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Mol Cancer Ther. 2006;5:400-410
© 2006 American Association for Cancer Research

c-Abl-independent p73 stabilization during gemcitabine- or 4'-thio-ß-D-arabinofuranosylcytosine–induced apoptosis in wild-type and p53-null colorectal cancer cells

Jaideep V. Thottassery1,2, Louise Westbrook1, Hitoshi Someya1 and William B. Parker1,2

1 Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research Institute and 2 University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, Alabama

Requests for reprints: Jaideep V. Thottassery, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205. Phone: 205-581-2846; Fax: 205-581-2877. E-mail: Thottassery{at}sri.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-ß-D-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53–/– HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53–/– and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis. [Mol Cancer Ther 2006;5(2):400–10]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clinically active nucleosides show their antitumor effects after conversion to triphosphates, followed by incorporation into the replicating strand and induction of apoptotic pathways. Interestingly, in certain tumors, despite being readily converted to triphosphates, nucleosides are not very effective. This suggests that the response of tumors to these agents is also likely to depend on their ability to turn on effectors of apoptosis. Both purine and pyrimidine nucleoside anticancer drugs are potent activators of p53 (1, 2). Gemcitabine, a pyrimidine nucleoside, activates p53 following its incorporation into DNA, and enforced expression of wild-type p53 in p53-null cells also seems to enhance gemcitabine cytotoxicity (3, 4). Enforced expression of wild-type p53 in p53–/– leukemia cells has also been shown to enhance their sensitivity to 1-ß-D-arabinofuranosylcytosine (ara-C), suggesting that p53 is required for efficient death from this trigger as well (5).

The activation of p53 is tightly coupled to its phosphorylation on certain NH2-terminal residues, which occurs in response to DNA damage-induced kinases and is predicted to block its interaction with murine double minute 2 (Mdm2), a protein ubiquitin ligase that normally targets p53 for rapid turnover. Gemcitabine seems to activate p53 following its incorporation into DNA by interacting with a complex containing DNA-dependent protein kinase and p53. DNA-dependent protein kinase phosphorylates p53 at Ser15 (3), which, although not sufficient to disrupt the p53-Mdm2 interaction, stimulates the transactivating function of p53 by enhancing the binding of this protein to the transcriptional coactivators p300/CREB binding protein and p300/CREB binding protein–associated factor (6–8). Phosphorylation of p53 at Ser15 might also set this protein up for secondary modifications that could alter the binding of Mdm2 to p53, thereby inhibiting p53 degradation (9). Phosphorylation of p53 at Ser20, on the other hand, interferes directly with the binding of p53 to Mdm2, thereby causing p53 accumulation in response to DNA damage (10, 11).

Previous studies have revealed a correlation between p53 status and sensitivity of tumor cells to certain chemotherapeutic drugs or a lack of such correlation in other cases (12–15). In chronic lymphocytic leukemia patients, TP53 mutation/deletion has been shown to correlate with poor response to therapy with nucleoside analogues (16, 17). However, in vitro studies examining the role of p53 in nucleoside cytotoxicity have shown conflicting results and it is known that drugs like gemcitabine and ara-C do induce apoptosis in cells with deficient p53 function (4, 18, 19). The pathways involved in the p53-independent cell death response to these drugs are, however, not well studied.

The p53 family member p73 has recently been implicated in p53-independent apoptosis (20). p73 has been reported to be involved in the cellular response to DNA damage induced by {gamma} radiation and chemotherapeutic agents such as cisplatin (21–24), a role that was previously attributed exclusively to p53. p73 is a nuclear protein that shares substantial sequence homology and functional similarities with p53 (25). Furthermore, mutant p53 can also inhibit p73 function in a dominant negative manner, suggesting that in cells with an inactive p53, p73-mediated apoptotic effects could also be impeded (26–28). This may explain why the attempt to correlate p53 status with prognosis and response to anticancer treatments like nucleosides has proved to be of limited clinical impact in the past.

4'-Thio-ß-D-arabinofuranosylcytosine (T-ara-C) is a new pyrimidine nucleoside in Southern Research Institute's ongoing anticancer drug development program. Unlike ara-C, from which it differs by a single substituent, T-ara-C is very effective against solid tumors, a property it shares with gemcitabine (29). In this report, we show that although the differential cytotoxicities of these drugs in the colon carcinoma line HCT 116 correlate with their differential abilities to induce p53 phosphorylation, stabilization, and activation, nucleoside-induced cytotoxicity is unperturbed in p53-deleted isogenic HCT 116 cells. Because in this line p73 would not be obviated by the presence of mutant p53 forms, we examined the effects of nucleosides on p73 in these cells. Interestingly, we find that T-ara-C and gemcitabine markedly induce p73 levels in p53–/– HCT 116 cells and also p73 DNA-binding activity in nuclear extracts from these cells, unlike ara-C. The upstream DNA damage-responsive kinase c-Abl has been shown to be activated by ara-C and plays a role in its apoptotic effects (30). In addition, c-Abl, which is also known to bind p53 and activate it, has recently been implicated in the phosphorylation, stabilization and activation of p73 as well (31). Using specific small interfering RNA to knockdown c-Abl expression, we show that nucleoside-induced p73 stabilization is independent of c-Abl. We also show that HCT 116 cell lines, wherein downstream p73 targets Bax and p53 up-regulated modulator of apoptosis (PUMA) are deleted, are relatively insensitive to these nucleosides. Thus, p73 stabilization could account for p53-independent apoptotic effects induced by pyrimidine nucleosides.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culture Conditions
HCT 116 cell lines (wild-type and the knockouts p53–/–, Bax–/–, and PUMA–/–) were all provided by Drs. Bert Vogelstein and Kenneth Kinzler (Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD). Cells were grown in Improved Modified Eagle's Medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Media and serum were purchased from Invitrogen (Carlsbad, CA). Cells were maintained in monolayer culture in a humidified 5% CO2 atmosphere at 37°C.

