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Clinical Cancer Research Vol. 9, 3204-3212, August 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Activation of a p53-mediated Apoptotic Pathway in Quiescent Lymphocytes after the Inhibition of DNA Repair by Fludarabine1

V. Ashutosh Rao and William Plunkett2

Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and The University of Texas Graduate School of Biomedical Sciences, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The inhibition of UV-initiated DNA repair by 9-ß-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), the nucleoside of fludarabine, induces apoptosis in quiescent human lymphocytes. The sensing and signaling mechanisms after DNA repair inhibition by F-ara-A are unknown. The purpose of this study was 2-fold: (a) determine the importance of the inhibition of DNA repair processes for F-ara-A cytotoxicity and (b) identify the apoptotic signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A.

Experimental Design: Lymphocytes were treated with F-ara-A to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Cell viability was quantitated with respect to the treatments alone and in combination to evaluate the actions of F-ara-A inhibition of DNA repair on p53 status and Fas death receptor ligand expression and function.

Results: Preincubation of lymphocytes with 3 µM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. After equivalent repair inhibition with 0.1 µM aphidicolin, there was apparently lesser p53 activation and significantly less apoptosis in irradiated lymphocytes than after 3 µM F-ara-A. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 µM aphidicolin lowered the apoptotic response to that observed with aphidicolin and UV. p53 serine 15 phosphorylation and protein accumulation were detected 2 h after treatment. Fas and Fas ligand mRNA expression and protein levels increased significantly after repair inhibition. Neutralizing antibodies against Fas or Fas ligand significantly reduced apoptosis.

Conclusions: These results suggest that inhibition of UV-induced DNA repair by F-ara-A is critical for cytotoxicity and that induction of apoptosis may be conducted by a p53-mediated signaling mechanism to the Fas death pathway.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fludarabine is most effective in the treatment of indolent diseases such as chronic lymphocytic leukemia and low-grade non-Hodgkin’s lymphomas (1 , 2) . In addition, the nucleoside of fludarabine, F-ara-A,3 is a potent inhibitor of cellular DNA synthesis in cycling cells (3) . This inhibition is brought about by the actions of its triphosphate metabolite, F-ara-ATP (4 , 5) , which is incorporated into the nascent strand causing DNA chain termination. Furthermore, the incorporated analogue is resistant to excision by DNA polymerase-associated exonuclease activity (6) . Also, self-potentiating inhibition of the repair process occurs via inhibition of ribonucleotide reductase, causing a decrease in the cellular deoxynucleotide triphosphate pools required for DNA replication and repair (7) . Such a reduction expedites the use of F-ara-ATP by DNA polymerases for the incorporation of F-ara-AMP into DNA. This incorporation has been shown to be a critical event in the mechanism for F-ara-A-mediated cell death in growing cells (3) .

The inhibition of unscheduled DNA synthesis by a nucleoside analogue in quiescent cells undergoing DNA repair has been studied as a means of sensitizing human lymphocytes and fibroblasts to undergo apoptosis (8 , 9) . In previous studies, ara-C triphosphate (8) and also F-ara-ATP (9) was shown to be incorporated into DNA during UV-induced NER. Such incorporation into repair patches blocked the DNA repair processes as shown by measurement of the uptake of radioactive thymidine. The inhibition of UV-initiated DNA repair correlated with the loss of clonogenicity in confluent fibroblasts (8) and a greater than additive increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive quiescent lymphocytes indicative of apoptosis (9) . This model was also tested using the comet assay in which repair inhibition by either fludarabine or the structurally related nucleoside analogue clofarabine initiated apoptosis in chronic lymphocytic leukemia cells (10) . However, little is known about processes for sensing DNA repair inhibition, whether analogue incorporation is requisite for cell death or the pathways by which signals for apoptosis are transduced. Stalled replication forks created after deoxynucleotide pool depletion by hydroxyurea have been observed to activate ATR (11 , 12) and BRCA1 (13 , 14) . The increased expression of wild-type ATR enhanced the phosphorylation of BRCA1 on serine 1423 after being exposed to hydroxyurea (14 , 15) . Also, the cytotoxic response to ara-C was shown to include the c-abl tyrosine (16) , p53 signaling (17 , 18) , caspase-3 activation (19) , and subsequent DNA fragmentation that leads to cellular demise (20 , 21) . Resting cells as well as phytohemagglutinin-activated lymphocytes treated with ara-C exhibited p53 accumulation and an increase in Fas and Fas ligand that was subsequently associated with cell death (18 , 22) .

