Frank Westermann
email: f.westermann@dkfz-heidelberg.de

Elena A Afanasyeva

Pieter Mestdagh

Candy Kumps

Jo Vandesompele

Volker Ehemann

Jessika Theissen

Matthias Fischer

Marc Zapatka

Benedikt Brors

Jessica Theissen

Larissa Savelyeva

Vitaliya Sagulenko

Frank Speleman

Manfred Schwab

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

Keywords: 3p25.3, miRNA, p53 stabilization, senescence

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

MicroRNAs (miRNAs) are non-coding short (18-24nt) RNAs, which control gene expression at a posttrancriptional level via inhibiting translation or triggering degradation of multiple target mRNAs.

They are considered as vitally important regulators of proliferation, differentiation and apoptosis.

Upregulated and downregulated miRNAs have been found in different malignancies, thus implicating that miRNAs act as oncogenes or tumor suppressors [START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF] . Moreover, an increasing body of evidence for miRNA involvement in tumorigenesis has prompted researchers to revisit cancer-associated genomic regions to search for miRNA loci [START_REF] Calin | Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[END_REF] . For example, miR-15 and miR-16, mapping to a deletion region at 13q14, are downregulated in B-cell chronic lymphocytic leukaemia. Both miR-15 and miR-16 negatively regulate BCL2 and act as positive regulators of apoptosis, which could explain their frequent inactivation in leukaemia [START_REF] Calin | Frequent deletions and down-regulation of microRNA genes mir-15 and mir-16 at 13q14 in chronic lymphocytic leukemia[END_REF] . Later, miR-34a at 1p36 emerged as a tumor suppressor gene (TSG). Several independent studies demonstrated the anti-proliferative and proapoptotic potential of miR-34a [START_REF] Chang | Transactivation of mir-34a by p53 Broadly Influences Gene Expression and Promotes Apoptosis[END_REF][START_REF] Welch | MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells[END_REF] . The potential is great for the involvement of many other, as yet unreported, miRNAs in cancer pathogenesis.

Neural crest-derived neuroblastoma, one of the most common solid tumors in children, is frequently characterized as an "enigmatic neoplasm". Indeed, neuroblastoma has the highest rate of spontaneous regression of all human malignancies. On the other hand, approximately 40% of neuroblastomas rapidly progress despite multimodal treatment regimes [START_REF] Westermann | Genetic parameters of neuroblastomas[END_REF][START_REF] Schwab | Neuroblastoma: biology and molecular and chromosomal pathology[END_REF] . Another interesting characteristic of neuroblastoma is the rarity of TP53 mutations; neuroblastoma retain very often functional p53. These facts suggest that the inhibitors of p53 pathway or a loss of p53 pathway positive regulators may be involved in neutralizing of p53 activity [START_REF] Mckenzie | Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated[END_REF][START_REF] Van Maerken | Escape from p53-mediated tumor surveillance in neuroblastoma: switching off the p14 ARF -MDM2-p53 axis[END_REF] . Neuroblastomas with poor outcome are subdivided into at least two biologically distinct groups. One group includes neuroblastomas harboring amplification of the MYCN oncogene, and a second group lacks amplified MYCN. It is known that this second group also frequently harbors segmental 3p deletions [START_REF] Plantaz | Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification[END_REF] , which implies that neuroblastoma suppressive transcripts are encoded from this region. Several efforts have been made to map and refine the critical region for 3p loss in neuroblastoma. Currently, a consensus region of loss is assigned to 3p25-26.2, which includes the VHL gene. Low VHL mRNA expression does correlate with poor prognosis, however, functional analyses could not reveal differences in pVHL protein expression nor pVHL pathway impairment [START_REF] Hoebeeck | The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma[END_REF] . To date, the identity of neuroblastoma-relevant tumor suppressor genes (TSGs) at 3p25-26.2 is unknown. In a previous study we detected several known and novel miRNA loci located within regions of neuroblastoma-relevant chromosomal aberrations [START_REF] Afanasyeva | New miRNAs cloned from neuroblastoma[END_REF] . We focus here on miR-885-5p, the only miRNA gene mapping to 3p25. 3. We show that miR-885-5p is able to interfere with cell proliferation and survival, promoting cellular senescence and apoptosis. Furthermore, we demonstrate that miR-885-5p downregulates CDK2 and MCM5, activates p53 and provide evidence that CDK2 and MCM5 are direct miR-885-5p targets. Our results suggest a tumor suppressive role for miR-885-5p in neuroblastoma.

Results

Low miR-885-5p expression in primary neuroblastomas with segmental 3p loss

In our previous work characterizing the neuroblastoma miRNA transcriptome we cloned miR-885-5p (MYCNSC_5_281) from a favorable tumor. miR-885-5p is a nonconserved miRNA that has no homologues in mouse and rat genomes (Supplementary Figure S1a). To evaluate rearrangements of the region encompassing miR-885-5p in primary neuroblastomas, we used array-based comparative genomic hybridization (aCGH) to analyze 193 neuroblastomas with different clinical and biological characteristics. The 3p25.3 region encompassing miR-885-5p was heterozygously deleted in the context of a segmental chromosomal 3p loss in 14% (n=27) of tumours (Figure 1a). Among the 3p deleted tumors, most were stage 4 (n=17). These data are consistent with previous reports on 3p25.3 status in neuroblastoma [START_REF] Plantaz | Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification[END_REF] . We profiled the expression of miR-885-5p by reverse transcription quantitative PCR (RT-qPCR) in 60 primary neuroblastomas. As a comparison, we profiled miR-331-3p, a miRNA that might be repressed by MYCN according to our previous data [START_REF] Afanasyeva | New miRNAs cloned from neuroblastoma[END_REF] and that might also have tumor suppressive functions. Assuming equal Cq-values for similar expression levels, expression of miR-885-5p appears to be lower than of miR-331-3p in neuroblastoma tumours (Figure 1b). No significant difference in miR-885-5p expression was detected between neuroblastomas with and without MYCN amplification, whereas miR-331-3p expression was lower in MYCN-amplified neuroblastomas (P<0.001, Figure 1b). Intriguingly, miR-885-5p expression was lower in aggressive tumors with segmental 3p deletions, as compared to those with intact 3p and favourable prognosis (P=0.02, Figure 1b). Expression of miR-885-5p and miR-331-3p was measured in nine neuroblastoma cell lines using RT-qPCR and northern blotting. Both MYCN-nonamplified cell lines as well as MYCN-amplified cell lines were assessed (Figure 1c). Expression of the precursor and mature miR-885-5p was low in all cell lines analyzed and lacked correlation with 3p25 status, while expression of precursor and mature form of miR-331-3p was higher and did not vary significantly (Figure 1d; Supplementary Figure S1b). In neuroblastoma cell lines, expression of miR-885-5p lacked correlation with 3p25 status, suggesting that also other miR-885-5p inactivating lesions in neuroblastoma cells exist.

