Justine Rudner
email: justine.rudner@med.uni-tuebingen.de

Simon J Elsaesser

Verena Jendrossek

Stephan M Huber

Anti-apoptotic Bcl-2 fails to form efficient complexes with pro-apoptotic Bak to protect from Celecoxib-induced apoptosis

Keywords: Antibiotics and Chemotherapeutics Intrinsic apoptosis, Celecoxib, Bcl-2, Bcl-xL, Mcl-1, Bak

The non-steroidal anti-inflammatory drug Celecoxib is a specific inhibitor of cyclooxygenase-2. Apart from its inhibitor function, Celecoxib induces apoptosis through the intrinsic pathway which is controlled by the Bcl-2 family members. In Jurkat T lymphoma cells, treatment with Celecoxib results in a rapid decline of the anti-apoptotic Bcl-2-related protein

Mcl-1. The depletion of Mcl-1 was sufficient for apoptosis induction and could be blocked by overexpression of Bcl-xL but not the close homologue Bcl-2. The present investigation analyzed the mechanism by which Bcl-xL prevents apoptosis induction whereas Bcl-2 failed to. Our data show that the involvement of the orphan nuclear receptor Nur77/TR3 specifically targeting Bcl-2 but not Bcl-xL was not involved in Celecoxib-induced apoptosis. Surprisingly, BH3-only proteins Bid, Bim, and Puma of the Bcl-2 family were not needed either. However, unlike Bcl-2, Mcl-1, and Bcl-xL sequestered Bak preventing it from activation through a direct interaction. Thus, when abundantly expressed, Bcl-xL can substitute for the loss of Mcl-1 whereas Bcl-2, incapable of forming a high affinity complex with Bak, could not.

A c c e p t e d M a n u s c r i p t

Introduction:

The non-steroidal anti-inflammatory drug Celecoxib (Cerebrex®) is a specific inhibitor of cyclooxygenase-2 (COX-2) with anti-neoplastic properties [START_REF] Grosch | COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib[END_REF]. COX-2 is involved in prostaglandin production during the inflammatory response [START_REF] Uchida | A lipid-derived endogenous inducer of COX-2: a bridge between inflammation and oxidative stress[END_REF]. The enzyme is also overexpressed in many human tumors and contributes to tumorigenesis [START_REF] Zha | Cyclooxygenases in cancer: progress and perspective[END_REF]. Thus, in addition to their anti-inflammatory activities, coxibes might interfere with tumor progression [START_REF] Phillips | A randomised, double blind, placebo controlled study of celecoxib, a selective cyclooxygenase 2 inhibitor, on duodenal polyposis in familial adenomatous polyposis[END_REF][START_REF] Steinbach | The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis[END_REF].

Previous experiments in COX-2-negative cell lines and with Celecoxib derivates lacking the COX-2 inhibitory function indicate that Celecoxib might have yet other targets through which it exerts cytotoxic effects [START_REF] Kardosh | Dimethylcelecoxib (DMC), a derivative of celecoxib that lacks cyclooxygenase-2-inhibitory function, potently mimics the anti-tumor effects of celecoxib on Burkitt's lymphoma in vitro and in vivo[END_REF][START_REF] Rudner | Differential effects of antiapoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on celecoxib-induced apoptosis[END_REF][START_REF] Pyrko | Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its noncyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo[END_REF][START_REF] Kardosh | Aggravated endoplasmic reticulum stress as a basis for enhanced glioblastoma cell killing by bortezomib in combination with celecoxib or its non-coxib analogue, 2,5-dimethylcelecoxib[END_REF][START_REF] Gao | OSU-03012, a novel celecoxib derivative, induces reactive oxygen species-related autophagy in hepatocellular carcinoma[END_REF]. We have recently shown that Celecoxib induced apoptosis through the intrinsic pathway [START_REF] Jendrossek | Celecoxib activates a novel mitochondrial apoptosis signaling pathway[END_REF][START_REF] Muller | Importance of Bak for celecoxib-induced apoptosis[END_REF][START_REF] Rudner | The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway[END_REF]. This apoptotic pathway involves permeabilization of the outer mitochondrial membrane and the dissipation of mitochondrial membrane potential [START_REF] Adams | Ways of dying: multiple pathways to apoptosis[END_REF][START_REF] Galonek | Upgrading the BCL-2 network[END_REF]. Apoptogenic factors which are found in the mitochondrial intermembrane space of healthy cells are released into the cytosol where they facilitate the activation of caspases, the executers of the apoptotic death program.

Members of the Bcl-2 protein family are the gate keepers of the mitochondrial homeostasis regulating the release of pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. The Bcl-2 protein family consists of pro-and anti-apoptotic members which are able to form heterodimers [START_REF] Adams | The Bcl-2 apoptotic switch in cancer development and therapy[END_REF][START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF]. Among others, the anti-apoptotic group encompasses Bcl-2, Bcl-xL, Mcl-1, and A1 which are often found to be over-expressed in tumor tissues.

The pro-apoptotic group is divided in multi-domain proteins (Bax, Bak, Bok) which share three Bcl-2 homologous domains (BH1-3) and the BH3-only proteins (Bim, Bid, Puma, Bad, Bik, Bmf, Noxa) which have only the BH3 domain in common.

The activation of the multi-domain proteins is absolutely essential for mitochondrial permeabilization and apoptosis induction. Currently, two theories exist which explain the involvement of the different anti-apoptotic and BH3-only proteins leading to activation of A c c e p t e d M a n u s c r i p t 4 Bak/Bax-like proteins. In the displacement model, the multidomain proteins are neutralized by the anti-apoptotic Bcl-2 family members in healthy cells [START_REF] Adams | The Bcl-2 apoptotic switch in cancer development and therapy[END_REF][START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF]. Upon apoptosis induction, BH3-only proteins bind to the anti-apoptotic ones thereby displacing Bax or Bak allowing them to be activated through spontaneous self-oligomerization.

The direct activation or hierarchical model discernes BH3-only protein activators (Bim, Bid, and sometimes Puma) and sensitizers (Bad, Bik, Bmf, Noxa) [START_REF] Kim | Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies[END_REF][START_REF] Letai | Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics[END_REF]. The former bind to all anti-apoptotic proteins with similar affinity as well as to the pro-apoptotic multidomain proteins whereas the latter do not interact with Bax/Bak-like proteins. Moreover, sensitizer BH3-only proteins display distinct binding preferences to the anti-apoptotic ones. So associated Noxa with Mcl-1 and A1 only while Bad interacts with Bcl-2 and Bcl-xL. In healthy cells, the activators are sequestered by the anti-apoptotic proteins. A certain apoptotic stimulus activates distinct set of sensitizer proteins which, in turn, bind their preferred antiapoptotic partners. The activator proteins, when released from their sequestration, bind to the Bax/Bak-like proteins to induce their oligomerization. Interestingly, overexpression of Bcl-xL but not Bcl-2 could prevent induction of apoptosis in response to Celecoxib [START_REF] Rudner | Differential effects of antiapoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on celecoxib-induced apoptosis[END_REF][START_REF] Jendrossek | Celecoxib activates a novel mitochondrial apoptosis signaling pathway[END_REF]. The mechanisms which lead to specific neutralization of Bcl-2 remain elusive. Because of their high sequence homology, Bcl-2 and Bcl-xL were thought to fulfill a redundant protective function. Their binding affinities to other BH3-only proteins are similar but they might also interact with specific partner [START_REF] Lin | Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3[END_REF][START_REF] Gary-Gouy | Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2[END_REF].

Our previous investigations have shown that

In this present investigation, we examined the mechanism leading to neutralization of Bcl-2 but not the closely related Bcl-xL during Celecoxib-induced apoptosis in Jurkat T cells. Downregulation of activator BH3-only proteins Bim and Puma by siRNA revealed that their presence is not essential for mitochondrial permeabilization and apoptosis induction by

Cells and cell culture

Jurkat E6.1 T-lymphoma cells were from ATCC (Bethesda, Maryland, USA). Jurkat cells stably expressing Bcl-xL or Bcl-2 and the respective Vector control were prepared as described before [START_REF] Rudner | Differential effects of antiapoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on celecoxib-induced apoptosis[END_REF]. Cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 37°C and 5% CO 2 .

A c c e p t e d M a n u s c r i p t 7

Transfection with siRNA

Cells were cultured at a low density to ensure log phase growth. For transfection 2x10 6 cells were resuspended in 200 µL RPMI-1640 without phenol red. Shortly before transfection, bim, puma, or non-targeting siRNA was added at indicated concentration. bim and puma ON-TARGET SMARTpool and the siCONTROL NON-TARGETING pool siRNA was purchased from Dharmacon (Chicago, IL, USA). Cells were electroporated in a 4 mm cuvette in an EPI2500 electroporator (Fischer, Heidelberg, Germany) at 370 V for 10 msec.

Immediately after transfection cells were resuspended in 6 mL pre-warmed medium and continued to be cultured as described above. Transfection efficiency and viability was determined by transfecting the cells with 400 nM green fluorescence siRNA (siGLO from Dharmacon, Chicago, IL, USA) followed by propidium iodide exclusion dye and flow cytometric analysis.

Flow cytometric analysis

The mitochondrial membrane potential (m) was analyzed using the m specific dye TMRE (Molecular Probes, Mobitech, Göttingen, Germany). At the indicated time points 10 5 cells were stained for 30 min in PBS containing 25 nM TMRE. Co-incubation with 1 µM of the cyanide derivate CCCP was used as a positive control to complete the mitochondrial depolarisation. Apoptosis induction was analyzed by Annexin V-PI double staining. In brief, Triton X-100, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM Na 3 VO 4 , 100 mM PMSF, 5 µg/mL Aprotinin, 5 µg/mL Leupeptin, and 3 µg/mL Pepstatin. After removing insoluble material by centrifugation for 10 min at 18 000 x g, the protein concentration was estimated in the supernatant using the Bio-Rad protein assay (Bio-Rad, Munich, Germany) according to the manufacturer's protocol. Protein was separated by SDS-PAGE under reducing conditions before transfer onto PVDF membranes (Roth, Karlsruhe, Germany).

Blots were blocked in TBS buffer containing 0.05% Tween-20 and 5% non-fat dry milk for 1 h at room temperature. The membrane was incubated overnight at 4°C with the respective primary antibodies. After repeated washings with TBS/Tween-20 (0.05%) the membranes were incubated with the secondary antibody for 1 h at room temperature before continuing to wash with TBS/Tween-20 (0,05%). Detection of antibody binding was performed by enhanced chemoluminescence (ECL Western blotting analysis system, GE Healthcare/Amersham-Biosciences, Freiburg, Germany). Equal loading was verified by antibodies against Tubulin, GAPDH, or -Actin. All Western blot experiments were repeated at least once.