Extensive work has also been done to understand the role of p53 in apoptotic signaling after DNA damage (23, 24, 25) . In replicating lymphoblast cell lines, the phosphorylation of p53 at serine 15 and the subsequent stabilization of the protein under DNA damaging conditions occur before apoptosis (24 , 26 , 27) . Mechanistically, the transcriptional activity of p53 increased the expression of Fas and Fas ligand after DNA damage conditions (28, 29, 30) . Additionally, the first intron of the promoter region of the human Fas receptor was shown to have a binding site for p53 (29) . This is vital in achieving maximal expression and proapoptotic signaling by wild-type p53. These responses were also investigated by comparing normal and temperature-sensitive mutant p53 cells exposed to DNA damaging drugs (30, 31, 32) . In replicating cells, the Fas pathway for the conduction of apoptotic signal has been identified while using ara-C (18 , 22) as well as other DNA damaging agents (33, 34, 35) . The signaling pathways activated after the inhibition of NER by fludarabine are largely unknown.

Given the absence of DNA replication, quiescent lymphocytes provide a model system to investigate the apoptotic mechanisms that are activated after F-ara-A inhibition of DNA repair. In this study, we have aimed to investigate the contribution of incorporation of the nucleoside analogue into the repair patches toward initiating apoptosis and to identify the proapoptotic signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
F-ara-A was supplied by Dr. Ven L. Narayanan (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD). [methyl, 1', 2'-3H]Thymidine (specific activity, 123 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Aphidicolin and hydroxyurea were purchased from Sigma Chemical Co. (St. Louis, MO).

Lymphocyte Collection and Cell Culture.
Whole blood was collected in heparinized tubes from healthy individuals, diluted 1:3 with cold PBS [0.135 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, (pH 7.4)] and layered onto Ficoll-Hypaque (specific gravity, 1.086; Life Technologies, Inc., Grand Island, NY). The blood was then centrifuged at 433 x g for 20 min, and mononuclear cells were removed from the interphase. Cells were washed twice with cold PBS and resuspended in 10 ml of RPMI 1640, without phenol red, supplemented with 10% fetal bovine serum and penicillin-streptomycin (Life Technologies, Inc.). The cells were then counted using a Coulter Counter (Beckman Coulter, Inc., Fullerton, CA), diluted to a concentration of 2 x 106 cells/ml, and incubated at 37°C for 4 h to remove the monocytes and acclimatize the cells to the culture conditions. The lymphocytes were then collected carefully, counted, and resuspended at a concentration of 2 x 106 cells/ml. Written informed consent, approved by the institutional review board, was obtained from all donors.

Drug Exposure and Irradiation.
Previous studies demonstrated that preincubation of lymphocytes with 3 µM F-ara-A alone for as long as 48 h did not initiate a significant apoptotic response (8) . Lymphocytes were transferred to 60-mm Petri dishes and irradiated with UV-C light (254 nm) using a UVGL-25 lamp (UVP, Inc., San Gabriel, CA). The radiation intensity was calibrated using a UVX-25 UV meter (UVP, Inc.). Immediately after irradiation, samples were incubated for indicated times at 37°C.

Analysis of DNA Repair Activity.
The incorporation [3H]thymidine into the DNA of lymphocytes exposed to UV radiation was used as a measure of unscheduled DNA synthesis. Briefly, 2 x 106 cells (F-ara-A treated or untreated in triplicate) were irradiated, then immediately incubated at 37°C for indicated times. Hydroxyurea (final concentration, 1 mM) was added to each sample 1 h before irradiation to suppress any potential anomalous DNA replication in the cells. Cell cultures (4 ml) were incubated for 90 min with [3H]thymidine (1 µCi/culture), the cells were collected, transferred onto a filter via vacuuming, and rinsed twice with 10 ml of cold PBS. The filter discs were then washed twice with 5 ml of 0.4 N perchloric acid and treated with 70% ethanol. After drying, the filter discs were removed into scintillation vials, 7 ml of scintillation fluid was added, and the level of radioactivity was quantitated using a Liquid Scintillation Analyzer (Packard Instrument Co., Downers Grove, IL).