Drugs, Antibodies, and Other Chemicals
Ara-C was purchased from Sigma Chemicals, Inc. (St. Louis, MO). T-ara-C was chemically synthesized in our laboratories as previously described (32, 33). Gemcitabine was provided by Dr. Larry W. Hertel (Eli Lilly, Indianapolis, IN). The following antibodies were purchased from Cell Signaling (Beverly, MA): anti–cleaved caspase-3, anti–cleaved poly(ADP-ribose) polymerase, anti–total p53, and anti–phospho-p53 (Ser15, Ser20, Ser46). The antiactin antibody was from Sigma and the anti-Mdm2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). For Western blot analysis of p73, the monoclonal antibodies IMG-259 and IMG-246 (Imgenex, San Diego, CA) were used; for p73 DNA binding ELISAs, the ER-15 monoclonal antibody (NeoMarkers) was used. For detecting c-Abl by Western blot analysis, the 8E9 monoclonal antibody (PharMingen, San Diego, CA) was used. All other reagents were from commercial sources.

Western Blot Analysis
Cells were washed with ice-cold PBS, scraped in lysis buffer (20 mmol/L Tris, 20 mmol/L ß-glycerophosphate, 150 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 1 mmol/L Na3VO4, 0.5% NP40, 1 mmol/L DTT) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, 4 µg/mL aprotinin and 1 µg/mL pepstatin A, and sonicated for 15 seconds. Cell debris was removed by centrifugation at 13,000 rpm for 15 minutes at 4°C. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Aliquots of cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with various primary antibodies followed by relevant horseradish peroxidase–conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence using reagents from the Supersignal West Pico kit (Pierce).

p53 and p73 DNA Binding ELISA Assays
The TransAM p53 Transcription Factor Assay kit (Active Motif, Carlsbad, CA) was used and the protocol of the manufacturer was followed. Nuclear extracts were prepared from treated cells using the Nuclear Extract kit (Active Motif) and were diluted to 2 µg/mL of total protein with lysis buffer. Extracts were applied to plates containing immobilized oligonucleotide containing the p53 consensus binding site (5'-GGACATGCCCGGGCATGTCC-3'). After 1-hour incubation at room temperature, plates were washed and incubated with diluted p53 antibody (1:1,000) for another hour. Diluted anti-rabbit horseradish peroxidase–conjugated antibody (1:1,000) was then added to previously washed plates and developing solution was added and incubated for 8 minutes to allow color development. The reaction was stopped and absorbance was read at 450 nm with a reference wavelength of 655 nm. To measure p73 DNA binding activity, nuclear extracts were applied to plates containing the p53-binding site oligonucleotide as above. However, the subsequent incubation was done with an anti-p73 monoclonal antibody (ER15; NeoMarkers). Diluted horseradish peroxidase–conjugated anti-mouse immunoglobulin G antibody was added to the plates after washing away the primary antibody. Developing solution was added and absorbance read as described above.

p73 Turnover Studies
Decay of constitutive and induced levels of p73 was measured after inhibiting new protein synthesis by adding cycloheximide at a concentration of 75 µg/mL to the cells 24 hours post a short-term treatment (2 hours) with the indicated nucleosides. Cellular levels of p73 or actin were determined by immunoblotting at increasing time intervals after cycloheximide addition. The amount of p73 relative to actin was normalized to that of the non-cycloheximide-treated control in each group of samples and expressed as a percentage.

3-(4,5-Dimethyl-Thiazol-2yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium Cell Viability Assays
Subconfluent HCT 116 cells (5 x 103 per well) were plated in Improved Modified Eagle's Medium in 96-well plates and were allowed to attach overnight. The next day, cells were treated with the indicated drugs for 2 hours, washed, and refed with 200 µL of complete medium. At 24, 48, and 72 hours after the acute treatment, 40 µL of 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit, Promega, Madison, WI) were added to each well and the plates were incubated at 37°C for 2 hours. Absorbance at 490 nm was read with a 96-well plate reader (Molecular Dynamics, Sunnyvale, CA).

Small Interfering RNA Knockdown of c-Abl
p53–/– and p53+/+ HCT 116 cells (2 x 105) were plated in 60-mm dishes. After overnight attachment, cells were transfected with either a c-Abl-specific double-stranded small interfering RNA oligonucleotide (5'-AGGUGAAAAGCUCCGGGUC-dTdT-3') or a control small interfering RNA with no known homology to mammalian genes using DharmaFECT reagent (Dharmacon, Inc., Lafayette, CO). After 24 hours of incubation, the transfected cells were subjected to a short-term treatment (2 hours) with nucleosides. Cells were then harvested at 24 hours post drug treatment, lysed, and the lysates were analyzed by immunoblotting.