Immunoprecipitation

Cells were lysed as described above using either 1% CHAPS or 1% Triton X-100. The protein concentration was adjusted to 2 mg/mL. 5 µg antibody and 50 µL slurry Dynabeads suspension (Dynal/Invitrogen, Karlsruhe, Germany) were added to 750 µL lysate. After the precipitation for 3h at 4°C the beads were washed thrice with 300 µL lysis buffer containing 0.2% of the respective detergent. Proteins were eluted by boiling the beads for 5 min in 100 µL SDS sample buffer with -Mercaptoethanol. 30 µL were separated by SDS gel Thus, the expression levels of the three BH3-only proteins were examined (Fig. 2A).

Role of activator BH3-only proteins during Celecoxib-induced apoptosis

Bim is expressed as an extra large, a large, or a small splice variant (Bim EL , Bim L , and Bim S respectively). Puma is expressed as Puma- and Puma  whereas Bid is expressed in an inactive p22 pro-form in healthy Jurkat cells which needs to be processed into a p15 fragment to be activated during apoptosis (Fig. 2A).

The protein levels of Bim remained unchanged during Celecoxib-induced apoptosis, but a strong reduction of pro-apoptotic Puma levels and cleavage of Bid were observed in Jurkat

Vector and Jurkat Bcl-2 cells (Fig. 2A). Since both of the events correlated with caspase activation, we tested whether the pan-caspase inhibitor zVAD could abrogate Bid cleavage and Puma decline. Treatment with zVAD blocked Celecoxib-induced exposure of Annexin V Full length Bid (p23) has to be processed to a p15 fragment to fully display its pro-apoptotic potential. In contrast, Puma can change its interaction partners before its degradation. To analyze its relevance for Celecoxib-induced apoptosis in Jurkat cells, Puma was downregulated by siRNA. Puma levels were reduced about 50% 72h after electroporation with 1µM siRNA into Jurkat cells (Fig. 3A). So, 72h after electroporation of 1 µM puma siRNA or the non-targeting control siRNA, the cells were treated with 50-100 µM Celecoxib for 6h. Apoptosis induction (Fig. 3B) and m dissipation (Fig. 3C) occurred with similar effectivity in cells transfected with non-targeting or puma siRNA. In addition, Puma could be The separate silencing of Bim and Puma showed that none of those two BH3-only proteins is essential for Celecoxib-induced apoptosis in Jurkat cell, but it does not exclude a redundant function of Bim and Puma. Therefore, the expression of both proteins was silenced by siRNA before treatment with 50-100 µM Celecoxib for 6h. However, simultaneous silencing of Bim and Puma was without effect on Celecoxib-induced apoptosis (Fig. 5A) and m dissipation (Fig. 5B).

Taken together, our experiments excluded a essential or redundant role of Bid, Bim, and Puma in mitochondrial permeabilization during apoptosis induction by Celecoxib.

Nur77/TR3 does not target Bcl-2 during Celecoxib-induced apoptosis

Since none of the examined BH3-only proteins were needed for Celecoxib-induced apoptosis, the different sensitivity, the regulation of Bcl-2 and Bcl-xL by those BH3-only proteins was implausible. There have to be other interaction partners of the anti-apoptotic proteins which explain the different sensitivity of Bcl-2-and Bcl-xL-overexpressing cells towards Celecoxib.

A c c e p t e d M a n u s c r i p t 14 Recent publications described the protein Nur77/TR3 which specifically binds to Bcl-2 but not Bcl-xL [START_REF] Li | Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3[END_REF]. In a complex with Nur77/TR3, Bcl-2 loses its protective function. Thus, in the next set of experiment we examined the role of Nur77/TR3 during Celecoxib-induced apoptosis. However, an upregulation of Nur77/TR3 in response to Celecoxib was not observed (Fig. 6A). Neither could we detect an interaction between Nur77/TR3 and Bcl-2 (Fig. 6B). Thus, an involvement of Nur77/TR3 during Celecoxib-induced apoptosis could be excluded.

Bcl-2 and Bcl-xL differ in their interaction with Bak

Since Bcl-2 and Bcl-xL showed different affinities for Bim, we hypothesized that those two Surprisingly, Bak was also co-precipitated with Bcl-2 in cells overexpressing Bcl-2 (Fig. 7A, middle panel). To estimate the affinity of the Bak interaction with the three different antiapoptotic proteins, we changed the lysis conditions. The use of the much stronger detergent Triton X-100 instead of the mild CHAPS averted complex formation between Bcl-2 and Bak (Fig. 7B). In contrast, Bcl-xL and Mcl-1 co-precipitated with Bak even under harsher lysis conditions. Similar results were obtained when Triton X-100 was reduced from 1% to 0.2%.

Celecoxib: an apoptosis-inducing drug

Celecoxib is a selective COX-2 inhibitor which effectively induces apoptosis by a mechanism yet unknown. The inhibitory and cytotoxic effects could be mapped to different structural characteristics of the molecule and therefore occur independently [START_REF] Schonthal | Celecoxib analogs that lack COX-2 inhibitory function: preclinical development of novel anticancer drugs[END_REF]. The mechanism by which Celecoxib induces apoptosis is not well understood. Celecoxib and its derivates without COX-2 inhibitory function were shown to induce aggravated endoplasmatic stress with subsequent caspase activation. Celecoxib and the related OSU-03012 could also interfere with the PKB/Akt survival pathway [START_REF] Zhu | From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1 inhibitors[END_REF][START_REF] Paik | Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt activation and suppresses hepatic fibrosis in rats[END_REF]. Moreover, Celecoxib, but not the other coxibes

Bcl-2 and Bcl-xL: similarities and differences

The Bcl-2 protein family was divided into three subgroups according to the similarities in structure and function: the anti-apoptotic proteins which sequester the pro-apoptotic ones, the pro-apoptotic multidomain proteins whose activation is required during intrinsic apoptosis, and the BH3-only proteins which regulate the activation of the multidomain and neutralize the anti-apoptotic ones [START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF]. Previous publications have shown that the Bcl-2 family members within a subgroup can fulfill a redundant role during apoptosis induction [START_REF] Jabbour | Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim[END_REF][START_REF] Hemmati | Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells[END_REF]. Newer data, however, point to a more complex mutual regulation of the pro-and anti-apoptotic Bcl-2 family members. Especially the anti-apoptotic Bcl-2 and its close relative Bcl-xL were thought to be exchangeable. Both proteins maintain mitochondrial homeostasis during apoptosis induction in response to ionizing radiation, hypoxia, cytotoxic drugs, and growth factor withdrawal, whereas both proteins did not affect death receptor induced apoptosis [START_REF] Belka | Differential role of caspase-8 and BID activation during radiation-and CD95-induced apoptosis[END_REF][START_REF] Weinmann | Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner[END_REF][START_REF] Reeve | Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL[END_REF][START_REF] Altman | Autophagy provides nutrients but can lead to Chop-dependent induction of Bim to sensitize growth factor-deprived cells to apoptosis[END_REF][START_REF] Ugarenko | ABT-737 overcomes Bcl-2 mediated resistance to doxorubicin-DNA adducts[END_REF]. Despite their similarities, Bcl-2-and Bcl-xL-deficient mice have a different phenotype indicating the regulation of distinct processes [START_REF] Veis | Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair[END_REF][START_REF] Motoyama | Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice[END_REF]. Expression of either Bcl-2

or Bcl-xL in hematopoietic progenitor cells commits to differentiation to erythroid or myolid cells respectively [START_REF] Haughn | BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation[END_REF]. Also, a switch of expression was observed during tumor progression in Our data emphasize the differences between the similar Bcl-2 and Bcl-xL showing that overexpression of Bcl-xL but not Bcl-2 inhibited Celecoxib-induced apoptosis in Jurkat T lymphoma cells.

Role of Nur77/TR3 during Celecoxib-induced apoptosis

The aim of the present study was the analysis of the mechanism leading to neutralization of Bcl-2 but not Bcl-xL during Celecoxib-induced apoptosis. In this regard, the specific interaction of the orphan nuclear receptor Nur77/TR3 was described. In response to some anti-neoplastic drugs, Nur77/TR3 translocates from nucleus to mitochondria where it interacts with Bcl-2 transforming the anti-apoptotic molecule into a pro-apoptotic one [START_REF] Lin | Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3[END_REF]. Nur77 is expressed and upregulated in response to phorbol acetate and calcium ionophore in Jurkat cells [START_REF] Li | Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3[END_REF]. Although having confirmed the expression of Nur77/TR3 protein in Jurkat cells, neither an upregulation of the orphan receptor nor its binding to Bcl-2 was observed during Celecoxib-induced apoptosis. Thus, the lack of protection of Bcl-2 during Celecoxib-induced apoptosis was not due to an interaction of Bcl-2 with Nur77/TR3.

BH3-only proteins during Celecoxib-induced apoptosis

The different protective properties of Bcl-2 and Bcl-xL might also be explained by distinct binding preferences to other Bcl-2 family members. Bcl-2 and Bcl-xL bind to the BH3-only proteins Bad and Bmf whereas they do not interact with Noxa [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF][START_REF] Labi | BH3-only proteins in cell death initiation, malignant disease and anticancer therapy[END_REF]. In this regard, the two survival-promoting proteins differ completely from the anti-apoptotic Bcl-2 family members

Mcl-1 and A1 which display complementary binding preferences. In contrast, activated Bid associates rather with Bcl-xL than with Bcl-2 [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF]. Bid must be cleaved by caspases to become activated. During apoptosis induction through the extrinsic pathway, caspases are A c c e p t e d M a n u s c r i p t activated at the death receptor signaling complex prior to m dissipation whereas, during induction of intrinsic apoptosis, they become activated after m dissipation [START_REF] Belka | Differential role of caspase-8 and BID activation during radiation-and CD95-induced apoptosis[END_REF][START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF]. Since the pan-caspase inhibitor blocked Bid activation without affecting Celecoxib-stimulated m dissipation we concluded that Bid plays no role in initial mitochondrial permeabilization.

Moreover, due to the low-affinity binding to Bcl-2, it is unlikely that tBid neutralized only Bcl-2 during Celecoxib-induced apoptosis [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF].

Puma was described as a BH3-only protein with broad binding spectrum to all anti-apoptotic proteins [START_REF] Labi | BH3-only proteins in cell death initiation, malignant disease and anticancer therapy[END_REF]. Although it is expressed in healthy Jurkat cells, we did not detect any association of Puma with anti-apoptotic proteins or with Bak during Celecoxib-induced apoptosis. Interestingly, Puma is downregulated during Celecoxib-induced apoptosis. The downregulation of Puma was caspase-dependent since it was blocked by the pan-caspase inhibitor zVAD. Thus, the decline of Puma is rather a consequence of apoptosis induction by Celecoxib and of no importance for the initial mitochondrial permeabilization.