Measurement of Caspase-8 and Caspase-3 Activity.
A microtiter plate was coated with either an anticaspase-8 (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) or anticaspase-3 monoclonal antibody (BD Biosciences, Lexington, KY), unspecific binding sites were blocked, and lysates from cells induced to undergo apoptosis were added. After washing, a caspase-8 (Ac-IETD-AFC; Biomol Research Laboratories, Inc.) or caspase-3 substrate (Ac-DEVD-AFC; Roche Molecular Biochemicals) was added. In the presence of active caspase, the substrate was cleaved into free fluorescent dye 7-amino-4-trifluormethyl coumarin that was detected by a FluoroCount Plate Reader (Packard Instrument Co.) at 505 nm. Values obtained from lysis buffer alone were used as the background and subtracted from raw data used for calculating activity. The relative change in fluorescence intensity was used as a measure of enzyme activity ({Delta}F/min).

Measurement of Apoptosis.
Cell death in lymphocytes was assessed via flow cytometry analysis with the use of annexin V (Annexin V-FLOUS staining kit; Roche Molecular Biochemicals, Indianapolis, IN), which allows for detection of the exposed phosphatidylserine moieties on the surface of apoptotic cells (using annexin V-FITC). After treatment, the cells were washed twice with 2 ml of PBS and incubated with annexin V-FITC in HEPES buffer containing propidium iodide for 15 min at room temperature while being protected from light. The staining was confirmed using a fluorescence microscope, and samples were analyzed using a BD FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Also, the raw data were analyzed using the Cell Quest 3.3 software program (Becton Dickinson Immunocytometry Systems). The apoptotic population was determined according to the percentage of total cells that were annexin V positive. Propidium iodide-positive cells were used as a control for necrotic cells (< 10% under experimental conditions).

Immunoblot Analysis of p53 and Serine 15-phosphorylated p53 Protein.
Each sample of cells was pelleted and washed twice with PBS. Each cell pellet was then washed with lysis buffer [10 mM HEPES (pH 7.4), 142 mM KCl, 1 mM EGTA, 1 mM DTT, 0.2% NP40, and protease inhibitor mixture; Roche Molecular Biochemicals] for 15 min at 4°C and centrifuged. The supernatant obtained was used for the immunoblot study. The total protein concentration in each lysate was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Aliquots of protein were boiled with Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for electrophoresis. Fractionated proteins were then transferred onto a nitrocellulose membrane by electroblotting. Nonspecific binding was blocked using 5% nonfat dry milk in Tris-buffered saline. A monoclonal antibody against p53 (Ab-6; Oncogene Research Products, Cambridge, MA) or serine 15-phosphorylated p53 (Cell Signaling Technology, Beverly, MA) was used to detect total p53 and the phosphorylated form of the p53 protein in each sample on separate gels, respectively. The blots were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Pierce Biotechnology, Inc., Rockford, IL) and normalized to the actin levels in each extract. A stripping buffer (Restore stripping buffer; Pierce Biotechnology, Inc.) was used to facilitate reprobing of the nitrocellulose membrane.

Real Time Reverse Transcriptase-PCR Analysis of Transcripts for Fas and Fas Ligand.
Total RNA was isolated from ~107 lymphocytes using the RNAzol B reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s instructions. The extracted RNA was then solubilized in diethylpyrocarbonate-treated RNase-free water and quantitated by measuring absorbance at 260 nm using a Pharmacia Biotech Ultraspec 3000 spectrophotometer (Amersham Pharmacia Biotech). TaqMan PCR core reagents (Applied Biosystems, Foster City, CA) were used according to the manufacturer’s instructions.

Reverse Transcriptase-PCR.
Forward and reverse primers for Fas, Fas ligand, and GAPDH were procured as TaqMan predeveloped reagents. Each probe was labeled using the 3'-quencher dye 6-carboxytetramethylrhodamine. The 5'-reporter dye for the Fas and Fas ligand probe was 6-carboxyfluorescein and for GAPDH was VIC. RNA samples at a concentration of 20 ng/µl were incubated with reverse transcriptase-PCR reagents in a 96-well optical plate and sealed with optical snap caps (Perkin-Elmer, Branchburgh, NJ). The optical plates were set to undergo 40 thermal cycles using a 7700 ABI Prism Sequence Detector (Perkin-Elmer). The fluorescence intensity measured at the end of each reverse transcriptase-PCR cycle was plotted against the cycle number. The first cycle in which fluorescence was detected above a threshold value (CT) was used as a measure of the final reverse transcriptase-PCR product.