Colony-Forming Assays
HCT 116 cells (5 x 102) or a similar number of p53–/–, Bax–/–, or PUMA–/– HCT 116 cells were plated in 100-mm dishes. After attachment, the cells were incubated with increasing doses of T-ara-C, ara-C, or gemcitabine for 2 hours, followed by a wash. The cells were then refed with fresh drug-free medium and colonies were allowed to form. Colonies were visualized at the end of a week after fixing and staining with 0.5% crystal violet. Colonies were washed with deionized water, photographed, and counted using a Scienceware colony counter (Bel-Art Scientific Poducts, Pequannock, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Acute Treatment of HCT 116 Colon Carcinoma Cells with Structurally Similar Cytosine Analogues Induces Differential Apoptotic and Cytotoxic Responses
Previous studies showed that a short-term treatment with ara-C, at concentrations of 1 or 10 µmol/L, induces massive apoptosis of human U937 myeloid leukemia and HL-60 promyelocytic leukemia cells, respectively (34–36). Ara-C, which is structurally similar to gemcitabine and T-ara-C (Fig. 1 ), does not, however, show significant in vivo antitumor activity against solid tumors (29). Ara-C-induced cytotoxicity has been associated with apoptotic DNA fragmentation and the activation of caspase-3 cleavage (35, 36). Caspase-3 is believed to be a critical effector of apoptosis and is itself activated after the cleavage of its pro-form to a 17-kDa form (37). We compared the accumulation of cleaved caspase-3 in ara-C-, T-ara-C-, and gemcitabine-treated HCT 116 cells at 24, 48, and 72 hours after an acute treatment and washout (Fig. 2A ). Ara-C and gemcitabine were employed at 10 µmol/L whereas T-ara-C was used at 100 µmol/L in this experiment. Because T-ara-C is not as good a substrate for deoxycytidine kinase as ara-C, a 10-fold higher concentration is required to generate roughly the same level of its intracellular triphosphate as that generated by ara-C (38).


Figure 1
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  		Figure 1. Structures of T-ara-C, ara-C, and gemcitabine.

 

Figure 2
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  		Figure 2. Differential apoptotic and cytotoxic responses of structurally similar deoxycytidine analogues. A, exponentially growing HCT 116 cells (wild-type) were exposed to T-ara-C (100 µmol/L), ara-C (10 µmol/L), or gemcitabine (10 µmol/L) for 2 h. Cells were then washed and incubated in fresh medium for an additional 24, 48, or 72 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and actin. B, exponentially growing HCT 116 cells were plated in 96-well plates and were exposed to the indicated concentrations of T-ara-C, ara-C, or gemcitabine for 2 h as described above in A. At 24, 48, or 72 h post washout, 40 µL of 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay reagent were added and absorbance at 490 nm was recorded after 1-h incubation. Columns, mean fold change in absorbance over control (CT) from triplicate determinations; bars, SD.

 
We found that gemcitabine (10 µmol/L) and T-ara-C (100 µmol/L) were very effective in inducing caspase-3 cleavage and concomitant cleavage of one of its substrates, poly(ADP-ribose) polymerase, at 48 and 72 hours after a mere 2-hour drug treatment in HCT 116 cells (Fig. 2A). Similar results were obtained in a 4-hour drug treatment experiment as well (data not shown). In contrast, ara-C (10 µmol/L), which is very effective in leukemia cells, showed minimal caspase-3 or poly(ADP-ribose) polymerase cleavage (Fig. 2A). Thus, acute ara-C treatment, unlike T-ara-C and gemcitabine, is not very effective in inducing classic hallmarks of apoptosis in HCT 116 colon carcinoma cells.

The effects of these treatments were also assessed in a short-term cell viability assay. Data shown in Fig. 2B show that a 2-hour treatment with T-ara-C (100 µmol/L) or gemcitabine (10 µmol/L) induced significant decrease in viability at 48 and 72 hours after washout. Treatment with ara-C (10 µmol/L), on the other hand, exhibited very little cytotoxicity in these cells, which was consistent with its modest effects on cleaved poly(ADP-ribose) polymerase and caspase-3 levels (Fig. 2A and B). We also found that a 10-fold lower dose of T-ara-C (10 µmol/L) was also very effective in causing cytotoxicity and was more effective than ara-C at the same concentration (data not shown). In sum, these data show that short-term in vitro treatments of HCT 116 cells with structurally similar pyrimidine nucleosides exhibit distinct cytotoxic efficiencies which largely reflect the responses generated in vivo in HCT 116 tumors (29).

Levels of Total p53, Phospho-p53(Ser15), Phospho-p53(Ser20), and Phospho-p53(Ser46) Are Induced in Response to T-ara-C and Gemcitabine
HCT 116 cells have wild-type p53, whose the function has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, replication blocks, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. We asked whether the distinct cytotoxic activities of T-ara-C, ara-C, and gemcitabine were related to their individual abilities to induce activation of p53 pathways. Genotoxic stress typically results in activation of damage-responsive kinases that phosphorylate p53 on several NH2-terminal sites, and phosphorylation of two of these sites, Ser15 and Ser20, in particular, have been implicated in stabilizing p53 directly or indirectly and increasing its transcriptional activity (39). Phosphorylation of p53 on Ser46 has also been implicated in the activation of p53-dependent apoptotic response (40, 41). Replication arrest has also been shown to increase phosphorylation of p53 on Ser15 and Ser46 and induce p53 accumulation (42). Because ara-C, T-ara-C, and gemcitabine can cause structural lesions in DNA, as well as replication stress, we tested whether these three drugs could induce p53 phosphorylation and total p53 accumulation.

