabine dose was injected i.p. into tumor-bearing mice, and, at various times later, were in the S phase at the time of fludarabine treatment. In addition, tumors were harvested and fixed in 10% neutral-buffered formalin. Paraffin-embedded sections were stained with hematoxylin and eosin, and apoptotic nuclei were counted unlabeled apoptotic nuclei were also found in the SA-NH tumors under light microscopy and expressed as a percentage of total nuclei. Each time point is following fludarabine administration, suggesting that fludarabine the mean Â±SE from three tumors. Five hundred nuclei were scored per tumor. )( 400. 6206

APOVrOSI5 AND CELL CYCLE SYNCHRONIZATION INDUCED BY F-ara-AMP

@ .,@ .@â€˜ ..â€” @@@ â€˜@. f- ., ,@.j_'#. 6h @ Â¶4@, @ .,, C @:

# :@ 4@;@'@ .f@ -5 @ .@ . a -.@

J@., .â€˜ * :@f â€¢1

, .@t 12h 24h

A ,@ I@. ,I â€˜,,.. â€˜@.s!e 4 ,

;@â€˜. @ â€¢@...../ I ,@ @)S @ v,, .@ @â€˜:@@- t@: @ ..p â€˜@r , @ p. .,.

.@- ,, ,@b .â€˜ @â€˜ Fig. 8. Light micrographs of SA-NH tumor sections after immunohistochemical staining of CldUrd-labeled nuclei. Tumor S-phase cells were labeled with CIdUrd injected i.p., and 30 mm later, an 800-mg/kg fludarabine dose was given. Tumors were harvested at 0, 6, 12, and 24 h after fludarabine administration, fixed in neutral-buffered 10% formalin, and paraffin-embedded sections were processed for immunohistochemicalstaining oflabeled nuclei using an antibody to CldUrd containing DNA. Labeled apoptotic nuclei, arrows. Labeled nonapoptotic nuclei, arrowheads. X 1000.

dose dependent, with DNA synthesis reinitiating at 12, 6, <6, and <3 through the cell cycle in vitro, with G2-M cells generally exhibiting a h after doses of 800, 400, 200, and 100 mg/kg, respectively (Fig. 2). 25â€”50%decrease in survival compared to exponentially growing These results indirectly suggest a dose-dependent concentra cells, depending on the radiation doses used (37). Thus, these results tion X time relationship for F-ara-ATP levels in the tumor after suggest that the basis of the increased tumor control observed at 24 h fludarabine administration. Since the degree of chromosome break after fludarabine administration was not inhibition of radiation-in repair inhibition is also dependent on the intracellular F-Ara-ATP duced chromosome break repair, but rather a partial synchronization concentration, these results help to explain the fludarabine dose of the tumor cells to a more radiosensitive phase of the cell cycle. dependent effect on radiation-induced tumor regrowth delay when Taken together, these results suggest that fludarabine can increase fludarabine was administered 3 h before irradiation, where fludarabine the radiosensitivity of tumor cells through at least two different doses of 800 and 400 mg/kg yielded similar degrees of normalized mechanisms, one involving inhibition of chromosome repair and tumor growth delays (i.e., that beyond the sum of fludarabine and another through synchronization to a more radiosensitive phase of the radiation effects alone) that were higher than that achieved by lower cell cycle. One could therefore hypothesize that both mechanisms fludarabine doses (25). could be therapeutically useful if fludarabine and radiation were given In the experiments described here, DNA synthesis in tumor cells in a fractionated schedule. Indeed, we have previously shown that the reinitiated at various times after a single fludarabine injection depend normalized regrowth delay effect is increased to an even greater ing on the dose. At all doses, DNA synthesis was evident by 24 h after degree when tumor-bearing mice were treated with four daily frac fludarabine treatment. Most likely the intracellular levels of F-ara tions of radiation, each preceded 3 h earlier by a 400 mg/kg fludara AlP had dropped to noninhibitory levels by 24 h after treatment. bine dose (25). Thus, each fludarabine dose inhibited chromosome Nevertheless, our previous studies showed that the most significant repair at the time of irradiation, induced S-phase cell kill, and impact of fludarabine on normalized radiation-induced growth delay synchronized the tumor cells for radiation the next day. occurred when fludarabine was given 24 h before irradiation, and this The studies decribed above helped to explain the basis for fludara effect was more prominent at higher fludarabine doses (i.e., 800 and bine enhancement. The studies described here also shed light on the 400 mg/kg) (25). The resultsshownin Figs. 1 and2 indicatethatat 24 mechanism of fludarabine-induced cytotoxicity in this tumor system. h after the higher fludarabine doses, there was a parasynchronous In a dose-dependent fashion, fludarabine was shown to effectively movement of tumor cells to the G2-M phase of the cell cycle. At lower inhibit DNA synthesis, and those cells in the S phase at the time of fludarabine doses, the degree of induced parasynchrony was less fludarabine administration were permanently unable to reinitiate DNA pronounced, and these cells had progressed beyond the G2-M bound synthesis even after intracellular levels of F-ara-ATP had fallen below ary into the next cell cycle. Radiosensitivity has been reported to vary inhibitory concentrations. Moreover, the cells that were caught in the 6207