Like Puma, Bim was described to have a similar broad binding spectrum to other antiapoptotic Bcl-2 family members [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF][START_REF] Labi | BH3-only proteins in cell death initiation, malignant disease and anticancer therapy[END_REF]. In our cell system, however, Bim preferred Bcl-2 to

Mcl-1 and Bcl-xL. Although Bim is released from its sequestration by Mcl-1 and Bcl-xL during Celecoxib-induced apoptosis, the interaction between Bcl-2 and Bim was not altered during the course. Binding of Bim to Bak in response to Celecoxib was not observed in our cell system. Moreover, siRNA-mediated downregulation of Bim and Puma showed convincingly that neither Bim nor Puma were essential for Celecoxib-induced apoptosis. In contrast, silencing of Bim or Puma improved the survival or Jurkat cells after growth factor withdrawal (Fig. S2) which is in accordance with observations made earlier [START_REF] Jabbour | Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim[END_REF][START_REF] Ekoff | The BH3-only protein Puma plays an essential role in cytokine deprivation induced apoptosis of mast cells[END_REF][START_REF] Erlacher | BH3only proteins Puma and Bim are rate-limiting for gamma-radiation-and glucocorticoidinduced apoptosis of lymphoid cells in vivo[END_REF].

Taken together, our data indicate that the BH3-only proteins Puma, Bid, and Bim are dispensable for the initial mitochondrial permeabilization during Celecoxib-induced apoptosis.

A c c e p t e d M a n u s c r i p t 20

Interaction with Bak during Celecoxib-induced apoptosis

Since the interaction between anti-apoptotic Bcl-2 family members and activator BH3-only proteins is of minor importance, we propose the displacement model for Celecoxib-induced apoptosis. This model suggests a sequestration of multidomain proteins by the anti-apoptotic Bcl-2 family members [START_REF] Galonek | Upgrading the BCL-2 network[END_REF][START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF][START_REF] Willis | Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak[END_REF][START_REF] Shimazu | NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition[END_REF]. Binding of the appropriate BH3-only proteins to antiapoptotic ones displaces the Bax/Bak-like proteins allowing their activation.

Jurkat T lymphoma cells do not express Bax but Bak [START_REF] Muller | Importance of Bak for celecoxib-induced apoptosis[END_REF][START_REF] Lopez-Royuela | Different contribution of BH3-only proteins and caspases to doxorubicin-induced apoptosis in p53-deficient leukemia cells[END_REF]. Thus, the activation of Bak is essential for Celecoxib-induced apoptosis [START_REF] Rudner | The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway[END_REF]. Our data show an interaction of Bak with

Mcl-1 or Bcl-xL in Jurkat Vector as well as in the Bcl-2 and Bcl-xL-overexpressing cells.

Using mild lysis condition, Bcl-2:Bak complexes were also detected in Bcl-2-overexpressing cells. However, the association of Mcl-1 or Bcl-xL with Bak was obviously different from that of Bcl-2 with Bak. In contrast to Bcl-2, Bcl-xL and Mcl-1 form complexes even under harsher lysis conditions when 0.2% and 1% Triton X-100 was used suggesting a much stronger interaction between the latter ones and Bak than between Bcl-2 and Bak.

The use of Triton X-100 is not unproblematic. Previous publications showed that immunoprecipitation of Bax and the heterodimerization with anti-apoptotic proteins depends on the detergent used [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF]. In addition, Hsu and Youle detected a heterodimerization of Bax with Bcl-2 and Bcl-xL in presence of Triton X-100 but not CHAPS [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF]. Contrary to this previous publication, using different concentrations of Triton X-100, our results show that the detergent did not facilitate the binding of the anti-apoptotic Mcl-1 and Bcl-xL to Bak but prevented interaction between Bcl-2 and Bak. Interestingly, Bak was easily precipitated in presence of Triton X-100, and the amount of precipitated Bak did not change over time after treatment with Celecoxib (data not shown). In presence of CHAPS, in contrast, we were hardly able to precipitate Bak in healthy cells. Probably, Triton X-100 interfered with intramolecular interactions of Bak facilitating the exposure of its N-terminus and, therefore, its precipitation with an antibody recognizing the N-terminus. This effect was not observed

  Celecoxib induced apoptosis through the Noxa/Mcl-1 axis in Jurkat T cell lymphoma cells leading to downregulation of Mcl-1 [7]. The depletion of Mcl-1 protein levels was sufficient to induce apoptosis in this cell system.

2 .

 2 was the activator BH3-only protein Bid which was converted into the apoptotic truncated Bid (tBid) by caspases downstream of m dissipation. We also excluded the involvement of Nur77/TR3 which targets Bcl-2 but not Bcl-xL to transfer it from an anti-apoptotic molecule into a pro-apoptotic one. However, we found a strong interaction of Mcl-1 and Bcl-xL with Bak in healthy Jurkat Vector control and Bcl-xLoverexpressing cells. A Bcl-2:Bak interaction was observed only when Bcl-2 was overexpressed. When harsher lysis conditions were applied the complex of Bcl-2 and Bak could not be detected any longer while Bcl-xL and Mcl-1 still associated with Bak. The present data clearly show that Bcl-2 cannot replace Bcl-xL in Jurkat T cells during Celecoxibinduced apoptosis. We concluded that Bcl-xL and Mcl-1 prevented activation of Bak through direct interaction. When sufficiently expressed, Bcl-xL can substitute for Mcl-1 loss in response to Celecoxib. Bcl-2, however, which is not able to form high affinity complexes with Bak, fails to inhibit Bak activation after Mcl-1 downregulation. A c c e p t e d M a n u s c r i p t 6 Material and methods:2.1 Reagents and antibodiesAll chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) unless otherwise specified. The pan-caspase inhibitor zVAD-fmk was purchased from Bachem (Weil/Rhein, Germany). Celecoxib was kindly provided by Pharmacia-Pfizer (Erlangen, Germany). Following antibodies were used for Western blotting and immunoprecipitation: mouse-anti caspase-9 and rabbit-anti Bak NT from Upstate (Millipore, Schwalbach, Germany), rabbitanti caspase-3, PARP, Mcl-1, Bcl-xL, Bid, Nur77, and Tubulin from Cell Signaling (NEB Frankfurt, Germany), mouse-anti caspase-8 from BioCheck (Münster, Germany), rabbit-anti Puma and Bim from Epitomics (Biomol, Hamburg, Germany), mouse-and rabbit-anti Bcl-2 from Santa Cruz Biotechnology (Heidelberg, Germany), mouse-anti Mcl-1 from Pharmingen (Becton Dickinson, Heidelberg, Germany), mouse-anti Bcl-xL from Transduction Lab (Becton Dickinson, Heidelberg, Germany), mouse-anti Bak (clone TC-100) from Calbiochem (Merck, Darmstadt, Germany), mouse-anti GAPDH from Abcam (Cambridge, UK), and mouse-anti -Actin was obtained from Sigma (Deisenhofen, Germany).

10 5

 5 cells were incubated in a solution containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 , 1:50 diluted Annexin-V-FLUOS (Roche Diagnostics, Mannheim, Germany), and 10 µg/mL propidium iodide. Cells stained with TMRE were detected in channel 2, cells stained with Annexin V-PI in channel 1 and 3 employing a FACS Calibur flow cytometer and the Cell Quest software from Becton Dickinson (Heidelberg, Germany). Flow cytometric analysis was performed using the FCS Express software (De Novo Software, Los Angeles, CA, USA). Data show mean values +/-S.D. of at least 3 independent experiments.

A c c e p t e d M a n u s c r i p t 8 2. 5

 85 Western blot analysisCells were lysed in 200 µL lysis buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1%

  by Western blotting as described above. Unless specified, immunoprecipitation studies were always performed in presence of the detergent CHAPS.2.7 Data analysisED50 values for cells with low m were calculated after fitting the flow cytometric results according to the equation y = (A 1 -A 2 )/(1+e (x-x0)/dx )+A2, ED50 values for healthy AnnV/PI double negative cells according to the equation y = 100-[(A 1 -A 2 )/(1+e (x-x0)/dx )+A2].Statistical significance between the ED50 values for different cell lines was calculated by ANOVA test using GraphPad Software (San Diego, CA, USA, www.graphpad.com).

A c c e p t e d M a n u s c r i p t 10 3. Results: 3 . 1 A c c e p t e d M a n u s c r i p t 11 1

 103111 Apoptosis induction by Celecoxib in Jurkat T cells overexpressing Bcl-2 and Bcl-xLFirst, we examined apoptosis induction in Jurkat Vector cells and in Jurkat cells overexpressing Bcl-2 or Bcl-xL. Celecoxib triggered apoptosis in Jurkat Vector cells in a concentration-dependent manner. 6h after treatment with Celecoxib the amount of Annexin V-positive cells was dramatically elevated (Fig.1A). 50 µM Celecoxib were sufficient to induce apoptosis in 30% of the cells. The dissipation of mitochondrial membrane potential (m) coincided with apoptosis induction (Fig.1B). Bcl-2-overexpressing cells were similar sensitive to Celecoxib-induced apoptosis and m dissipation while overexpression of Bcl-xL was strongly protective (Fig.1A and 1B). The calculated ED50 values for Celecoxibinduced apoptosis (healthy, AnnV/PI double negative cells) and m dissipation (m low) in Vector (ED50(AnnV-/PI-) = 63.5+/-2.2 µM), ED50(m low) = 61.4+/-2.3 µM) and Bcl-2-overexpressing cells (ED50(AnnV-/PI-) = 71.4+/-2.2 µM, ED50(m low) = 67.6+/-0.2 µM) were very similar (Fig.1C and FigS1). In constrast, significantly higher concentrations were calculated for Bcl-xL-overexpressing cells (ED50(AnnV-/PI-) = 166.4+/-11.3 µM, ED50(m low) = 132.1+/-11.4 µM). Caspase activation, a hallmark of apoptosis induction downstream of m dissipation, could be detected in Jurkat Vector and Jurkat Bcl-2 cells as early as 3 h after treatment with 75 µM Celecoxib (Fig.1D). The initiator caspase-9, executing caspase-3, the caspase-3 substrates PARP, and caspase-8 were fully active 6 h after administration of Celecoxib in those cells, while no cleavage fragments were observed in cells overexpression of Bcl-xL.The downregulation of Mcl-1 is essential for Celecoxib-induced apoptosis[START_REF] Rudner | Differential effects of antiapoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on celecoxib-induced apoptosis[END_REF]. We observed a drastic reduction of Mcl-1 protein levels as early as 3 h after treatment with 75 µM Celecoxib whereas levels of Bcl-2, Bcl-xL, and Bak remained unchanged (Fig.1D). The decline of Mcl-shows a similar profile in Jurkat Vector cells and in Bcl-2-and Bcl-xL-overexpressing cells does not correlate with caspase activation suggesting that Mcl-1 protein level is not regulated by caspases. Indeed, treatment with 30 µM of the pan-caspase inhibitor zVAD did not prevent the initial decline of Mcl-1 protein levels 3h after treatment with Celecoxib but attenuated the total removal during the executive phase of apoptosis (Fig.2C).

  So far, the results from this experiments confirm previous observations demonstrating the early downregulation of Mcl-1 during Celecoxib-induced apoptosis, the protection by Bcl-xL overexpression and the lack thereof by Bcl-2 overexpression [7, 12]. To explore the mechanism of Celecoxib-induced apoptosis further, BH3-only proteins of the Bcl-2 family and their preferred interaction partners were analyzed. The focus was on the activator BH3only proteins which include Bid, Bim and sometimes Puma because a direct interaction of activator BH3-only proteins with Bax/Bak is thought to be prerequisite for activation of the multi-domain proteins. According to the sequestration model the binding preferences of Bcl-2 and Bcl-xL to different BH3-only proteins might change during Celecoxib-induced apoptosis.