Western blot analysis shown in Fig. 3A shows that lysates from HCT 116 cells extracted at 24 hours following a short-term treatment with either T-ara-C or gemcitabine shows marked increases in p53 phosphorylation at Ser15, Ser20, and Ser46 relative to untreated controls. Concomitantly, there is also substantial accumulation of total p53 in these cells (Fig. 3A). Ara-C, on the other hand, following an acute treatment in HCT 116 cells, did not cause any detectable phosphorylation at Ser15 and Ser20 and only modest phosphorylation at Ser46 (Fig. 3A). However, there was significant, yet relatively modest, p53 accumulation in ara-C-treated cells at 24 hours (Fig. 3A). Extracts from p53–/– HCT 116 cells treated similarly were also run alongside the samples from wild-type cells as controls. As expected, neither total p53 nor phospho-p53-specific bands were detected in these lanes (Fig. 3A). Substantial phosphorylation and accumulation of p53 was also observed in wild-type cells at 48 and 72 hours posttreatment with T-ara-C and gemcitabine, at which times significant decrease in viability is also observed (data not shown).


Figure 3
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  		Figure 3. T-ara-C and gemcitabine induce stabilization of p53 and increase phospho-p53(Ser15), phospho-p53(Ser20), phospho-p53(Ser46), and p53 DNA-binding activity. A, exponentially growing HCT 116 cells (wild-type p53) were exposed to T-ara-C (100 µmol/L), ara-C (10 µmol/L), and gemcitabine (10 µmol/L) for 2 h. Cells were then washed and incubated in fresh medium for an additional 24 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against phosphospecific p53, total p53, actin, and Mdm2. B, nuclear extracts were prepared as described in Materials and Methods. The level of p53 that can bind to its consensus recognition sequence was determined by the TransAM p53 ELISA. Columns, mean fold increase relative to untreated cells at 0 h from triplicate determinations; bars, SD.

 
T-ara-C and Gemcitabine Treatment Enhances DNA-Binding Activity of Nuclear Extracts to a Specific p53-Responsive Consensus Element
The transcriptional activity of p53 is important in its apoptotic functions (43). We therefore assessed the ability of p53 in nuclear extracts from HCT 116 cells treated with the three nucleoside analogues to bind to a p53 consensus DNA binding site as a read-out of its transcriptional activation. We used the TransAM p53-DNA binding ELISA assay on nuclear extracts made 24 hours after a 2-hour drug treatment. As shown in Fig. 3B, the levels of p53 DNA-binding activities were markedly increased in T-ara-C- and gemcitabine-treated p53+/+ cells. The ~6.5- to 8-fold increases in p53 DNA-binding activity induced by T-ara-C and gemcitabine were markedly higher than those in nuclear extracts from ara-C-treated cells (Fig. 3B). Similar increases were also observed at 48 hours posttreatment in T-ara-C- or gemcitabine-treated cells (not shown). These results suggested that in nuclear extracts from cells treated with ara-C, the lack of detectable phosphorylation of p53 at Ser15, Ser20, and Ser46, coupled with modest p53 accumulation, results in low DNA-binding ability for p53. We also confirmed that the nucleoside-induced increases in binding to a p53 consensus element indeed showed bona fide p53 activity by examining these responses in p53–/– HCT 116 cells. Nuclear extracts from T-ara-C- or gemcitabine-treated p53–/– HCT 116 cells did not show any increased p53-DNA binding over background (Fig. 3B).

We also measured the expression of a p53 target, Mdm2 (Fig. 3A). The Mdm2 gene contains a p53 binding site composed of two consensus sequences linked by a 17-bp spacer (44). Mdm2 is transcriptionally activated by p53 and is part of an autoregulatory feedback loop that mediates p53 ubiquitination and degradation through the proteasome pathway. As shown in Fig. 3A, Mdm2 protein was induced to higher levels by T-ara-C and gemcitabine as compared with ara-C, which largely correlates with higher p53-DNA binding ability of nuclear extracts and higher total and phosphorylated p53 in cells treated with these two drugs. We also showed that the Mdm2 protein increases induced by nucleosides were p53 dependent by examining these responses at the 24-hour time point in p53–/– HCT 116 cells. As shown in Fig. 3A, T-ara-C- and gemcitabine-induced Mdm2 increases in p53+/+ cells were higher than that induced by ara-C and correlated with their effects on p53 accumulation, and furthermore, Mdm2 increases by all three agents were abrogated in p53–/– cells.

Similar Sensitivities of p53+/+ and p53–/– HCT 116 Cells to T-ara-C, Ara-C, and Gemcitabine
Previous studies suggested that the difference in drug response between tumors with wild-type p53 and those harboring p53 loss-of-function mutations can be explained in part by p53-mediated apoptosis. However, mutant p53 proteins can have other effects, such as a dominant negative effect with respect to p73, which could obfuscate studies of the role of p73 in apoptosis in such lines. The p53–/– HCT 116 line, however, is an isogenic line where p53 has been deleted by homologous recombination which can be used to assess whether the induction of p53 is essential in the apoptotic/cytotoxic effects of these drugs (14). We measured the dose-dependent decrease in colony outgrowth following acute treatment with these drugs in wild-type versus p53–/– HCT 116 cells. As shown in Fig. 4 , T-ara-C and gemcitabine potently inhibited clonogenecity of wild-type HCT 116 cells, and this response was essentially intact in p53–/– cells. Acute ara-C treatment, on the other hand, had only a modest effect on clonogenecity in these cells, and loss of p53 also did not markedly alter the response to ara-C. Therefore, it seems that p53 deficiency in these cells does not alter the cytotoxic effects of these drugs when used in short treatments at these doses.