Standard Curve and Quantitation.
The relative quantitation method was used to determine levels of transcript expression. For reference, CCRF-CEM cells were treated with doxorubicin (10 µg/ml) for 2 h to stimulate Fas and Fas ligand (36) . A standard curve was constructed using CT values from known concentrations of CCRF-CEM RNA (2–200 ng). Relative levels of lymphocyte transcripts were obtained by extrapolation. The levels of Fas and Fas ligand gene expression in this analysis were normalized to the GAPDH levels in the same sample and used as a measure of Fas and Fas ligand gene expression.

Accumulation of Fas, Soluble Fas Ligand, and Cellular Fas Ligand Protein.
A Fas or Fas ligand ELISA was used to measure the amount of protein in each sample. Total cell lysates were used to assay the Fas receptor. Also, culture media and cell pellets were used to measure soluble and cellular Fas ligand, respectively. A sandwich enzyme immunoassay using a mouse monoclonal antibody was used according to the manufacture’s instructions (Oncogene Research Products, Boston, MA). The monoclonal antibody was immobilized onto the surface of microtiter wells. Sample and biotinylated detector monoclonal antibody were pipetted into the wells and allowed to incubate for 3 h, during which any Fas ligand present was bound to the antibodies. After washing, horseradish peroxidase-conjugated streptavidin was added, which bound to the detector antibody. Horseradish peroxidase catalyzed the conversion of chromogenic substrate tetramethylbenzene from a colorless solution to a blue solution. Fas or Fas ligand was expressed as a measure of the absorbance of each sample at a wavelength of 450 nm analyzed by a microplate reader (Dynatech Laboratories, Inc., Chantilly, VA). Quantitation was achieved by comparing experimental samples of Fas or Fas ligand with known standards.

Inhibitor Studies.
Requirement of Fas or Fas ligand for induction of apoptosis was challenged using antagonizing antibodies. The ZB-4 antibody (Beckman Coulter, Inc.) neutralized the human cell surface antigen Fas on peripheral blood cells. Also, the NOK-1 antibody (PharMingen, San Diego, CA) antagonized both membrane-bound and soluble human Fas ligand. Lymphocytes that were treated as indicated were preincubated with either 10 ng of ZB-4 or 1 µg of NOK-1 for 1 h, and apoptosis was measured as described above. The importance of caspase-3 enzyme in executing cell death was assayed using the specific peptide inhibitor Z-DEVD-fmk (Enzyme System Products, Livermore, CA).

Statistical Analysis.
The effect of the combination of irradiation and F-ara-A versus that of either agent alone was calculated using the two-tailed Student’s t test for paired samples with the GraphPad Prism 3.0 software program (GraphPad Software, Inc., San Diego, CA). Statistical significance was defined as P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of Unscheduled DNA Synthesis by F-ara-A in Human Lymphocytes.
To extend the understanding of the mechanism of inhibition of unscheduled DNA repair by F-ara-A, we used a lymphocyte model that has been characterized previously (9) . A detailed time course analysis of the onset of DNA repair using 2 J/m2 of UV radiation and its inhibition by 3 µM F-ara-A was performed. Cellular uptake of radioactive thymidine was used as a measure of the ongoing repair resynthesis. The exposure of cells to 2 J/m2 of UV radiation increased radioactive thymidine incorporation in quiescent lymphocytes over time (0–72 h; Fig. 1Citation ). The extent of UV-induced DNA repair at 24 h was reduced by 71% in cells pretreated with 3 µM F-ara-A. Previous work has shown that the intracellular F-ara-ATP accumulated after 2 h of incubation with lymphocytes is efficiently inserted into DNA repair patches (9) ; here, we demonstrate repair inhibition 72 h after continuous incubation with drug. Thus, F-ara-A inhibited unscheduled DNA repair triggered by UV radiation.



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  		Fig. 1. Time course of unscheduled DNA repair inhibition by F-ara-A in UV-treated lymphocytes. DNA repair was quantitated by measuring cellular uptake of [3H]thymidine. Quiescent lymphocytes were treated using 3 µM F-ara-A ({blacktriangleup}), 2 J/m2 UV radiation ({blacktriangledown}), combination of the two ({circ}), or were left untreated ({square}) for 0–72 h. Thymidine incorporation is expressed in disintegrations/min (dpm)/106 cells and is representative of two donor samples in triplicate. Bars, SD.