A c c e p t e d M a n u s c r i p t 12 while

 12 m dissipation was unaffected (Fig.2Bupper and lower panel, respectively). Furthermore, zVAD interfered with caspase-9, caspase-3, and caspase-8 activation as well as PARP and Bid cleavage and inhibited Puma decline (Fig.2C). The results indicate that the regulation of Bid and Puma occurs in the executive phase of apoptosis upon caspase activation and plays a minor role before m dissipation.

M a n u s c r i p t 13 and

 13 co-precipitated neither with Mcl-1 nor Bcl-2, Bcl-xL, or Bak (Fig. 3D) indicating that this BH3-only protein plays no role during Celecoxib-induced apoptosis. Since activation of Bid turned out to be downstream of caspase activation and Puma was not essential for Celecoxib-induced apoptosis, we next analyzed the role of Bim. Bim preferentially interacted with Bcl-2 and Mcl-1, but less with Bcl-xL in Jurkat cells (Fig 4A, left panel). Reverse precipitation of Bcl-2, Bcl-xL, and Mcl-1 confirmed the binding of Bim to the analyzed anti-apoptotic proteins.(Fig. 4A, middle and right panels). In Bcl-2overexpressing cells, an association of Bim with Mcl-1 or Bcl-xL is hardly detectable (Fig 4B). Bcl-2 crowded Mcl-1 and Bcl-xL from its interaction with Bim. In contrast, overexpression of Bcl-xL did not affect the binding of Bcl-2 to Bim but Bcl-xL was able to supplant Mcl-1 to lesser extent. After stimulation with 75 µM Celecoxib, no change of interaction could be observed between Bcl-2 and Bim in Jurkat cells (Fig. 4A). Although a reduced interaction of Bcl-xL with BimA c c e p t e d Mcl-1 with Bim was noticed in response to Celecoxib, an enhanced association of the released Bim with the multi-domain protein Bak could not be detected. The results point to an ancillary role of Bim during Celecoxib-induced Bak activation and m dissipation.Silencing of Bim by siRNA should reassess the assumption. Successful downregulation of Bim by siRNA was verified 48h later by Western blotting (Fig.4C). Thus, 48h after electroporation of Jurkat cells with bim or the non-targeting control siRNA, cells were stimulated with 50-100 µM Celecoxib for 6h. Surprisingly, Celecoxib triggered apoptosis (Fig.4D) and m dissipation (Fig.4E) with similar sensitivity in Jurkat cells irrespective of Bim levels. A slight protection by bim siRNA was only observed when cells were treated with 75 µM Celecoxib. The experiments indicate that, similar to Puma, Bim was not needed either for Celecoxib-induced apoptosis.

  similar anti-apoptotic proteins could also vary in their binding to Bak. Coimmunoprecipitation studies with an antibody that preferably recognized the active conformation of Bak as well as with antibodies against Mcl-1, Bcl-2, and Bcl-xL revealed that Bak interacted primarily with Mcl-1 and Bcl-xL (Fig.7A, left panel). Bcl-2:Bak complexes were not detected in healthy Jurkat vector cells, nor in cells treated with Celecoxib. In Bcl-xLoverexpressing cells, more Bak co-precipitated with Bcl-xL than in Jurkat Vector controls (Fig.7A, right panel). In total, however, less Bak was precipitated with the activation specific antibody when compared to Jurkat vector or Bcl-2 overexpressing cells confirming previous observations that Bcl-xL inhibits Celecoxib-induced Bak activation and m dissipation.

A c c e p t e d M a n u s c r i p t

  The last experiments indicate that the interaction of Bak with Bcl-xL or Mcl-1 differs from that of Bak with Bcl-2.Taken together, the results show that Bcl-2 and Bcl-xL do not interact in the same way with Bak in Jurkat cells. The different affinities to Bak might also explain why Bcl-2, in contrast to Bcl-xL, did not protect from Celecoxib-induced apoptosis.Members of the Bcl-2 protein family are important regulators of survival and death during apoptosis induction through the intrinsic pathway. Many cytotoxic drugs as well as the COX-2 inhibitor Celecoxib, ionizing radiation, growth factor withdrawal, and severe hypoxia initiate apoptosis through the mitochondrial pathway[START_REF] Jendrossek | Celecoxib activates a novel mitochondrial apoptosis signaling pathway[END_REF][START_REF] Belka | Differential role of caspase-8 and BID activation during radiation-and CD95-induced apoptosis[END_REF][START_REF] Weinmann | Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner[END_REF][START_REF] You | FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal[END_REF][START_REF] Mcgrogan | Taxanes, microtubules and chemoresistant breast cancer[END_REF]. Overexpression of antiapoptotic proteins or inefficient activation of the pro-apoptotic ones improves cellular survival and accounts for resistance against diverse anti-cancer therapies.Here we show that the Celecoxib-induced apoptosis could be blocked by overexpression of Bcl-xL but not by the closely related Bcl-2 in Jurkat cells. Mcl-1 and Bcl-xL tightly associated with Bak in healthy Jurkat cells. In contrast, Bcl-2:Bak complexes were detectable in Bcl-2-overexpressing cells and under mild lysis conditions only. We conclude that the strong interaction between Bcl-xL and Bak kept Bak in an inactive conformation thereby protecting from mitochondrial permeabilization and apoptosis induction by Celecoxib in Bcl-xL-overexpressing cells whereas Bcl-2, incapable of such interaction, did not interfere with Celecoxib-induced apoptosis. Our data provide strong evidence that Bcl-xL and Bcl-2 do not use the same mechanism to interfere with apoptosis induction in Jurkat cells.

A c c e p t e d M a n u s c r i p t

  Rofecoxib and Valdecoxib, can inhibit protein translation transiently with subsequent downregulation of short-lived proteins[START_REF] Pyrko | Celecoxib transiently inhibits cellular protein synthesis[END_REF]. Previous result in our lab revealed that Celecoxib facilitated a rapid downregulation of the anti-apoptotic Mcl-1. Lowering the expression levels of the anti-apoptotic Mcl-1 was sufficient for apoptosis induction through the intrinsic pathway[START_REF] Rudner | Differential effects of antiapoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on celecoxib-induced apoptosis[END_REF]. In addition, using an antibody in immunoprecipitation studies which recognized preferentially the active conformation of Bak, we now show that Celecoxib induced a rapid activation of Bak in Jurkat Vector and Bcl-2-overexpressing cells but not in Bcl-xLoverexpressing cells.

A c c e p t e d M a n u s c r i p t 18 melanoma

 18 cells when Mcl-1 and Bcl-xL were up-regulated whereas Bcl-2 was downregulated[START_REF] Zhuang | Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma[END_REF].

A c c e p t e d M a n u s c r i p t 21 when

 21 the milder detergent CHAPS was used. The N-terminal exposure is a step during Bak activation that precedes Bak oligomerization. In this case, Triton X-100 would allow the association of Mcl-1 and Bcl-xL, but not Bcl-2, with a "partially activated" Bak.The specificity of Bak for Mcl-1 and Bcl-xL was described earlier[START_REF] Willis | Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins[END_REF][START_REF] Zhai | Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1[END_REF]. Both publications did not detect any interaction of Bcl-2 with Bak. Thus, Mcl-1 and Bcl-xL protected from apoptosis by sequestration of the pro-apoptotic Bak whereas Bcl-2 did not. Yet, Bcl-2 appears to use other mechanism to protect from apoptosis induced by overexpression of Bax and Bak[START_REF] Zhai | Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1[END_REF]. Interestingly, our Jurkat cells overexpressing Bcl-2 and Bcl-xL also inhibited apoptosis induction in response to ionizing radiation in earlier experiments[START_REF] Belka | Differential role of caspase-8 and BID activation during radiation-and CD95-induced apoptosis[END_REF][START_REF] Rudner | Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis[END_REF]. Although Bcl-2 is not capable of effective Bak sequestration, still it could bind to and neutralize other proapoptotic BH3-only family members including Bim, Puma, Bad, and Bmf. Regarding our data, we suggest following mechanisms for Celecoxib-induced apoptosis: in Jurkat T lymphoma cells, pro-apoptotic Bak is sequestered by Bcl-xL and Mcl-1. Treatment with Celecoxib induces a rapid downregulation of Mcl-1 protein levels which is sufficient to activate Bak. Overexpression of Bcl-xL protects from apoptosis because Bcl-xL can substitute for Mcl-1 loss by sequestering Bak which was released after Mcl-1 downregulation. Overexpression of Bcl-2 fails to inhibit Celecoxib-induced apoptosis because of inaptness to interact with Bak. The different association preferences of Bcl-2 and Bcl-xL with other proapoptotic Bcl-2 family members observed in our experiments allow the conclusion that Bcl-xL and Bcl-2 use different mechanisms to protect from apoptosis in response to distinct stimuli. and early cleavage of Bid was detected in Jurkat Vector and Bcl-2overexpressing cells. (B, C) Jurkat Vector cells were co-treated with 30 µM of caspase inhibitor zVAD-fmk and 75 µM Celecoxib. (B) Treatment with zVAD-fmk prevented Celecoxib-induced apoptosis (B, left panel) and processing of caspase-9, caspase-3, caspase-8, and PARP (C) but had no effect on m dissipation (B, right panel). (C) Decline of Puma and Bid cleavage were prevented by inhibition of caspases whereas downregulation of Mcl-1 was still observed in presence of zVAD-fmk. Tubulin was used as loading control.

Figure 3 :

 3 Figure 3: BH3-only Puma is not essential for Celecoxib-induced apoptosis. (A) Jurkat

Figure 4 :

 4 Figure 4: BH3-only Bim is not essential for Celecoxib-induced apoptosis. (A) Jurkat E6.1

Figure 5 :

 5 Figure 5: Bim and Puma have no redundant function during Celecoxib-induced

Figure 6 :

 6 Figure 6: The orphan nuclear receptor Nur77/TR3 does not bind to Bcl-2 during

Figure 7 :

 7 Figure 7: Mcl-1 and Bcl-xL but not Bcl-2 associate tightly with Bak to prevent its

Figure S1 :

 S1 Figure S1: Apoptosis induction and m dissipation in response to Celecoxib. Jurkat

Figure S2 :

 S2 Figure S2: Silencing of Bim and Puma inhibits apoptosis induction after serum

  Figure 1

Figure 3 A

 3 Figure 3

Figure 4 A

 4 Figure 4

Acknowledgments

The work was supported by grants from the fortüne-Program of the University of Tübingen (1806-1-0) and from the German Research Foundation DFG (1641/1-1) to JR. Pharmacia Pfizer kindly provided Celecoxib for our study.

Disclosure of potential conflicts of interests

The authors declare no conflict of interests.