Figure 4
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  		Figure 4. Loss of p53 does not alter T-ara-C- or gemcitabine-induced decreases in colony formation. Five hundred wild-type or p53–/– HCT 116 cells were plated in 100-mm dishes and exposed to the indicated concentrations of T-ara-C, ara-C, and gemcitabine for 2 h. Cells were then washed and incubated in fresh medium. Colonies were allowed to form for an additional 1 wk. The incubation was terminated and the colonies were stained with crystal violet and enumerated. Points, mean of triplicate determinations; bars, SD.

 
T-ara-C or Gemcitabine Treatment Enhances p73 Protein Levels and Induces p73-Specific DNA-Binding Activity in p53+/+ and p53–/– HCT 116 Cells
The p73 protein shares significant sequence similarity with p53 and can transactivate p53 target genes such as PUMA (45). It has also been shown that p73 is induced in response to a wide variety of chemotherapeutic drugs and that p73 is a determinant of chemotherapeutic efficacy (24). We therefore assessed whether the ability of nucleosides to induce p53-independent cell death could be mediated by p73 instead. Figure 5A shows a Western blot analysis of lysates from both p53–/– and wild-type HCT 116 cells treated for 2 hours with T-ara-C and harvested at 24 and 48 hours. Using the monoclonal antibody IMG-259, which recognizes all isoforms of p73, we observed that T-ara-C robustly induced full-length p73 (TA p73{alpha}) at 24 and 48 hours posttreatment in both p53+/+ and p53–/– HCT 116 cells (Fig. 5A). We also confirmed these results with another monoclonal antibody, IMG-246, which recognizes all the TAp73 isoforms (isoforms with the intact NH2-terminal transactivation domain). The smaller TA isoforms, TAp73ß, TAp73{delta}, and TAp73{gamma}, were not detectable in these lysates (not shown). Changes in the levels of the NH2-terminal deleted dominant negative p73 isoforms were also not observed (not shown). T-ara-C-treated lysates from both p53+/+ and p53–/– cells also showed increases in PUMA, which correlated with p73 increases (Fig. 5A).


Figure 5
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  		Figure 5. T-ara-C and gemcitabine induced increases in p73 and p73 DNA binding activity. A, exponentially growing wild-type cells (p53+/+) and p53–/– HCT 116 cells were exposed to T-ara-C (100 µmol/L) for 2 h. Cells were then washed and incubated in fresh medium for an additional 24 or 48 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting. B, exponentially growing wild-type HCT 116 cells (p53+/+) and p53–/– HCT 116 cells were exposed to ara-C (10 µmol/L) or gemcitabine (10 µmol/L) and analyzed as in A. C, nuclear extracts were prepared as described in Materials and Methods. Level of p73 that can bind to a p53-DNA binding site was determined by an ELISA assay. Columns, mean fold increase relative to untreated cells at 0 h from triplicate determinations; bars, SD.

 
We also showed p73 increases at 24 and 48 hours posttreatment in p53+/+ and p53–/– HCT 116 cells treated with gemcitabine. The p73 increases induced by the less effective pyrimidine nucleoside analogue ara-C were modest at best in both genotypes (Fig. 5B). We also tested p73 binding to a p53 consensus DNA binding site as a read-out of its transcriptional activation. There is significant identity between p73 and p53 in the primary sequence especially in the DNA-binding domain and, to a lesser extent, in the transactivation domain. As a result, p73 is able to transactivate many target genes that have p53 binding sites in their promoter proximal regions. We used the immobilized p53-DNA binding site oligonucleotide with p73 antibodies in an ELISA assay on nuclear extracts made from p53+/+ and p53–/– HCT 116 cells. As previously mentioned, nuclear extracts from drug-treated p53–/– HCT 116 cells did not show any increased p53-DNA binding over background (Fig. 3B). In contrast, when nuclear proteins bound to the DNA sequence were probed with a p73-specific monoclonal antibody (ER-15, Ab2; NeoMarkers), we found that p73-specific DNA binding increases were exhibited by both p53+/+ and p53–/– HCT 116 cells treated with T-ara-C or gemcitabine (Fig. 5C). These data suggest that pyrimidine nucleosides effective in HCT 116 cells can potentially mediate their apoptotic effects by modulating p73 activities.

T-ara-C or Gemcitabine Treatment Decreases p73 Protein Turnover in HCT 116 Cells
The mechanisms by which p73 is activated in response to genotoxic stress is currently an active area of study by different laboratories. Several reports lend support to the idea that p73 is predominantly regulated at the level of protein degradation (22, 46–48). p73 is a protein that is rapidly turned over and, in cultured cells, exhibits a half-life of 1 to 2 hours (49, 50). To test whether T-ara-C or gemcitabine treatment can alter p73 protein turnover, we treated control or drug-treated cells with cycloheximide at 1, 2, or 4 hours before harvest at the 24-hour time point to prevent new protein synthesis. As shown in Fig. 6A , p73 in untreated cells was almost undetectable within 2 hours following incubation with cycloheximide, indicating rapid turnover. T-ara-C- or gemcitabine-induced p73 levels were, however, maintained at high levels even at 4 hours post cycloheximide treatment. Densitometric analysis of p73 revealed that nearly 75% to 80% of p73 was remaining in T-ara-C- or gemcitabine-treated cells even after a 4-hour incubation with cycloheximide, indicating that nucleoside treatment increased p73 protein stability (Fig. 6B).