 
Induction of Apoptosis after DNA Repair Inhibition.
The consequences of inhibition of DNA repair were evaluated by measuring caspase-3 activity and by flow cytometric analysis of annexin V-stained cells. When lymphocytes were treated using this experimental design and assayed for activation of caspase-3, it was clear the combination exhibited substantially greater caspase-3 activation at 24 h than did those treated using either F-ara-A or UV radiation alone (Table 1)Citation . After these doses were combined, the cells also exhibited significantly greater than additive annexin V-positive apoptosis at 24 h relative to the sum of actions by UV and F-ara-A alone. This effect was dependent upon time as it was not seen 12 h after combination treatment. These responses suggest a mechanistic interaction between F-ara-A and a DNA repair-inducing agent.


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  		Table 1 Induction of cell death in lymphocytes undergoing DNA repair

Caspase-3 activity was measured using the peptide substrate Ac-DEVD-AFC as described in "Materials and Methods." The percentage of the total population of cells that were annexin V positive was used as a marker for apoptosis. Data represent the mean ± SD of three separate experiments.

 
Inhibition of Repair Synthesis Is Necessary but not Sufficient to Induce Apoptosis.
To examine the contribution of F-ara-A nucleotide incorporation into repair patches to the initiation of apoptosis in lymphocytes, we compared the actions of F-ara-A with those of aphidicolin, a known DNA polymerase inhibitor that is not incorporated into DNA (37) . A concentration response study of aphidicolin was conducted to determine its inhibition of UV-induced NER in lymphocytes (Fig. 2)Citation . Preincubation of lymphocytes with 0.1 µM aphidicolin inhibited repair comparable with 3 µM F-ara-A (~61% inhibition) and, as with F-ara-A alone (Fig. 3Citation , Lane 2), did not show significant cell death (Fig. 3Citation , Lane 4). F-ara-A or UV radiation produced significantly greater than additive cell death when used in combination (Fig. 3Citation , Lane 6). In contrast, aphidicolin, when combined with 2 J/m2 UV, was not able to cause greater than additive cell death (Fig. 3Citation , Lane 7). Thus, when normalized to the extent of repair inhibition, F-ara-A elicited a greater apoptotic response than did aphidicolin, suggesting that incorporation of the nucleotide analogue contributed to the cell death response.



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  		Fig. 2. Concentration-dependent inhibition of DNA repair by aphidicolin. Cells were pretreated using the indicated concentrations of aphidicolin for 90 min and then exposed to UV radiation (2 J/m2). DNA repair was quantitated at 24 h by [3H]thymidine incorporation as described in "Materials and Methods." Repair inhibition was also caused by a 2 h incubation with 3 µM F-ara-A before irradiation. Results were obtained from two lymphocyte donors assayed in triplicate. Bars, SD.

 


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  		Fig. 3. Contribution of F-ara-A incorporation into repair patches to the initiation of apoptosis in lymphocytes. Apoptosis during UV-induced DNA repair was compared in lymphocytes irradiated with UV alone or in combination with either F-ara-A or aphidicolin at 24 h. After indicated exposures, lymphocytes were stained with annexin V. Values for annexin V positivity are expressed as fold increases over untreated sample values in Lane 1. The results are the mean of three experiments. *, P < 0.05 between Lane 7 and Lane 6, P = nonsignificant between Lane 7 and the sum of Lanes 3 and 4; **, P < 0.05 between Lane 9 and Lane 6, P = nonsignificant between Lane 9 and the sum of Lanes 2, 3, and 5; Bars, SD.

 
To additionally evaluate the impact of the nucleotide analogue incorporation on the cell death processes, we preincubated lymphocytes with 30 µM aphidicolin before addition of F-ara-A to block F-ara-A incorporation. At this concentration of aphidicolin, repair synthesis was inhibited by >95% (Fig. 2)Citation in the absence of significant apoptosis (Fig. 3Citation , Lane 5). After irradiation, the inhibition of resynthesis activity by 30 µM aphidicolin reduced cell death (Fig. 3Citation , Lane 9) to a level similar to aphidicolin alone in UV-treated lymphocytes (Fig. 3Citation , Lane 8). These results suggest that F-ara-A incorporation into repair patches is essential for induction of apoptosis in irradiated lymphocytes.

Phosphorylation and Accumulation of p53.
As p53 is involved in cellular responses to cytotoxic DNA damage, we considered that it might also be associated with apoptotic signaling when DNA repair was inhibited in quiescent cells. Phosphorylation of endogenous p53 at serine 15 after DNA damage by various protein kinases has been associated with its stabilization (23 , 24 , 27) and may be critical for its stabilization and transcriptional activity. Treatment of lymphocytes with the F-ara-A and UV combination caused an increase in both serine 15-phosphorylated p53 and total p53 protein levels (Fig. 4A)Citation . p53 phosphorylation on serine 15 occurred within 2 h. Elevated p53 protein levels were detected as early as 2 h in the absence of apoptosis, suggesting that p53 stabilization is an early event in the cellular response to inhibition of DNA repair.