Contributions

JR designed the study and contributed most of the experimental work, analyzed and interpreted the data, drafted and revised the manuscript and gave the final approval. SJE contributed significantly to data acquisition and analysis. VJ and SMH helped drafting and revising the manuscript. In addition, SMH helped with data analysis and interpretation.


Philipp Saiko

Geraldine Graser

Benedikt Giessrigl

Andreas Lackner

Michael Grusch

Georg Krupitza

Barij Arijit Basu

Nayan Sinha

Venkatesan Jayaprakash

Walter Jaeger

Monika Fritzer-Szekeres

Thomas Szekeres
email: thomas.szekeres@meduniwien.ac.at

Nayan Barij

Sinha

A novel N-Hydroxy-N'-aminoguanidine derivative inhibits ribonucleotide reductase activity: Effects in human HL-60 promyelocytic leukemia cells and synergism with arabinofuranosylcytosine

Keywords: N-Hydroxy-N'-aminoguanidines, ribonucleotide reductase, cell cycle arrest, arabinofuranosylcytosine, synergistic combination effects

come

A c c e p t e d M a n u s c r i p t

Introduction

Various compounds with hydroxyguanidine, thiosemicarbazide, and substituted benzohydroxamic acid functional groups have shown promising antitumor activity [START_REF] Koneru | Synthesis and testing of new antileukemic Schiff bases of N-hydroxy-N'-aminoguanidine against CCRF-CEM/0 human leukemia cells in vitro and synergism studies with cytarabine (Ara-C)[END_REF][START_REF] Ren | Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents[END_REF][START_REF] Tai | Novel N-hydroxyguanidine derivatives as anticancer and antiviral agents[END_REF][START_REF] T'ang | Optimization of the Schiff bases of N-hydroxy-N'aminoguanidine as anticancer and antiviral agents[END_REF][START_REF] Van't Riet | Synthesis of hydroxy-and aminosubstituted benzohydroxamic acids: inhibition of ribonucleotide reductase and antitumor activity[END_REF]. Hydroxyguanidines and hydroxysemicarbazides were especially active against human CCRF-CEM/0 and murine L1210 leukemia cells as well as against human HT-29 colon cancer cells [START_REF] Koneru | Synthesis and testing of new antileukemic Schiff bases of N-hydroxy-N'-aminoguanidine against CCRF-CEM/0 human leukemia cells in vitro and synergism studies with cytarabine (Ara-C)[END_REF][START_REF] Ren | Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents[END_REF][START_REF] Tai | Novel N-hydroxyguanidine derivatives as anticancer and antiviral agents[END_REF][START_REF] T'ang | Optimization of the Schiff bases of N-hydroxy-N'aminoguanidine as anticancer and antiviral agents[END_REF][START_REF] Matsumoto | Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives[END_REF]. These agents inhibited DNA synthesis as a consequence of inhibiting ribonucleotide reductase (RR; EC 1.17.4.1) activity.

RR is significantly upregulated in tumor cells in order to meet the increased need for deoxyribonucleoside triphosphates (dNTPs) of these rapidly proliferating cells for DNA synthesis [START_REF] Takeda | Role of ribonucleotide reductase in expression in the neoplastic program[END_REF]. The enzyme is an α22 complex consisting of two subunits [START_REF] Kolberg | Structure, function, and mechanism of ribonucleotide reductases[END_REF].

The effector binding R1 subunit possesses an α2 homodimeric structure with substrate and allosteric effective sites that control enzyme activity and substrate specificity. The nonheme iron R2 subunit, a 2 homodimer, forms two dinuclear iron centers each stabilizing a tyrosyl radical. The inhibition of the nonheme iron subunit can be caused, for instance, by iron chelation or radical scavenging of the tyrosyl radical [START_REF] Shao | Ribonucleotide reductase inhibitors and future drug design[END_REF]. Additionally, a p53-inducible R2-homologue (p53R2) has been described recently [START_REF] Shao | Ribonucleotide reductase inhibitors and future drug design[END_REF]. Expression of the R2 and p53R2 subunits is induced by DNA damage and it has been reported that p53R2 supplies dNTPs for DNA repair in G 0 /G 1 cells in a p53-dependent manner [START_REF] Bourdon | Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion[END_REF]. Hydroxyurea (HU) is the first RR inhibitor that has been used in clinical practice and is given to treat chronic myeloid leukemia and many other neoplastic diseases [START_REF] Saban | Hydroxyurea and hydroxamic acid derivatives as antitumor drugs[END_REF][START_REF] Tennant | Chronic myelogenous leukemia: an overview[END_REF]. Difluorodeoxycytidine (Gemcitabine; dFdC) is applied in chemotherapy regimens against non-small cell lung cancer and pancreatic cancer [START_REF] Noble | Gemcitabine. A review of its pharmacology and clinical potential in non-small cell lung cancer and pancreatic cancer[END_REF][START_REF] Toschi | Role of gemcitabine in cancer therapy[END_REF].

A c c e p t e d M a n u s c r i p t 4 HU is believed to destabilize R2 iron centers by scavenging the tyrosyl radical which is essential for enzyme activity [START_REF] Shao | Ribonucleotide reductase inhibitors and future drug design[END_REF][START_REF] Hatse | Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation[END_REF]. Several newer iron chelating agents including tachypyridine [START_REF] Abeysinghe | p53-independent apoptosis mediated by tachpyridine, an anti-cancer iron chelator[END_REF][START_REF] Samuni | Mechanisms underlying the cytotoxic effects of Tachpyr--a novel metal chelator[END_REF][START_REF] Turner | Tachpyridine, a metal chelator, induces G2 cell-cycle arrest, activates checkpoint kinases, and sensitizes cells to ionizing radiation[END_REF][START_REF] Torti | Tumor cell cytotoxicity of a novel metal chelator[END_REF][START_REF] Greene | Activation of caspase pathways during iron chelator-mediated apoptosis[END_REF] and a number of thiosemicarbazones such as triapine [START_REF] Shao | Ribonucleotide reductase inhibitors and future drug design[END_REF][START_REF] Tsimberidou | Evolving role of ribonucleoside reductase inhibitors in hematologic malignancies[END_REF] were shown to interact with the iron-containing R2 subunit. These compounds are currently under (pre)clinical development.

Modern drug design uses qualitative and quantitative structure-activity relationship (QSAR) studies as an approach to find relationships between chemical structures or structure-related properties and biological activities of distinct compounds. Based on the prediction of the best QSAR model, we synthesized 13 novel compounds (ABNM-1 to ABNM-13) with potential RR inhibitory capacities. Five of these agents were active in human HL-60 promyelocytic leukemia cells and ABNM-13 was chosen as lead substance because of its pronounced growth inhibitory effects which were up to 10-fold stronger than those of HU. The HL-60 cell line is an excellent in vitro model and has been extensively used by our group and others, especially with regard to investigate RR activity after treatment with various drugs. Additionally, growth inhibition and cytotoxicity caused by ABNM-13 were also investigated in human AsPC-1 pancreatic cancer cells. To study the mechanisms by which ABNM-13 influences cell cycle transit in HL-60 cells, we examined the effects on RR and the cell cycle regulators downstream of checkpoint kinase activation.

In general, anticancer drugs are more effective when used in combination. The major advantage of drug combinations is the achievement of additive or synergistic effects through intimidation of distinct molecular pathways and, accordingly, a decrease of drug resistance. For example, administration of leucovorin increases the binding of an active 5-fluorouracil metabolite to its target, thymidylate synthase, thus increasing the antineoplastic effects [START_REF] Erlichman | A randomized trial of fluorouracil and folinic acid in patients with metastatic colorectal carcinoma[END_REF]. In addition, various RR inhibitors caused synergism A c c e p t e d M a n u s c r i p t 5 together with arabinofuranosylctosine (Ara-C), a first line antileukemia drug affecting intracellular dCTP pools [START_REF] Saiko | N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxy-benzamidine, a novel resveratrol analog, inhibits ribonucleotide reductase in HL-60 human promyelocytic leukemia cells: synergistic antitumor activity with arabinofuranosylcytosine[END_REF][START_REF] Horvath | Synergistic action of resveratrol, an ingredient of wine, with Ara-C and tiazofurin in HL-60 human promyelocytic leukemia cells[END_REF][START_REF] Horvath | Cytotoxic and biochemical effects of 3,3',4,4',5,5'-hexahydroxystilbene, a novel resveratrol analog in HL-60 human promyelocytic leukemia cells[END_REF][START_REF] Fritzer-Szekeres | Trimidox, an inhibitor of ribonucleotide reductase, synergistically enhances the inhibition of colony formation by Ara-C in HL-60 human promyelocytic leukemia cells[END_REF][START_REF] Fritzer-Szekeres | Biochemical effects of piceatannol in human HL-60 promyelocytic leukemia cells--synergism with Ara-C[END_REF]. Following this strategy, we combined ABNM-13 with Ara-C in order to test potential additive or synergistic properties.

A c c e p t e d M a n u s c r i p t 6

Materials and methods

Chemicals and supplies

ABNM 1-13 were synthesized and provided by the Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, India. Structural formulas are shown in figure 1. Ara-C, HU and all other chemicals and reagents were commercially available (Sigma-Aldrich, Vienna, Austria) and of highest purity.

Cell culture

The human HL-60 promyelocytic leukemia and human AsPC-1 pancreatic adenocarcinoma cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Both lines were grown in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 1% L-Glutamine, and 1% Penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 using a Heraeus cytoperm 2 incubator (Heraeus, Vienna, Austria). AsPC-1 cells were grown in a monolayer culture using 25cm 2 tissue culture flasks and were periodically detached from the flask surface by 0.25% trypsinethylenediaminetetraacetic acid (trypsin-EDTA) solution. All media and supplements were obtained from Life Technologies (Paisley, Scotland, UK). Cell counts were determined using a microcellcounter CC-110 (SYSMEX, Kobe, Japan). Cells growing in the logarithmic phase of growth were used for all experiments described below.

Growth inhibition assay

HL-60 cells (0.1x10 6 per ml) were seeded in 25cm 2 Nunc tissue culture flasks and incubated with increasing concentrations of ABNM 1-13 and HU at 37°C under cell

Clonogenic assay

AsPC-1 cells (2x10 3 per well) were plated in 24-well plates and allowed to attach overnight at 37°C in a humidified atmosphere containing 5% CO 2 . Then cells were incubated with increasing concentrations of ABNM-13 for 6 days. Subsequently, the medium was carefully removed from the wells and the plates were stained with 0.5% crystal violet solution for 5 minutes. Colonies of more than 50 cells were counted using an inverted microscope at 40-fold magnification.

MTT chemosensitivity assay

AsPC-1 or HL-60 cells (5x10 3 per well) were seeded in 96-well microtiter plates in supplemented RPMI 1640 medium. AsPC-1 cells were allowed to attach overnight.