Figure 6
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  		Figure 6. T-ara-C and gemcitabine induce p73 stabilization by decreasing protein turnover. A, exponentially growing HCT 116 cells were exposed to the indicated concentrations of T-ara-C (100 µmol/L) or gemcitabine (10 µmol/L) for 2 h. Cells were then washed and incubated in fresh medium for an additional 24 h, at which point they were treated with cycloheximide (CHX; 75 µg/mL) for 1, 2, or 4 h. Cells were then harvested and cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against p73 or actin. B, densitometric analysis was done to determine p73 levels relative to actin and are graphically presented as a function of time after cycloheximide treatment.

 
T-ara-C- or Gemcitabine-Induced p73 Protein Stabilization in HCT 116 Cells Is c-Abl Independent
Ara-C has been shown to activate the tyrosine kinase c-Abl and to stimulate the complexation of c-Abl with p53 (51). Inhibition of the tyrosine kinase activity of c-Abl with STI-571 has also been shown to decrease the apoptotic response to ara-C (30). c-Abl also interacts with and enhances p73 stability in the apoptotic response to DNA damaging agents such as cisplatin. Therefore, we tested whether the nucleoside-induced increases in p73 stability were mediated by c-Abl. Knockdown of c-Abl was accomplished by specific double-stranded small interfering RNA. As shown in Fig. 7A , near-complete silencing of c-Abl expression did not affect T-ara-C- or gemcitabine-induced p73 levels in either p53+/+ or p53–/– cells.


Figure 7
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  		Figure 7. T-ara-C- and gemcitabine-induced p73 stabilization is c-Abl independent. A, exponentially growing p53+/+ and p53–/– HCT 116 cells were transfected with a c-Abl-specific double-stranded small interfering RNA and exposed to T-ara-C (100 µmol/L) or gemcitabine (10 µmol/L) for 2 h. Cells were then washed and incubated in fresh medium for an additional 24 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against c-Abl, p73, and p53. B, exponentially growing p53+/+ and p53–/– HCT 116 cells were pretreated with STI-571 (10 µmol/L) for 1 h and exposed to T-ara-C (100 µmol/L) or gemcitabine (10 µmol/L) for 2 h. Cells were then washed and incubated in STI-571-containing medium for an additional 24 hours. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against p73, p53, and actin.

 
To further confirm this, we examined whether T-ara-C- or gemcitabine-induced p73 stabilization would be affected by the c-Abl kinase inhibitor STI-571. HCT 116 cells (p53+/+ and p53–/–) were pretreated with STI-571 (10 µmol/L) for 1 hour, which completely inhibits c-Abl tyrosine kinase activity (30). The cells were then treated with T-ara-C or gemcitabine for 2 hours, followed by further incubation in the presence of STI-571 for an additional 24 hours. As shown in Fig. 7B, STI-571-treated p53+/+ or p53–/– cells showed p73 increases in response to T-ara-C or gemcitabine similar to that in cells that were not exposed to the inhibitor. In sum, these results suggest that c-Abl does not regulate p73 in the apoptotic response to these nucleosides in HCT 116 cells.

HCT 116 Cells with Deleted p53/p73 Targets, PUMA and Bax, Are Less Sensitive to T-ara-C and Gemcitabine
The BH3-only protein PUMA was initially identified as a p53 transcriptional target (52). PUMA is also transcriptionally induced during p73-mediated cell death and also induces Bax conformational change and relocalization of Bax to the mitochondria (53). Therefore, we tested the sensitivity to T-ara-C or gemcitabine of isogenic HCT 116 lines wherein PUMA or Bax was individually deleted by homologous recombination. We find that Bax–/– HCT 116 and PUMA–/– HCT 116 cells were relatively resistant to T-ara-C and gemcitabine (Fig. 8A ). To address whether some of these differences could be due to clonal variation, we tested an additional Bax–/– clone which also showed essentially identical sensitivity as the above-mentioned clone (data not shown). The differences in cytotoxicities to T-ara-C or gemcitabine among PUMA–/–, Bax–/–, and wild-type cells are also not likely to be due to differences in nucleoside metabolism, nucleotide half-lives, or the initial sensing of genotoxic/replication stresses between these genotypes because equivalent increases in p73 and p53 were exhibited in response to T-ara-C and to gemcitabine in all three cell types (Fig. 8B). This suggests that the activation of the proximal/upstream apoptotic pathways by nucleoside-induced genotoxic/replication stress is similar between these lines. Thus, T-ara-C- and gemcitabine-induced cytotoxicity in HCT 116 cells is dependent partly on PUMA and Bax.