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  		Fig. 4. Serine 15 phosphorylation and total p53 protein accumulation after DNA repair inhibition in human lymphocytes. Lymphocytes were incubated with 3 µM F-ara-A for 120 min before irradiation with 2 J/m2 UV. A, the levels of the total p53 protein and serine 15-phosphorylated p53 were measured in whole cell extracts by immunoblot analysis 0–48 h after treatment. B, the actions of 3 µM F-ara-A or 0.1 µM aphidicolin in UV-treated lymphocytes were compared after 4 h of treatment. Pretreatment with either agent at this concentration displayed comparable inhibition of DNA repair (Fig. 2)Citation . Actin levels were used as a loading control for each immunoblot experiment. Two independent experiments were performed with similar results.

 
The importance of incorporation of the nucleoside analogue for cytotoxicity was shown by comparison of its action with aphidicolin (Fig. 3)Citation . Additionally, to evaluate the mechanism for cell death after the inhibition of DNA repair, phosphorylation and stabilization of p53 were compared subsequent to the actions of 3 µM F-ara-A and 0.1 µM aphidicolin in UV-treated lymphocytes (Fig. 4B)Citation . Cells were pretreated with either F-ara-A or aphidicolin before irradiation. The combination of F-ara-A and 2 J/m2 UV produced a greater response in cells than observed with individual agents. p53 phosphorylation and accumulation appeared to be greater in irradiated cells treated with F-ara-A than in those incubated with aphidicolin. This result suggests that the inhibition of DNA repair may not be sufficient for activating a p53-mediated cell death signal. Rather, the presence of the nucleoside analogue appears to facilitate p53 phosphorylation associated with cell death.

Expression of Fas and Fas Ligand after DNA Repair Inhibition.
Although cytotoxic DNA damage induces accumulation of the p53 target gene Fas and its ligand (33 , 36) , their role in the cellular response to inhibition of the repair induced by nonlethal irradiation is not known. After exposure to 3 µM F-ara-A and 2 J/m2 UV, an increase in the expression of both Fas and Fas ligand mRNA was seen 2 h after treatment (Fig. 5)Citation . Also, the treatment of these cells was associated with an increase in the levels of Fas receptor as well as both soluble and cellular Fas ligand protein (Fig. 6)Citation . Significant accumulation of soluble Fas ligand, cellular Fas ligand, and Fas receptor protein was first seen 6, 4, and 6 h after combination treatment, respectively. Lymphocytes treated with both F-ara-A and UV induced significantly greater protein accumulation than they did after exposure to the single agents (Fig. 7, A and B)Citation . The activity of caspase-8, the enzyme associated with the death receptor ligand pathway (35) , was also augmented after treatment with UV and F-ara-A (Fig. 7C)Citation . This suggests that inhibition of DNA repair in lymphocytes triggered signaling that may involve interaction between the death receptor and its ligand.



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  		Fig. 5. Real-time reverse transcriptase-PCR analysis of Fas and Fas ligand transcripts. Total RNA extracted from lymphocytes was used for gene expression analysis as described in "Materials and Methods." The relative values of (A) Fas or (B) Fas ligand transcripts were standardized to GAPDH levels. The analysis plotted in the figure was obtained from a single lymphocyte donor in triplicate (±SD) and is representative of three sample donors.

 


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  		Fig. 6. Fas, cellular Fas ligand, and soluble Fas ligand accumulation after the inhibition of DNA repair. An enzyme immunoassay was used to quantitate Fas or Fas ligand in cell lysates as described in "Materials and Methods." Lymphocytes were treated with F-ara-A and UV, and samples were taken between 0–48 h. A, the culture medium was used for determining Fas ligand accumulation. B, whole cell extracts were used for cellular Fas ligand protein determination. C, Fas receptor was assayed in whole cell. Results were obtained in duplicate from two lymphocyte donors and are expressed relative to those from untreated samples. Bars, SD.