Cells were then incubated with various concentrations of ABNM-13 for 96 hours at 37°C under cell culture conditions. After that period, the reduction of the yellow tetrazolium compound 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazoliumbromide

Simultaneous growth inhibition assay using ABNM-13 and Ara-C

HL-60 cells (0.1x10 6 per ml) were simultaneously incubated with various concentrations of ABNM-13 (12.5, 15, 17.5, and 20 µM) and Ara-C (10, 15, and 20 nM) for 72 hours. After that period, cells were counted using a microcellcounter CC-110.

Sequential growth inhibition assay using ABNM-13 and Ara-C

HL-60 cells (0.1x10 6 per ml) were first incubated with different concentrations of ABNM-13 (2.5, 5, 7.5, and 10 µM) for 24 hours. Then ABNM-13 was washed out and cells were further exposed to various concentrations of Ara-C (10, 15, and 20 nM) for another 48 hours. After that period, cells were counted using a microcellcounter CC-110.

Cell cycle distribution analysis

Cells (0.4x10 6 per ml) were seeded in 25cm 2 Nunc tissue culture flasks and incubated with increasing concentrations of drugs at 37°C under cell culture conditions. After 24 hours, cells were harvested and suspended in 5 ml cold PBS, centrifuged, resuspended and fixed in 3 ml cold ethanol (70%) for 30 minutes at 4°C. ModFit LT software (Verity Software House, Topsham, ME, USA).

Western blotting

After incubation with 15µM ABNM-13 and/or 15nM Ara-C, HL-60 cells (2x10

Incorporation of 14 C-labeled cytidine into DNA (DNA synthesis assay)

To analyze the effect of ABNM-13 treatment on the activity of DNA synthesis, an assay was performed as described previously [START_REF] Szekeres | Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase[END_REF]. Radiolabeled 14 C-cytidine has to be reduced by RR in order to be incorporated into the DNA of HL-60 cells following incubation with ABNM-13 and/or the RR-inhibitor Ara-C. HL-60 cells (0.4x10 6 cells per ml) were incubated with various concentrations of ABNM-13 for 24 hours. After that, cells were counted and pulsed with 14 C-cytidine (0.3125 µCi, 5 nM) for 30

Determination of deoxyribonucleoside triphosphates (dNTPs)

Cells were seeded in 175 cm 2 tissue culture flasks (5x10 7 per flask) and then incubated with increasing concentrations of ABNM-13 for 24 hours. The cells were then centrifuged at 1800 g for 5 min, resuspended in 100 µl of PBS, and extracted with 10 µl of trichloracetic acid (90%). The lysate was allowed to rest on ice for 30 min and neutralized by the addition of 1.5 volumes of freon containing 0.5 mol/l tri-noctylamine. Concentrations of dNTPs were then determined using the method described by Garrett and Santi [START_REF] Garrett | A rapid and sensitive high pressure liquid chromatography assay for deoxyribonucleoside triphosphates in cell extracts[END_REF]. Aliquots (100 µl) of the samples were analyzed using a Merck ''La Chrom'' high-performance liquid chromatography (HPLC) system

Hoechst dye 33258 and propidium iodide double staining

The Hoechst staining was performed according to the method described by our group [START_REF] Grusch | Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside[END_REF]. HL-60 cells (0.2x10 6 per ml) were seeded in 25cm nuclear PI uptake without chromatin condensation or nuclear fragmentation [START_REF] Huettenbrenner | The evolution of cell death programs as prerequisites of multicellularity[END_REF].

Cells were judged according to their morphology and the integrity of their cell membranes, counted under the microscope and the number of apoptotic cells was given as percentage value.

Statistical calculations

Dose-response curves were calculated using the Prism 5.01 software package (GraphPad, San Diego, CA, USA) and significant differences between controls and each drug concentration applied were determined by unpaired t-test. The calculations of dose response curves and combination effects were performed using the "Calcusyn" software designed by Chou and Talalay (Biosoft, Ferguson, MO) [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF].

The analytical method of Chou and Talalay [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF][START_REF] Chou | Generalized equations for the analysis of inhibitions of Michaelis-Menten and higher-order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors[END_REF] describes the interaction among drugs in a given combination. A combination index (CI) of <0.9 indicates synergism, a CI of 0.9-1.1 indicates additive effects, and a CI of >1.1 indicates antagonism.

A c c e p t e d M a n u s c r i p t 14

Results

Effect of ABNM 1-13 on the growth of HL-60 and AsPC-1 cells

HL-60 cells (0.1x10 6 per ml) were seeded in 25cm inhibited the growth of AsPC-1 cells with an IC 50 of 76±4 µM.

Effect of ABNM-13 on the growth of HL-60 cellsalone and in combination with Ara-C

HL-60 cells were seeded at a concentration of 0.1x10 6 per ml and incubated with increasing concentrations of ABNM-13. After 48 and 72 hours, the cell number of viable leukemia cells was determined. ABNM-13 inhibited the growth of HL-60 cells with IC 50 values (IC 50 = 50% growth inhibition of tumor cells) of 15±0.3 and 11±1.1 µM, respectively (figure 2a). Exposure to ABNM-13 for 24 hours resulted in a cell count of 67±0.6% (33% growth inhibition). Treatment with HU, a RR inhibitor currently used in the clinic for 48 and 72 hours resulted in IC 50 values of 143±0.2 and 88±0.2 µM, respectively (figure 2b). These findings are consistent with those hours of incubation [START_REF] Szekeres | Synergistic growth inhibitory and differentiating effects of trimidox and tiazofurin in human promyelocytic leukemia HL-60 cells[END_REF].

To investigate the effect of ABNM-13 in combination with Ara-C, HL-60 cells were seeded at a concentration of 0.1x10 6 per ml and simultaneously or sequentially incubated with increasing concentrations of drugs (ABNM-13 first for 24 hours and then Ara-C for 48 hours as described in the methods section). All twelve drug combinations yielded additive effects when ABNM-13 and Ara-C were applied simultaneously (data not shown). Moreover, all twelve combinations led to highly synergistic effects when applied sequentially (cells were first incubated with 2.5, 5, 7.5, and 10 µM ABNM-13 followed by the addition of 5, 10, and 20 nM Ara-C, respectively) (table 1).

Effect of ABNM-13 on the growth of AsPC-1 cell colonies

AsPC-1 cells were seeded at a concentration of 2x10 3 per well and incubated with increasing concentrations of ABNM-13. Colonies were counted after 6 days of treatment. ABNM-13 inhibited the growth of AsPC-1 cell colonies with an IC 50 value of 11.5±1.4 µM (figure 2c) being almost identical to the IC 50 seen in HL-60 cells (11±1.1 µM).

MTT chemosensitivity assay

AsPC-1 or HL-60 cells (5x10 3 per well) were seeded in 96-well microtiter plates and exposed to increasing concentrations of ABNM-13 as described in the methods section. After 96 hours of incubation, ABNM-13 reduced the absorbance (viability) of AsPC-1 and HL-60 cells with IC 50 values of 40±3.4 and 9±1.7 µM, respectively.

A c c e p t e d M a n u s c r i p t 16

Inhibition of incorporation of 14 C-cytidine into DNA of HL-60 cells (DNA synthesis assay) and dNTP alterations after treatment with ABNM-13 and/or

Ara-C

Incorporation of 14 C-cytidine into nascent DNA was measured in HL-60 cells after incubation with increasing concentrations of ABNM-13. Exposure to 10, 20, and 40 µM ABNM-13 for 24 hours significantly decreased 14 C-cytidine incorporation to 52±13.9%, 17±6.1%, and 4±2.8%, respectively (figure 3a). Constitutive RR activity maintains balanced dNTP pools, whereas RR inhibition tilts this balance. In line with this, ABNM-13 treatment caused also an imbalance of dNTPs in HL-60 cells after 24 hours, which was determined by HPLC analysis. Incubation of cells with 40 µM ABNM-13 resulted in a significant depletion of intracellular dGTP pools to 36±15.7%.

Treatment with 10, 20, and 40 µM ABNM-13 significantly increased dTTP pools to 134±8.0%, 200±22.7%, and 237±21.3% of control values, respectively. Regarding dCTP and dATP pools, treatment with ABNM-13 led to insignificant changes (figure 3b).

To analyze the immediacy of DNA synthesis inhibition, HL-60 cells were exposed to 15 nM Ara-C, 15 µM ABNM-13, and the simultaneous combination of both compounds for only 30 minutes. Even this short incubation period reduced the incorporation of 14 C-cytidine to 93±33.8%, 27±6.3%, and 4±5.7% of controls, respectively (figure 3c).

Expression of RR subunits R1, R2, and p53R2 after treatment with ABNM-13 and/or Ara-C

To monitor the effect of RR inhibitors on the expression of RR subunits, HL-60 cells were incubated with 15 nM Ara-C and/or 15 µM ABNM-13 for 0.5, 2, 4, 8, and 24

A c c e p t e d M a n u s c r i p t hours and subjected to western blot analysis. The protein level of the constitutively expressed R1 subunit remained unchanged during the whole time course. R2 levels showed an increase after 8 and 24 hours, and p53R2 levels were elevated after 24 hours of incubation (figure 3d). Both R2 and p53R2 are S-phase specific.

Cell cycle distribution in HL-60 cells after treatment with ABNM-13 and/or

Ara-C

HL-60 cells were simultaneously incubated with 15 µM ABNM-13 and/or 15 nM Ara-C for 24 hours. Treatment of HL-60 cells with 15 µM ABNM-13 caused cell cycle arrest in S-phase, increasing this cell population from 34±0.4% to 62±0.0%, whereas G0-G1 phase cells decreased from 46±0.1% to 21±0.1%. 15 nM Ara-C likewise caused an accumulation of 69±1.6% HL-60 cells in S-phase and a concomitant decrease of G0-G1 cells to 12±0.8%. Simultaneous incubation of HL-60 cells with 15 µM ABNM-13 and 15 nM Ara-C led to an even more pronounced growth arrest in the S-phase, increasing this cell population from 34±0.4% to 94±0.5% while decreasing cells in the G0-G1 phase from 46±0.5% to 6±0.5% (figures 4a-c). No subG1 peaks could be observed by FACS at the time points measured.

Expression of checkpoint and cell cycle regulating proteins after treatment with ABNM-13 and/or Ara-C

To investigate whether S-phase inhibition caused activation of cell cycle checkpoint kinases, HL-60 cells were simultaneously treated with 15 nM Ara-C and/or 15 µM ABNM-13 for 0.5, 2, 4, 8, and 24 hours and subjected to western blot analysis (figures 4d-e). Chk1 was phosphorylated at the activating Ser317 site within 30 minutes (Ara-C), 2 hours (ABNM-13), and 30 minutes (Ara-C/ABNM-13). Chk2 was (ABNM-13), and 30 minutes (ABNM-13/Ara-C). Chk1 protein levels remained unchanged, whereas Chk2 protein levels increased transiently, in particular when using the combination of ABNM-13 and Ara-C (figure 4d). In addition, ABNM-13 caused phosphorylation at Ser75 and Ser177 of the dual-specificity phosphatase Cdc25A, which are target sites of Chk1 and Chk2, respectively, resulting in its downregulation after 8 and 24 hours. On the other hand, ABNM-13 upregulated Cdc25B protein levels after 24 hours (Ara-C after 8 and 24 hours), resulting in the dephosphorylation of Tyr15 of Cdk1 after 24 hours, which is indicative for its activation (figure 4e). Ara-C treatment did not cause dephosphorylation of Cdk1.