Figure 8
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  		Figure 8. Loss of p53/p73 targets PUMA and Bax makes HCT 116 cells less sensitive to T-ara-C and gemcitabine. A, five hundred wild-type HCT 116 cells, PUMA–/– HCT 116 cells, or Bax–/– HCT 116 cells were plated in 100-mm dishes and exposed to the indicated concentrations of T-ara-C, ara-C, and gemcitabine for 2 h. Cells were then washed and incubated in fresh medium. Colonies were allowed to form for an additional 1 wk. The incubation was terminated and the colonies were stained with crystal violet and enumerated. Points, mean of triplicate experiments; bars, SD. *, P < 0.05, versus wild-type cells. B, wild-type HCT 116 cells, PUMA–/– HCT 116 cells, or Bax–/– HCT 116 cells were exposed to T-ara-C (100 µmol/L) or gemcitabine (10 µmol/L) for 2 h, washed, and incubated in fresh medium for an additional 24 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against p73, p53, and actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have examined the p53 and p73 activation responses induced by nucleoside drugs after an acute treatment in HCT 116 colon carcinoma cells to determine whether it would provide us with some insight into their apoptotic mechanisms in solid tumor lines. We find that T-ara-C and gemcitabine are robust inducers of p53 accumulation and phosphorylation at Ser15/Ser20 in these cells (Fig. 3A). These results also correlate with T-ara-C- and gemcitabine-induced increases in p53-DNA binding activity in nuclear extracts from wild-type HCT 116 cells (Fig. 3B). Ara-C, a close structural analogue, was, however, less efficient in inducing p53 activation under these conditions (Fig. 3A and B). Despite the activation of p53 by T-ara-C and gemcitabine, we find that the sensitivity of p53–/– HCT 116 cells to these drugs as measured by decrease in colony outgrowth was not significantly different from that in the wild-type cells (Fig. 4). This prompted us to ask whether p73 might be involved in eliciting the p53-independent cytotoxic response.

p73 is a p53 family member and has a modular structure similar to that of p53 and a high degree of sequence homology, particularly in the DNA binding region. We find that T-ara-C and gemcitabine induce marked increases in p73 levels and p73 DNA binding activity (Fig. 5). Because p73 is able to transactivate the promoters of several p53-responsive genes involved in apoptosis and because no functionally significant p73 mutations have been reported in cancers, unlike p53, we suggest that this could play a major role in nucleoside-mediated cytotoxicity at least in p53-independent situations. T-ara-C differs from ara-C in a single substituent (Fig. 1) and is more effective than ara-C against a broad range of both leukemias and solid tumor lines both in vitro and in vivo (29). The engagement of apoptotic effector pathways by ara-C is controlled by upstream kinases such as c-Abl and p38 mitogen-activated protein kinase (MAPK; refs. 30, 36, 54, 55). Gemcitabine has also been shown to activate p38 MAPK (56). c-Abl phosphorylates, stabilizes, and activates p73 and, moreover, the p38 MAPK cascade mediates this response (23, 57). Because p73 has also recently been implicated in c-Abl induction of apoptosis following DNA damage (23), it was formally possible that c-Abl plays a role in T-ara-C- or gemcitabine-mediated p73 increases as well. c-Abl has also been shown to activate p53 in response to genotoxic stress (51, 55, 58). However, our studies reveal that neither p73 nor p53 increases induced by T-ara-C or gemcitabine are substantially altered either by silencing of c-Abl expression or by inhibiting its tyrosine kinase activity (Fig. 7A and B).

A number of alternative explanations could account for this finding. The promyelocytic leukemia protein modulates p73 half-life by inhibiting its ubiquitination and degradation in a promyelocytic leukemia nuclear body–dependent manner and p38 MAPK-mediated phosphorylation of p73 is required for p73 recruitment into the promyelocytic leukemia nuclear body and subsequent promyelocytic leukemia–dependent p73 stabilization (47). Interestingly, p38 MAPK is activated by c-Abl-independent mechanisms as well (55). Thus, it is possible that T-ara-C and gemcitabine could mediate activation of p38 MAPK independent of c-Abl activation, which could subsequently induce p73 stabilization. Both Chk1 and Chk2 also play a role in p73 induction after DNA damage (59). We have found that T-ara-C induces both Chk1 and Chk2 activation in these cells.3 Similarly, Chk1 activation by gemcitabine has also been reported by other laboratories (60). Therefore, it is possible that Chk1/2 activation by these drugs is the predominant mode for p73 stabilization in these cells. Both of these issues are currently being investigated.

The downstream p73 targets that mediate the apoptotic response to these nucleosides are also of interest. In this regard, it is important to note that caspase-3 cleavage is activated by T-ara-C and gemcitabine. This suggests, therefore, that the intrinsic mitochondrial pathways of apoptosis are likely to be activated by cytotoxic nucleosides. The BH3-only protein PUMA, which was originally characterized as a p53 target, is also transcriptionally induced during p73-mediated cell death (53). PUMA induces Bax conformational change and relocalization of Bax to the mitochondria (53). Bax is a proapoptotic member of the Bcl-2 protein family that is predominantly localized in the cytosol of healthy cells and translocates to mitochondria after a variety of death stimuli (61). Once translocated to the outer membranes of the mitochondria, Bax forms oligomers or clusters that cause mitochondrial dysfunction and apoptotic cell death (61). We have observed PUMA increases in T-ara-C-treated p53+/+ and p53–/– cells (Fig. 5). However, because PUMA–/– and Bax–/– cells are not completely resistant to the effects of nucleosides, we suggest that other p73-induced pathways also contribute to nucleoside effects (Fig. 8). It is also possible that in Bax–/– cells, other PUMA-dependent effects are operative and vice versa. It is also important to note that both Bax–/– and PUMA–/– cells also have wild-type p53. Thus, although the cytotoxic effects of nucleosides are not diminished in p53–/– cells, and in that sense not dependent on p53, it is likely, however, that p53 does contribute to the full manifestation of the apoptotic effects of nucleosides in those cells that do express wild-type p53. In a similar vein, it is also likely that nucleoside-induced p73 effects that are Bax and PUMA independent can be operative in p53–/– cells.