 


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  		Fig. 7. Comparison of Fas receptor and soluble or cellular Fas ligand protein expression after inhibition of DNA repair by F-ara-A. Protein levels of (A) the Fas death receptor and (B) soluble ({square}) or cellular ({blacksquare}) Fas ligand were obtained as in Fig. 6Citation . The effect of either UV or F-ara-A alone was compared with that of a combination of the two at 24 h. Protein values are expressed as fold increases over untreated samples. Mean values for Fas receptor, cellular, and soluble Fas ligand protein in untreated samples were 18.9 ng/106 cells, 19.5 ng/ml media, and 152.2 ng/106 cells, respectively (*, P < 0.05, between UV and F-ara-A combination treatment and the sum of increase in protein after UV or F-ara-A alone). C, activity of caspase-8 was measured using a fluorochrome-conjugated substrate, IETD, in cells treated as indicated for 24 h. Increase in caspase-8 activity after combination treatment compared with the sum of single agents, *, P < 0.01. n = 3; Bars, SD.

 
Inhibition of Fas and Fas Ligand.
Neutralizing antibodies against Fas and Fas ligand were used to determine whether either was required for apoptosis in this system. The NOK-1 antibody recognizes both soluble and membrane-bound Fas ligand, whereas the ZB4 antibody recognizes cell surface antigen Fas. Although, the combination of F-ara-A and UV induced annexin positivity in 43% of the cell population by 24 h, the presence of antagonistic antibodies against either Fas or Fas ligand reduced this apoptotic effect of F-ara-A and UV by 73 and 67%, respectively (Fig. 8)Citation . Coincubation with the caspase-3 inhibitor Z-DEVD-fmk reduced this cytotoxicity to background levels. These results suggest that apoptosis of lymphocytes induced by DNA repair inhibition requires, at least in part, the interaction of Fas and Fas ligand, thus engaging the extrinsic pathway for apoptosis (34) .



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  		Fig. 8. Actions of neutralizing antibodies against Fas or Fas. Cells were preincubated for 1 h with either 10 ng/ml ZB-4 or 2 µg/1 million cells of NOK-1 before exposure to F-ara-A and UV. The requirement of caspase-3 was assayed using the caspase-3 inhibitor Z-DEVD-fmk at 10 µM along with the treatment conditions. Results were obtained from three experiments (*, P < 0.05, between UV and F-ara-A combination treatment alone and the combination treatment with inhibitors). Bars, SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The precise mechanisms of F-ara-A-induced apoptosis in both growing and quiescent cells are still unknown. Previous studies presented experimental systems in quiescent lymphocytes and in growing cell lines wherein the combination of F-ara-A and an agent that causes DNA repair such as UV radiation (9) , cyclophosphamide (10) , or cisplatin (38) initiated apoptosis. UV radiation is a well-characterized agent that activates the machinery for NER; its cytotoxic effect at high doses has been studied extensively (12 , 23) . The experimental design of the present investigations was to use a concentration of F-ara-A and a dose of UV radiation that alone were not toxic to lymphocytes, thereby allowing us to study the mechanistic interaction of the combination that produces greater than additive toxicity. At 3 µM, a clinically achievable concentration, F-ara-A was nontoxic to lymphocytes even after 48 h and did not produce significant Fas/Fas ligand accumulation or caspase-3 activity. Also, DNA damage caused by 2 J/m2 UV triggered unscheduled DNA repair in the absence of significant apoptosis at 24 h. However, in combination, F-ara-A and UV radiation induced greater than additive cell death, suggesting a mechanistic interaction of the two modalities.

To evaluate the role of the incorporated nucleotide analogue in initiating cell death processes, we used a concentration of aphidicolin that inhibited UV-induced repair to a level similar to that caused by 3 µM F-ara-A. After UV-initiated DNA repair, we observed a lower degree of p53 activation (Fig. 4)Citation and cell killing (Fig. 3)Citation with aphidicolin than with F-ara-A. Experiments conducted to determine the effects of aphidicolin in irradiated cells on changes in Fas or Fas ligand protein displayed a lesser response in irradiated cells pretreated with aphidicolin than in those preincubated with F-ara-A (data not shown). Unlike F-ara-A, which is metabolized and incorporated into the DNA repair patches, aphidicolin acts solely via inhibition of DNA polymerases required for NER (37) . This aspect of the actions of F-ara-A has also been shown to be critical for its toxicity in growing cells (3 , 5) . To further test the contribution of analogue incorporation in quiescent cells, we coincubated 30 µM aphidicolin with F-ara-A before UV irradiation. The mechanistic interaction of such a combination would have allowed the buildup of F-ara-ATP while preventing the polymerases from incorporating the triphosphate into repairing DNA. Completely blocking the repair DNA synthesis with this high dose of aphidicolin significantly lowered the cell killing observed with UV and F-ara-A. Thus, we conclude that the incorporation of the analogue into repairing DNA is likely to initiate processes that increase cytotoxicity to a level greater than that caused by inhibiting polymerase activity. Studies in model systems have demonstrated that pol {epsilon}, an enzyme that participates in the resynthesis step of NER (39 , 40) , is inactivated as its 3'-5' exonuclease activity attempts to excise F-ara-AMP from the 3'-terminus of a primer-template complex (6) . If this also occurs at sites of incorporation in DNA repair patches, it may serve to attract sensors of DNA damage more effectively than do the stalled replication patches caused by aphidicolin.