S phase at the time of fludarabine administration were eliminated these mechanistic studies provide additional insights for new from the tumor within 24 h, apparently through an apoptotic strategies for clinical trials combining fludarabine and radiation. mechanism. Fludarabine-induced apoptosis in SA-NH mouse tumors was a ACKNOWLEDGMENTS somewhat unexpected finding. Although fludarabine has been shown to induce apoptosis in rapidly proliferating lymphoblasts in vitro (38, We aregratefulto LaneWatkinsandhis stafffor the supplyandexemplary 39) and in slowly proliferating lymphocytes from patients with care of the animals used in these studies and to Nancy Hunter and Susy Cwerin for technical assistance. We also thank Nicholas Terry for helpful discussions chronic lymphocytic leukemia (40), fludarabine-induced apoptosis regarding the labeling technique. has not been previously reported in solid tumors. Moreover, the SA-NH tumors used in these experiments exhibited low apoptotic activity in the unperturbed state and are relatively resistant to induced REFERENCES apoptosis by radiation (41) and some other chemotherapeutic agents 1. Fertil, B., and Malaise, E. P. Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. tnt. J. Radiat. Oncol. Biol. Phys., 7: 621â€”629,1981. such as cyclophosphamide.6 These studies therefore suggest that 2. Weichselbaum, R. R., Dahlberg, W., and Little, 3. B. Inherently radioresistant cells apoptosis may be triggered by various initiating events to which exist in some human tumors. Proc. Natl. Acad. Sci. USA, 82: 4732â€”4735,1985. different cell types may react with differential sensitivity. In the case 3. Weichselbaum, R. R., Dahlberg, Beckett, M., Karrison, T., Miller, D., Clark, J., and Ervin, T. J. Radiation-resistant and repair-proficient human tumors cells may be of SA-NH tumor cells, termination of DNA strand elongation, perhaps associated with radiotherapy failure in head- and neck-cancer patients. Proc. Nail. through the chain-terminating activity of incorporated F-Ara-ATP, Acad. Sci. USA, 83: 2684-2688, 1986. 4. Weichselbaum, R. R., Bcckett, M. A, Schwartz, J. L, and Dritschilo, A. Radiore activated a program of cell death associated with the up-regulation of sistant tumor cells are present in head and neck carcinomas that recur after DNA degradation activity in the S-phase cells. radiotherapy. Int. J. Radiat. Oncol. Biol. Phys., 15: 575â€”579,1988. When mice bearing SA-NH tumors were pretreated with CldUrd to 5. West, C. M. L., Davidson, S. E., Hendry, J. H., and Hunter, R. D. Prediction of cervical carcinoma response to radiotherapy (Letter). Lancet, 338: 818, 1991. label the S-phase cells before fludarabine administration and then 6. Bmck, W. A., Baker, F. L, Wike, J. L, and Peters, L 3. Cellular radiosensitivity of histological sections were immunocytochemically stained and exam primary head and neck squamous cell carcinomas and local tumor control. mt. J. Radiat. Oncol. Biol. Phys., 18: 1283â€”1286,1990. med for apoptotic bodies after fludarabine treatment, a fraction of the 7. WIOdek,D.,andHittelman,W.N.Therepairofdouble-strandDNAbreakscorrelates apoptotic cells were not labeled by the anti-CldUrd-labeling proce with radiosensitivityofL5178Y-Sand L5178-Rcells@RadiaLRes., 112: 146-155, 1987. dure. This would suggest that some cells from outside of the S phase 8. Wlodek, D., and Hittelman, W. N. The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells: II. Repair. Radiat. Res., 115: might also be susceptible to fludarabine-induced apoptosis. One pos 566â€”575,1988. sibility is the cells just entering the S phase during fludarabine 9. Schwartz, J. L, Rotmensch J., Giovanazzi, S., Cohen, M. B., and Weichsclbaum, R. R. Faster repair of DNA double-strand breaks in radioresistant human tumor cells. exposure. The origin of these cells is not known. Close examination of tnt. J. Radiat. OncoL Biol. Phys., 15: 907â€”912,1988. Fig. 1 does not show evidence of a preferential pattern of DNA 10. Kelland, L R., Edwards, S. M., and Steel, G. G. Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radioscnsitiv degradation from any fraction of the cell cycle. Future experiments ity. Radiat. Rca., 116: 526â€”538,1988. utilizing an end-labeling assay on cells prepared for flow cytometry 11. Schwartz, J. L, and Vaughan, A. T. M. Association among DNA/chromosome break may help to address this question. rejoining rates, chromatin structure alterations, and radiation sensitivity in human tumor cell lines. Cancer. Res., 49: 5054â€”5057, 1989. The S-phase specific mechanism of fludarabine action might sug 12. Downes, C. S., Collins, A. R. S., and Johnson, R. 1. International workshop on gest that with a fractionated schedule of administration, one dose of inhibition of DNA repair. Mutat. Res., 112: 75â€”83,1983. 