Cdc25C levels remained unchanged throughout the time course.

Induction of apoptosis in HL-60 cells by ABNM-13 and/or Ara-C

HL-60 cells were exposed to 12.5, 15, 17.5, and 20 µM ABNM-13 and/or 15 nM Ara-C for 24 and 48 hours and double stained with Hoechst 33258 and propidium iodide to analyze whether apoptotic cell death was induced. The nuclear morphology of 16±0.9% and 22±2.4% HL-60 cells showed early or late apoptosis stages upon treatment with 15 µM ABNM-13 for 24 and 48 hours, respectively (figure 5a).

Incubation with 15 nM Ara-C or the combination of 15 µM ABNM-13 and 15 nM Ara-C for 24 hours resulted in only 8.2±0.5% and 13±2.7% apoptotic cells, respectively.

Even the exposure of cells to 15 nM Ara-C or the combination of 15 µM ABNM-13 and 15 nM Ara-C for 48 hours led to no more than 10±0.6% and 28±4.8% apoptotic cells, respectively, suggesting that cell death is at best additive but not synergistic after simultaneous application of both compounds (figure 5b). The induction of apoptosis was further substantiated by the cleavage and therefore activation of

Discussion

3D molecular space modeling techniques were used to design in silico structures specifically to inhibit the activity of ribonucleotide reductase (RR), which is the ratelimiting enzyme of de novo DNA synthesis. From a panel of 13 compounds, we found that ABNM-13 is the most active agent with regard to growth inhibition of HL-60 cells.

The analysis of the in situ RR activity evidenced that ABNM-13 is a powerful RR inhibitor even after a short incubation time and at low concentrations. In addition, ABNM-13 caused alterations of deoxyribonucleoside triphosphate (dNTP) pool balance: dGTP pools were significantly depleted while dTTP pools were elevated. By misbalancing the concentration of precursors for de novo DNA synthesis, the latter is blocked in proliferating cells. Cell cycle perturbations, growth arrest and induction of apoptosis are the consequences, as it was observed in the course of ABNM-13 treatment.

The prime effect of ABNM-13 was a strong S-phase arrest which is consistent with the role of RR as the rate limiting enzyme for S-phase transit and the fact that inhibition of RR leads to inhibition of cells in S-phase [START_REF] Chimploy | E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin[END_REF]. It has been suggested that cells in which RR was inhibited by HU may enter the early S-phase at a normal rate and accumulate there until they undergo apoptosis [START_REF] Yarbro | Mechanism of action of hydroxyurea[END_REF][START_REF] Maurer-Schultze | An in vivo study on the synchronizing effect of hydroxyurea[END_REF]. The protein level of the constitutively expressed R1 subunit of RR remained unchanged. In contrast, the Sphase specific R2 subunit and also the p53R2 subunit of the enzyme were elevated although HL-60 cells are p53 deficient, indicating a compensatory up-regulation through which the cells try to rebalance their dNTP production. However, these findings are in line with the observations made by Yanomoto et al [START_REF] Yanamoto | Silencing of the p53R2 gene by RNA interference inhibits growth and enhances 5-fluorouracil sensitivity of oral cancer cells[END_REF] who demonstrated that basal levels of p53R2 are expressed regardless of the cellular p53 shown to induce cell cycle arrest by abrogating entry into mitosis [START_REF] Varmeh-Ziaie | The dual specificity phosphatase Cdc25B, but not the closely related Cdc25C, is capable of inhibiting cellular proliferation in a manner dependent upon its catalytic activity[END_REF]. Furthermore, Cdk2, as being regulated by Cdc25A, is required for S-phase progression [START_REF] Donzelli | Dual mode of degradation of Cdc25 A phosphatase[END_REF].

Therefore, the combined effect of Cdc25A degradation and Cdc25B overexpression most likely caused the almost complete S-phase arrest induced by ABNM-13 alone and together with Ara-C. Apoptosis upon treatment with ABNM-13 occurred in only 22% of cells (after 48 hours), indicating that cell cycle inhibition rather than induction of programmed cell death seems to be the primary antineoplastic effect of ABNM-13.

We therefore believe that a portion of treated cells was growing much slower than untreated controls, but did not undergo necrosis or apoptosis. The latter was further determined by the expression of cleaved caspase-3 (after 8 hours) which in turn led to elevation of γH2AX protein levels (after 24 hours), suggesting that treatment with ABNM-13 was not the primary cause for DNA double strand breaks but the consequence of caspase-3 induced DNAse activation. This was supported by the fact that constitutive phospho-ATM levels were not elevated, either. Cell death via mitotic catastrophe (i.e. the formation of giant cells with two or more nuclei) being promoted by Chk2 inhibition [START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF][START_REF] Portugal | Mechanisms of drug-induced mitotic catastrophe in cancer cells[END_REF] could not be observed at any time point.

Combination treatment is expected to produce fortified antitumor effects, if the pharmacokinetic and pharmacological properties are different from each other.

Accordingly ABNM-13, which disregulated dTTP and dGTP pools and Ara-C, which is known to affect dCTP pools [START_REF] Gandhi | Incorporation of fludarabine and 1-beta-D-arabinofuranosylcytosine 5'-triphosphates by DNA polymerase alpha: affinity, interaction, and consequences[END_REF][START_REF] Wills | Ara-C differentially affects multiprotein forms of human cell DNA polymerase[END_REF][START_REF] Seymour | Influence of fludarabine on pharmacokinetics and pharmacodynamics of cytarabine: implications for a continuous infusion schedule[END_REF] inhibited cell proliferation synergistically. Using a sequential combination of ABNM-13 and Ara-C, all twelve concentrations applied yielded highly synergistic antineoplastic effects.

Taken together, we demonstrate that the novel RR inhibitor ABNM-13 exerts pronounced anticancer activity both as single agent and as enhancer of another

Figure 2c. Inhibition of colony formation of AsPC-1 cells after incubation with

ABNM-13

AsPC-1 cells (2x10 3 per well) were plated in 24-well plates and allowed to attach overnight at 37°C in a humidified atmosphere containing 5% CO 2 . After 24 hours, the cells were incubated with increasing concentrations of ABNM-13 for 6 days.

Subsequently, the medium was carefully removed from the wells and the plates were stained with 0.5% crystal violet solution for 5 minutes. Colonies of more than 50 cells were counted using an inverted microscope at 40-fold magnification. Data are means ± standard errors of three determinations.

A c c e p t e d M a n u s c r i p t 7 culture

 7 conditions. Cell counts and IC 50 values (IC 50 = 50% growth inhibition of tumor cells) were determined after 24, 48, and 72 hours using a microcellcounter CC-110.In another set of experiments, AsPC-1 cells were seeded in 25cm 2 Nunc tissue culture flasks and allowed to attach overnight. Cells were then incubated with increasing concentrations of ABNM-13 for 72 hours. After that period, cells were detached from the flask surface by 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) solution. After removal of trypsin-EDTA by centrifugation and suspension of the pellet in RPMI 1640 medium, cells were counted using a microcellcounter CC-110. Viability of cells was determined by staining with trypan blue. Results were calculated as number of viable cells.

A c c e p t e d M a n u s c r i p t 8 (

 8 MTT) by the mitochondrial dehydrogenases of viable cells to a purple formazan product was determined using an assay kit from Promega® according to the supplier's manual. The change in absorbance at 550nm was tracked on a Wallac 1420 Victor 2 multilabel plate reader (PerkinElmer Life and Analytical Sciences). Drug effect was quantified as the percentage of control absorbance of reduced dye at this wavelength.

A c c e p t e d M a n u s c r i p t 9 After

 9 two washing steps in cold PBS RNAse A and propidium iodide were added to a final concentration of 50 µg/ml each and incubated at 4°C for 60 minutes before measurement. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and cell cycle distribution was calculated with

A c c e p t e d M a n u s c r i p t 10 Chemoluminescence

 10 was developed by the ECL detection kit (Amersham, Buckinghamshire, UK) and then membranes were exposed to Amersham Hyperfilms.Equal numbers of cells were lysed for each sample, protein content was measured by the Bradford method, and PVDF membranes were checked by Ponceau S staining. Equal sample loading was controlled by β-actin expression which appeared to be stable when inspected in short term exposures to x-ray films. Each western blot experiment was performed at least twice, and specific experimental points were done more often as they served as internal controls. Antibodies directed against p(Ser1981)-ATM, p(Ser317)-Chk1, Chk1, p(Thr68)-Chk2, Chk2, p(Tyr15)-Cdc2, cleaved Caspase-3 (Asp175) and anti-rabbit IgG were from Cell Signaling (Danvers, MA, USA), against p(Ser75)-Cdc25A from Abcam (Cambridge, MA, USA), against p(Ser177)-Cdc25A from Abgent (San Diego, CA, USA), against R1 (T-16), R2 (I-15), p53R2 (N-16), Cdc25A (F-6), Cdc25B (C-20), Cdc25C (C-20), Cdc2 p34 (17), and donkey anti-goat IgG from Santa Cruz (Santa Cruz, CA, USA), against ph(Ser139)-γH2AX from Merck (Darmstadt, Germany), against β-actin from Sigma (St. Louis, MO, USA), and anti-mouse IgG was from Dako (Glostrup, Denmark).

A c c e p t e d M a n u s c r i p t 11 minutes

 11 at 37°C. In another set of experiments, cells were treated with ABNM-13 and/or Ara-C for 30 minutes and simultaneously pulsed with 14 C-cytidine (0.3125 µCi, 5 nM). Afterwards, cells were collected by centrifugation and washed with PBS. Total DNA from 5x10 6 cells was purified by phenol-chloroform-isoamyl alcohol extraction and specific radioactivity of the samples was determined using a Wallac 1414 liquid scintillation counter (PerkinElmer, Boston, MA) whose read out was normalized by a Hitachi U-2000 Double Beam Spectrophotometer to ensure equal amounts and purity of DNA.

(

  Merck, Darmstadt, Germany) equipped with D-7000 interface, L-7100 pump, L-7200 autosampler, and L-7400 UV detector. Detection time was set at 80 min, with the detector operating on 280 nm for 40 min and then switched to 260 nm for another 40 min. Samples were eluted with a 3.2 M ammonium phosphate buffer (pH 3.6, adjusted by the addition of 3.2 mM H 3 PO 4 ) containing 20 M acetonitrile using a 4.6x250 mm PARTISIL 10 SAX column (Whatman Ltd., Kent, UK). Separation was performed at constant ambient temperature and a flow rate of 2 ml per minute. TheA c c e p t e d M a n u s c r i p t 12 concentration of each dNTP was calculated as percentage of the total area under the curve for each sample.