Genetic deficiencies in the NER mechanism lead to well-known human syndromes (39, 40, 41, 42) . Moreover, the consequences of deficient NER machinery on UV-induced cellular responses has been extensively investigated (43, 44, 45, 46, 47) . Fibroblasts derived from xeroderma pigmentosum patients are hypersensitive to lethal doses of UV radiation and show different kinetics of p53 stabilization when compared with normal fibroblasts (48) . Correction of the repair defect by repletion of a wild-type XP gene leads to a normal p53 response in these cells (49) . Nevertheless, how lymphocytes that have otherwise competent repair mechanisms respond when the completion of NER is inhibited is not fully clear. The p53 protein has been studied for its association with DNA repair mechanisms (46 , 50, 51, 52, 53) , as well as apoptosis (23 , 54 , 55) . It is well known that the level of p53 protein level increases in response to toxic treatment with DNA damaging agents and that is followed by cell cycle arrest or apoptosis (24 , 27 , 54, 55, 56, 57) . We have demonstrated early serine 15 phosphorylation, as well as accumulation of total p53 after treatment with fludarabine and UV radiation. Although the exact consequences of site-specific phosphorylation are still unknown, p53 serine 15 phosphorylation and subsequent apoptosis have been linked with the activity of upstream sensor kinases such as ATM, ATR, or DNA-PK (26 , 58 , 59) .

The transcription activity of p53 is known to increase expression of the Fas death receptor (25 , 28 , 29 , 32) . Our studies demonstrated increased levels of Fas and Fas ligand transcription and accumulation of the proteins after DNA repair inhibition by fludarabine. The absence of a high level of expression induced by fludarabine or UV alone supports the conclusion that the mechanistic interaction of the two agents initiates signals for increased expression of the death receptor and its ligand. Interaction of the Fas and Fas ligand activates an intracellular caspase cascade that involves the engagement of active caspase-8 (34) . Proapoptotic caspase-8 then cleaves, among other substrates, caspase-3 and is responsible for mediating cellular demise (35) . Antibodies that were antagonistic to the receptor-ligand interaction significantly reduced the percentage of dying cells, implicating the Fas death receptor pathway as a means of executing apoptosis after inhibition of DNA repair. The activation of p53 and requirement for Fas provide additional biochemical evidence for the toxicity of the mechanistic interaction produced by the combination of a nucleoside analogue with a DNA repair-initiating agent. Exploiting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of human lymphocytes. The ability of quiescent cells to respond to such a combination provides a rationale for scheduling therapies that use similar means of action (60 , 61) .

Important questions regarding the role of upstream sensor kinases and p53 signaling remain to be answered. The lymphocyte model has its limitations in genetic manipulations that allow the evaluation of their role in signaling. Future studies should be aimed at developing models that would provide isogenic comparisons of sensing and signaling responses in the presence or absence of these proteins (62) .


   ACKNOWLEDGMENTS
 
We thank Min Du and Brenita Tyler for their assistance with blood sample collection.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported, in part, by NIH Grants CA32839 and CA81534 and Cancer Center Support Grant P30 CA16672 from the National Institutes of Heath, Department of Health and Human Services. Back

2 To whom requests for reprints should be addressed, at Department of Experimental Therapeutics, Box 71, The University of Texas M. D. Anderson Cancer Center; 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3335; Fax: (713) 794-4316; E-mail: wplunket{at}mail.mdanderson.org Back

3 The abbreviations used are: F-ara, 9-ß-D-arabinofuranosyl-2-fluoroadenine; F-ara-ATP, 5' triphosphate of F-ara; ara-C, 1-ß-D-arabinofuranosylcytosine; NER, nucleotide excision repair; GAPDH, glyceraldehye-3-phosphate dehydrogenase. Back

Received 1/ 6/03; revised 3/20/03; accepted 3/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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