13. Bryant, P. E., and iliakis, G. Possible correlations between cell killing, chromosome fludarabine might both effectively eliminate S-phase cells from the damage and DNA repair after X-irradiation. Nucleic Acids Symp. Set., 13: 291-308, tumor and partially synchronize cells for the next fludarabine dose. 1984. Moreover, when combined with radiation, the drug might reduce or 14. Iliakis, G., Pantelias, 0. E., and Seaner, R. Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau phase CHO cells measured by even control tumor proliferation between radiation doses. Indeed we premature chromosome condensation: implications for repair and fixation of a-PU). previously reported that when fractionated schedules of radiation and Radiat. Rca., 114: 361-378, 1988. 15. Iliakis, 0., Pantelias, 0., Okayasu, R., and Seaner, R. Comparative studies on repair fludarabine were used, no tumor growth was observed during the inhibition by araA, araC and aphidicolin of radiation induced DNA and chromosome treatment, whereas during radiation treatment alone, the tumors in damage in rodent cells: comparison with fixation of PU). tnt. J. Radiat. Oncol. Biol. creased in size (25). In some tumors such as head and neck squamous Phys., 16: 1261â€”1265,1989. 16. Malaise, E. P., Debieu, D., Chavaudra, N., and Grinfeld, S. The effect of the oxic cell cell carcinoma, proliferation between radiation doses has been con sensitizer @-Ara-Aonhuman fibroblasts in plateau and in exponential growth phases. sidered a limiting factor for local tumor control after radiotherapy Int. J. Radiat. Oncol. Biol. Phys., 16: 1257â€”1259,1989. 17. Chavaudra, N., Halimi, M., Parmentier, C., Gaillard, N., Grinfeld, S., and Malaise, (42). Thusstrategiesaimedat reducingtheoveralltreatmenttime E.P.Theinitialslopeofhumantumorcellsurvivalcurves:itsmodificationbythe (e.g., accelerated fractionation or concomitant boost) have been pro oxic cell sensitizer @-arabinofuranosyladenine. Int. J. Radiat. Oncol. Biol. Phys., 16: posed to overcome this problem (43, 44). The mechanistic studies 1267â€”1271,1989. 18. Montgomery, J. A., Clayton, S. D., and Shortnacy, A. T. An improved procedure for presented here suggest that the combination of fludarabine and radi the preparation of 9-@-D-arabinofuranosyl-2-fluoroadenine. J. Hetemcycl. Chem., 16: ation treatment might represent another approach to overcome tumor 157â€”160,1979. 19. Catapano, C. V., Perrino, F. W., and Fernandes, D. J. Primer RNA chain termination proliferation during radiation treatment. Since more actively prolifer induced by 9-f3-r-arabinofuranosyl-2-fluoradenine 5'-triphosphate. J. Biol. Chem., ating and more radioresistant tumor cells are likely to be progressively 268: 7179â€”7185,1993. selected during a standard radiotherapy treatment course, a clinical 20. Huang, P., Chubb, S., and Plunkett, W. Termination of DNA synthesis by 9-@-D- arabinofuranosyl-2-fluoroadenosine: a mechanism for cytotoxicity. 1. Biol. Chem., strategy combining fludarabine and radiotherapy during the end of a 265: 16617â€”16625,1990. standard treatment course might offer one way to exploit the different 21. Tseng, W. C., Derse, D., Clung, Y. C., Brockman, R. W., and Bennett, L L Jr. In mechanisms of interaction between fludarabine and radiation. This vitro biological activity of 9-@-D-arabinofuranosyl-2-fluoroadenosine and the bio chemical actions of its triphosphate on DNA polymerases and ribonucleotide strategy would have increased attractiveness if the tumor cells still reductase from HeLa cells. Mol Pharmacol., 21: 474â€”477,1982. surviving at the end of the radiotherapy schedule were resistant due to 22. Yang, S. W., Huang, P., Plunkett, W., Becker, F. F., and Chan, J. Y. H. Dual mode of inhibition of purified DNA ligase I from human cells by 9-@-o-arabinofuranosyl an increased capacity for repair, a process preferentially poisoned by 2-fluoroadenosine. J. Biol. Chem., 267: 2345â€”2349,1992. fludarabine. 23. Jayanth, R. V., and Hittelman, W. N. 9-@-o-Arabinofuranosyl-2-fluoroadcrnne This study demonstrates that fludarabine-induced inhibition of re (F-ara-A) inhibits both the fast and slow components of chromosome repair (Abstract). In: J. D. Chapman, W. C. Dewey, and 0. F. Whitmore (eds.), Radiation pair, fludarabine-induced apoptosis, and cell cycle synchronization all Research: A Twentieth-Century Perspective, Vol 1, p. 41 1. San Francisco: Academic contribute to the enhancement of tumor radiation response. Moreover, Press, 1991. 6208