Data presented herein also suggest that differences in the efficiency of activation of p73 pathways could account for the differential sensitivities of these cells to structurally similar nucleosides, at least in the absence of functional p53. Given the structural similarities of the three pyrimidine nucleosides examined in this study and the fact that they are readily converted to their respective triphosphates at the concentrations used, it is intriguing that they exhibit large differences in activation of p73 pathways. One reason for these differences could be that molecular sensors respond differently to structurally distinct lesions caused in DNA by the incorporation of the individual nucleosides. It is also possible that the differences in biochemical properties of the drug triphosphates could further contribute to distinct p73 activation responses. In a previous report, we showed that T-ara-C is a poorer substrate than ara-C for deoxycytidine kinase, and as a result, at equimolar concentrations, less T-ara-C monophosphate is formed versus ara-C monophosphate (62). However, we have also recently reported that the half-life of T-ara-C triphosphate was 10-fold over that of ara-C triphosphate in HCT 116 cells (63). It is clear from our data that after an acute treatment, T-ara-C at 10 µmol/L is more cytotoxic than ara-C at the same concentration (Fig. 2B). This suggests that at the 48- and 72-hour time points, the longer intracellular retention of T-ara-C triphosphate compensates for the low rate of formation of T-ara-C monophosphate. Thus, longer retention of T-ara-C triphosphate, as well as gemcitabine triphosphate (as compared with ara-C triphosphate), could also account for the differences in p73 activation among the three drugs.

In this regard, it is interesting that Robinson et al. (64) have found that after a 24-hour incubation with gemcitabine, HCT 116 p53–/– lines showed less sensitivity as compared with wild-type cells in a colony outgrowth assay. These investigators attributed this difference to a lower retention of gemcitabine triphosphate in the p53–/– cells, which was shown by using low concentrations of the drug (100–500 nmol/L) in a 24-hour incubation assay. In that study, triphosphate retention at micromolar concentrations was not examined (64). In contrast, our study shows that wild-type HCT 116 and their p53–/– counterparts exhibit similar decreases in colony outgrowth following a short-term exposure to gemcitabine with doses raging from 100 nmol/L to 10 µmol/L.

It is also interesting to note that in response to T-ara-C and gemcitabine, p53 DNA-binding activity and the expression of the p53 target Mdm2 are enhanced and, moreover, the Mdm2 increases induced by these nucleosides are abrogated in p53–/– cells (Fig. 3). Therefore, Mdm2 increases in HCT 116 cells are not a product of nucleoside-induced p73 increases but instead a reflection of nucleoside-induced transcriptionally active p53. These data are in contrast with a report that shows that Mdm2 is also a p73 target, although in that study only the induction of Mdm2 by transfected p73 was studied (65). Structural lesions caused by nucleoside incorporation, the frank termination of chain elongation following incorporation, and the replication stress as a result of inhibiting ribonucleotide reductase or DNA polymerases can all play causal roles in p53 accumulation and activation. In contrast, however, Gottifredi et al. (42) have shown that although p53 accumulates in response to other replication stressors like hydroxyurea and aphidicolin, it is transcriptionally impaired in these situations. Thus, whereas some of the effects of stalled replication forks induced by nucleosides are likely to overlap those elicited by HU and aphidicolin, others might be distinct, resulting in different effects on p53 properties. In conclusion, despite inducing a transcriptionally active p53, nucleoside-induced cytotoxic effects are not absolutely dependent on p53. Instead, p73 stabilization could also play a role in nucleoside apoptotic effects. This could be of therapeutic importance in those tumors where p53 is mutated, provided of course that the p53 mutants do not hinder the function of p73.


   Acknowledgments
 
We thank Drs. Bert Vogelstein and Kenneth Kinzler for providing the p53 -/-, PUMA -/- and Bax -/- HCT 116 lines; and Drs. Marshall Urist and Carol Prives for help regarding use of p73 antibodies; and Dr. Gerard P. Zambetti for helpful discussions while this work was in progress.


   Footnotes
 
Grant support: NIH grant P01-CA34200. J.V. Thottassery is the recipient of a Susan G. Komen. Breast Cancer Grant (BCTR 2000-000456).

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.

3 Thottassery et al., unpublished studies. Back

Received 10/ 6/05; revised 11/29/05; accepted 12/16/05.


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 Materials and Methods
 Results
 Discussion
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M. L. Clarke, V. L. Damaraju, J. Zhang, D. Mowles, T. Tackaberry, T. Lang, K. M. Smith, J. D. Young, B. Tomkinson, and C. E. Cass
The Role of Human Nucleoside Transporters in Cellular Uptake of 4'-Thio-beta-D-arabinofuranosylcytosine and beta-D-Arabinosylcytosine
Mol. Pharmacol., July 1, 2006; 70(1): 303 - 310.
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