  A c c e p t e d M a n u s c r i p t 13 it from non-apoptotic cell death by means of necrosis. The latter is characterized by

A c c e p t e d M a n u s c r i p t 15 obtained

 15 by Szekeres and coworkers who determined an IC 50 of 73 µM after 96

A c c e p t e d M a n u s c r i p t 18 phosphorylated

 18 at the activating Thr68 site within 24 hours (Ara-C), 30 minutes

caspase- 3

 3 after 8 and 24 hours of treatment with 15 µM ABNM-13 or the combination of 15 µM ABNM-13 and 15 nM Ara-C which in turn led to increased protein levels of γH2AX after 24 hours (figure 5c). In contrast, 15 nM Ara-C induced activated caspase-3 and γH2AX levels only marginally. Constitutive phospho-ATM levels were not enhanced upon treatment with ABNM-13 and/or Ara-C. Examples of the cellular morphology are provided in figure 5d.

A c c e p t e d M a n u s c r i p t 20

 20

A c c e p t e d M a n u s c r i p t 21 status

 21 and of Zhang et al[START_REF] Zhang | Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response[END_REF] who showed that up-regulation of the R2 protein levels occurs in response to DNA damage and involves up-regulation and activation of Chk1.DNA damage or disrupted dNTP balance and incomplete DNA synthesis activate cell cycle checkpoints to prevent DNA synthesis and cell cycle progression[START_REF] Kastan | Cell-cycle checkpoints and cancer[END_REF][START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF][START_REF] Bartek | Chk1 and Chk2 kinases in checkpoint control and cancer[END_REF] and to provide time for repair before the damage gets passed on to daughter cells or to allow for the reconstitution of the dNTP pools. These regulatory pathways govern the order and timing of cell cycle transitions to ensure completion of one cellular event prior to commencement of another. Before mitosis, cells have to pass G1-S, intra-S, and G2-M cell cycle checkpoints, which are controlled by their key regulators, ATR and ATM protein kinases, through activation of their downstream effector kinases Chk1 and Chk2, respectively[START_REF] Bartek | Chk1 and Chk2 kinases in checkpoint control and cancer[END_REF][START_REF] Abraham | Cell cycle checkpoint signaling through the ATM and ATR kinases[END_REF]. Activated Chk1 and Chk2 phosphorylate the Cdc25A phosphatase at Ser75 and Ser177, respectively, and target it for proteasomal degradation. Cdc25A is an oncogene and required for cell cycle transit.Treatment with ABNM-13 activated both Chk1 and Chk2, the latter being phosphorylated within as little as 30 minutes.Both Cdc25B and Cdc25C induce mitosis by activating Cdk1/cyclin B[START_REF] Donzelli | Regulating mammalian checkpoints through Cdc25 inactivation[END_REF], and Cdc25B has been implicated as the initial phosphatase to activate Cdk1/cyclin B[START_REF] Nishijima | A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea[END_REF]. Activated Cdk1/cyclin B then phosphorylates and activates Cdc25C, which in turn keeps Cdk1/cyclin B active, creating a positive feedback loop that drives the cell through mitosis[START_REF] Hoffmann | Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis[END_REF]. Cdc25B protein levels were upregulated by ABNM-13, leading to dephosphorylation and activation of Cdk1. Cdc25C levels remained unchanged. In contrast, Ara-C induced Cdk1 protein expression, and co-treatment with Ara-C and ABNM-13 resulted in both an increase of Cdk1 levels and subsequent increase of its activity. Undue overexpression of Cdc25B i.e. when Cdc25A is unavailable, and A c c e p t e d M a n u s c r i p t 22 consequent dephosphorylation of Cdk1/cyclin B, as observed in this study, was

A c c e p t e d M a n u s c r i p t 23 antitumor

 23 drug such as Ara-C. Due to these promising results, ABNM-13 may support conventional chemotherapy of human malignancies and therefore deserves further preclinical and in vivo testing. A c c e p t e d M a n u s c r i p t 24

A c c e p t e d M a n u s c r i p t

Figure legends Figure 1 .

 legends1 Figure legends

A c c e p t e d M a n u s c r i p tFigure 3a .

 3a Figure 3a. Inhibition of incorporation of 14 C-cytidine into DNA of HL-60 cells

Figure 3b .

 3b Figure 3b. Concentration of dNTP pools in HL-60 cells upon treatment with ABNM-13. HL-60 cells (0.4x10 6 cells per ml) were incubated with 10, 20, and 40 µM ABNM-13 for 24 hours. Afterwards, 5x10 7 cells were separated for the extraction of

Figure 3c :

 3c Figure 3c: Inhibition of incorporation of 14 C-cytidine into DNA of HL-60 cells

Figure 3d :Figure 4d :

 3d4d Figure 3d: Expression levels of RR subunits R1, R2 and p53R2 in HL-60 cells

Figure 4e :

 4e Figure 4e: Expression levels of Cdc25B, Cdc25C, p(Tyr 15)Cdk1, and Cdk1 after

Figure 5c :

 5c Figure 5c: Expression levels of cleaved caspase-3 and γH2AX after incubation

Figure 5d :

 5d Figure 5d: Examples of the cellular morphology. After incubation with increasing

Figure 6 :

 6 Figure 6: Proposed mechanism of action of ABNM-13 and Ara-C

Figure 1 :

 1 Figure 1: Structure and biological activity of ABNM 1-13 in HL-60 cells

Figure A c c e p t e d M a n u s c r i p t

  Figure

Figure 2a :

 2a Figure 2a: Growth inhibition of HL-60 cells after incubation with ABNM-13

Figure

  Figure

Figure 2b :

 2b Figure 2b: Growth inhibition of HL-60 cells after incubation with HU

Figure

  Figure

Figure 2c :

 2c Figure 2c: Inhibition of colony formation of AsPC-1 cells after incubation with

Figure

  Figure

Figure 3a :

 3a Figure 3a: Inhibition of 14 C-incorporation into DNA of HL-60 cells after

Figure 3b :ABNM

 3b Figure 3b: Concentration of dNTP pools in HL-60 cells after treatment with

Figure

  Figure

Figure 3c :

 3c Figure 3c: Inhibition of 14 C-incorporation into DNA of HL-60 cells after

Figure

  Figure 4a: HL-60 cell cycle distribution after incubation with ABNM-13

Figure A c c e p t e d M a n u s c r i p t

  Figure

Figure

  Figure 4b: HL-60 cell cycle distribution after incubation with Ara-C

Figure A c c e p t e d M a n u s c r i p t

  Figure

Figure 5a : 13 for

 5a13 Figure 5a: Induction of apoptosis in HL-60 cells after treatment with ABNM-13

Figure 5b :

 5b Figure 5b: Induction of apoptosis in HL-60 cells after treatment with 15 µM

Figure

  Figure

  2 Nunc tissue culture flasks and exposed to increasing concentrations of ABNM-13 for 24 and 48 hours.

	Hoechst 33258 (HO, Sigma, St. Louis, MO, USA) and propidium iodide (PI, Sigma,

St. Louis, MO, USA) were added directly to the cells to final concentrations of 5 µg/ml and 2 µg/ml, respectively, followed by 60 minutes of incubation at 37°C. Cells were examined on a Nikon Eclipse TE-300 Inverted Epi-Fluorescence Microscope (Nikon, Tokyo, Japan) equipped with a Nikon DS-5M-L1 Digital Sight Camera System including appropriate filters for Hoechst 33258 and PI. This method allows distinguishing between early apoptosis, late apoptosis, and necrosis and is therefore superior to TUNEL assay which fails to discriminate among apoptosis and necrosis

[START_REF] Grasl-Kraupp | In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note[END_REF][START_REF] Rosenberger | The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells[END_REF]

and does not provide any morphological information. In addition, the HO/PI staining is more sensitive than a customary FACS based Annexin V binding assay

[START_REF] Rosenberger | The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells[END_REF][START_REF] Grusch | Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox[END_REF][START_REF] Fritzer-Szekeres | Trimidox, an inhibitor of ribonucleotide reductase, induces apoptosis and activates caspases in HL-60 promyelocytic leukemia cells[END_REF]

. The Hoechst dye stains the nuclei of all cells and thus allows monitoring cellular changes associated with apoptosis, such as chromatin condensation and nuclear fragmentation. In contrast, PI is excluded from viable and early apoptotic cells; consequently, PI uptake indicates loss of membrane integrity being characteristic of late apoptotic and necrotic cells. In combination with fluorescence microscopy to evaluate the morphologies of nuclei, the selective uptake of the two dyes enables studying the induction of apoptosis in intact cultures and to distinguish

Table 1 : Synergistic combination effects of ABNM-13 and Ara-C in HL-60 cells employing a sequential growth inhibition assay

 1

	Compound	Concentration	Cell number ± SD	Predicted value*	Combination
		(µM/nM)	(% of control)		Index**
	ABNM-13 (A)	2.5	88.7 ± 0.78
	(µM)	5.0	61.8 ± 0.16
		7.5	50.9 ± 0.78
		10.0	32.6 ± 0.31
	Ara-C (B)	10	72.4 ± 0.94
	(nM)	15	71.8 ± 3.61
		20	59.3 ± 3.45
	ABNM-13	2.5
	+ Ara-C	10	49.2 ± 4.55	64.2	0.607***
	ABNM-13	2.5
	+ Ara-C	15	25.2 ± 4.87	63.7	0.305***
	ABNM-13	2.5
	+ Ara-C	20	39.8 ± 2.35	52.6	0.607***
	ABNM-13	5
	+ Ara-C	10	15.5 ± 7.85	44.7	0.329***
	ABNM-13	5
	+ Ara-C	15	20.0 ± 5.65	44.4	0.418***
	ABNM-13	5
	+ Ara-C	20	32.1 ± 1.10	36.6	0.692***
	ABNM-13	7.5
	+ Ara-C	10	17.3 ± 5.02	36.9	0.514***
	ABNM-13	7.5
	+ Ara-C	15	26.4 ± 0.00	36.6	0.740***
	ABNM-13	7.5
	+ Ara-C	20	23.2 ± 1.41	30.2	0.695***
	ABNM-13	10
	+ Ara-C	10	17.2 ± 2.04	23.6	0.670***
	ABNM-13	10
	+ Ara-C	15	16.8 ± 1.73	23.4	0.676***
	ABNM-13	10
	+ Ara-C	20	17.9 ± 2.67	19.3	0.727***
	Cells were sequentially incubated with (1) ABNM-13 for 24h and (2) Ara-C for 48h, and then the cell
	number was determined. Data are means of two determinations ± standard deviations (SD).
	* Predicted Value: (%A x %B) / 100
	** Combination indices according to the equation of Chou and Talalay [36]
	*** Synergistic combination effect

Acknowledgements

This investigation was supported by the "Fonds zur Foerderung der Wissenschaftlichen Forschung des Buergermeisters der Bundeshauptstadt Wien", grant #09059 to M.F.-S., and the "Hochschuljubilaeumsstiftung der Stadt Wien", grant #H-756/2005 to T.S. The authors wish to thank Toni Jaeger for preparing the western blotting figures.