24. Keating, M. J., McLaughlin, P., Plunkett, W., Robertson, L E., O'Brien, S., Gandhi, and Van Dierendonck, J. H. A new method to detect apoptosis in paraffin sections: in V., GrÃ©goire,V.,Yang, L., and Cabanillas, F. Fludarabineâ€”presentstatus and future situ end-labeling of fragmented DNA. 1. Histochem. Cytochem., 41: 7â€”12,1993. developments in chronic lymphocytic leukemia and lymphoma. Ann. Oncol., 5 35. Dow, L W., Bell, D. E., Poulakos, L, and Fridland A. Differences in metabolism and (Suppl. 2): S79-583, 1994. cytotoxicity between 9-@3-o-arabinofuranosyladenincand9-@-r-arabinofuranosyl-2- 25. GrÃ©goire,V., Hunter, N., Milas, L, Bmck, W. A., Plunkett, W., and Hittelman, W. N. fluoroadenine in human leukemic lymphoblasts. Cancer Rca., 40: 1405â€”1410,1980. Potentiation of radiation-induced regrowth delay in murine tumors by fludarabine. 36. Plunkett, W., Chubb, S., Alexander, L, and Montgomery, 1. A. Comparison of the Cancer Res., 54: 468â€”474,1994. toxicity and metabolism of 9-@-r-arabinofuranosyl-2-fluoroadenine and 9-fl-n-ar 26. GrÃ©goire,V., Hunter, N., Brock, W. A., Milas, L, Plunkeu, W., and Hittelman, W. N. abinofuranosyladenine in human lymphoblastoid cells. Cancer Res., 40: 2349â€”2355, Fludarabine improves the therapeutic ratio of radiotherapy in mouse tumors after 1980. single dose irradiation. Int. J. Radiat. Oncol. Biol. Phys., 30: 363â€”371,1994. 37. Sinclair, W. K. Cyclic x-ray response in mammalian cells in vitro. Radiat. Res., 33: 27. Avramis, V. I., and Plunkeu, W. Metabolism and therapeutic efficacy of 9-j3-r- 620-643, 1968. arabinofuranosyl-2-fluomadenine against murine leukemia P388. Cancer Rca., 42: 38. Huang, P., and Plunkett, W. Cell cycle-dependent apoptosis induced by nucleoside 2587â€”2591,1982. analogues (Abstract). Proc. Am. Assoc. Cancer Res., 33: 151, 1992. 28. Volpe, 1. P., Hunter, N., Basic, I., and Miles, L Metastatic properties of murine 39. Huang, P., and Plunkett, W. A quantitative assay for fragmented DNA in apoptotic sarcomas and carcinomas. I. positive correlation with lung colonization and lack of cells. Anal. Biochem., 207: 163â€”167,1992. correlation with s.c. tumor take. Clin. Exp. Metastasis, 3: 281â€”294,1985. 40. Robertson, L E., Chubb, S., Meyn, R. E., Story, M., Ford, R., Hittelman, W. N., and 29. Milas, L, Hunter, N., Mason, K., and Withers, H. R. Immunological resistance to Plunkett, W. Induction of apoptotic cell death in chronic lymphocytic leukemia by pulmonarymetastasesinC3Hf/Bumicebearingsyngeneicfibrosarcomaofdifferent 2-chloro-2'-deoxyadenosine and 9-fl-n-arabinosyl-2-fluoroadenine. Blood, 81: sizes. Cancer Rex., 34: 61â€”71,1974. 143â€”150,1993. 30. Begg, A. C., and Hofland, I. Cell kinetic analysis of mixed populations using 41. Meyn, R. E., Stephens, L C., Mg, K. K., Hunter, N. R., Brock, W. A., Milas, L, and three-color fluorescence flow cytometry. Cytometry, 12: 445â€”454,1991. Peters, L J. Heterogeneity in the development of apoptosis in irradiated murine 31. Pollack, A., Terry, N. H. A., Van, N. T., and Meistrich, M. L Flow cytometry tumours of different histologies. Int. J. Radial. Biol., 64: 583â€”591,1993. analysis of two incorporated halogenated thymidine analogues and DNA in a mouse 42. Withers, H. R., Taylor, J. M. 0., and Maciejewski, B. The hazard of accelerated tumor mammary tumor grown in vivo. Cytometry, 14: 168â€”172,1993. clonogen repopulation during radiotherapy. Acts Oncol., 27: 131â€”146,1988. 32. Stephens, L C., Mg, K. K., Schultheiss, 1. E., Milas, L, and Meyn, R. E. Apoptosis 43. Knee, R., Fields, R. S., and Peters, L .1.Concomitant boost radiotherapy for advanced in irradiated murine tumors. Radiat. Res., 127: 308â€”316,1991. squamous cell carcinoma of the head and neck. Radiother. Oncol., 4: 1â€”7,1985. 33. Gabrieli, Y., Sherman, Y., and Ben-Sasson, S. Identification of programmed cell 44. Honot, 3. Cl., van den Bogaert, W., Mg, K. K., van der Schueren, E., Bartelink, H., death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119: Gonzalez, D., de Pauw, M., and van Glabbeke, M. European Organization for 493â€”501,1992. Research on Treatment of Cancer trials using radiotherapy with multiple fractions per 34. Wijsman, J. H., Jonker, R. R., Keijzer, R., Van Dc Velde, C. J. H., Cornelisse, C. J., day. A 1978â€”1987survey.Front. Radiat. Ther. Oncol., 22: 149â€”161,1988.

6209

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. The Role of Fludarabine-induced Apoptosis and Cell Cycle Synchronization in Enhanced Murine Tumor Radiation Response in Vivo

Vincent Grégoire, Nguyen T. Van, L. Clifton Stephens, et al.

Cancer Res 1994;54:6201-6209.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/23/6201

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/54/23/6201. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.