Our data support a tumor suppressive role of miR-885-5p at least in a group of neuroblastomas with intact 3p25.3. miR-331-3p data are less supportive, however, we included miR-331-3p further in our study as a positive control to monitor the specificity of miR-885-5p-mediated effects.

miR-885-5p inhibits neuroblastoma proliferation and survival

For functional analysis of miR-885-5p in neuroblastoma cells, we introduced miR-885-5p mimics into KELLY, IMR32, SK-N-BE(2)c, SH-EP and HDN33 cell lines. SH-EP, KELLY and IMR32 cell lines have wild-type TP53, while p53 in SK-N-BE(2)c lacks transcription factor activity due to mutation in the DNA-binding domain [START_REF] Tweddle | Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line[END_REF] . The p53 pathway may not be intact in HDN33 based on the presence of centrosome amplification, multinuclear cells [START_REF] Fukasawa | Abnormal centrosome amplification in the absence of p53[END_REF][START_REF] Carroll | Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression[END_REF] and heterozygous mutation leading to an amino acid change at codon 15 (Ser to Cys) (Supplementary Figure S2). Cell proliferation was assessed in cells transiently transfected with either the miR-885-5p mimic, the miR-331-3p mimic or a nontargeting control miRNA using Alamar Blue assay. Enforced miR-885-5p expression reduced proliferation capacity in all cell lines analyzed, whereas transfection of miR-331-3p only inhibited proliferation in the HDN33 and SK-N-BE(2)c cell lines (Figure 2a). SH-EP, KELLY, IMR32 and SK-N-BE(2)c cells were also less capable of anchorage-independent growth in soft agar after enforced miR-885-5p expression (Figure 2b). Importantly, suppression of anchorage-independent growth after miR-885-5p introduction was more pronounced in cell lines with wild-type TP53 (KELLY, IMR32 and SH-EP) than in SK-N-BE(2)c cells with mutant TP53. Enforced miR-331-3p expression only reduced colony growth of SK-N-BE(2)c cells in soft agar. We also tested whether proliferation inhibition by miR-885-5p was associated with senescence. Senescence-associated acidic β-galactosidase (SA-β-Gal) activity was detected in miR-885-5p-transfected MYCN nonamplified HDN33 and SH-EP cells (Figure 2a, insert), but not in MYCN amplified KELLY, IMR32, and SK-N-BE(2)c cells (data not shown).

Cell cycle profiles were also analyzed after enforced expression of either miR-885-5p or miR-331-3p using flow cytometry (FACS). Accumulation in G1/G0 after miR-885-5p transfection was observed in cell lines with wild-type TP53 (KELLY, IMR32 and SH-EP) (Figure 2c), whereas enforced miR-885-5p expression in SK-N-BE(2)c and HDN33 resulted in accumulation of cells in S and G2/M phase, respectively. In contrast, enforced miR-331-3p expression did not lead to pronounced cell cycle changes (>±3%) in IMR32, KELLY and SH-EP cells. Enforced miR-331-3p expression in HDN33 resulted in G1/G0 and in SK-N-BE(2)c in G2/M accumulation. To ascertain whether the observed cell cycle changes were accompanied by cell death, we analyzed the sub-G1 fraction. Enforced miR-885-5p expression slightly increased the sub-G1 fraction of SH-EP and IMR32 and moderately in SK-N-BE(2)c and HDN33 cells, whereas the sub-G1 fraction after miR-331-3p transfection was only increased in SK-N-BE(2)c cells (Figure 2d). The amount of Annexin V-positive cells was elevated after miR-885-5p treatment in HDN33 and SK-N-BE(2)c but not in IMR32, KELLY and SH-EP (Figure 2d, insert; data not shown).

Taken together, these data suggest that miR-885-5p mediated inhibition of neuroblastoma proliferation is preferentially via G0/G1 arrest in TP53 wild-type cells (IMR32, KELLY and SH-EP) and by apoptotic cell death in neuroblastoma cells with disabled p53 pathway (SK-N-BE(2)c and HDN33).

miR-885-5p induces p53 and downregulates CDK2 and MCM5

To further analyze the role of miR-885-5p on p53 regulation and function, we measured protein levels of p53 and its downstream targets after enforced miR-885-5p expression. We observed a marked p53 induction in KELLY and IMR32 cells, and a moderate p53 induction in SH-EP cells after miR-885-5p transfection (Figure 3a). Transfection of miR-885-5p also induced p53 in TP53 mutant SK-N-BE(2)c cells. Enforced miR-885-5p expression resulted in elevated p21 waf1 levels in KELLY, IMR32 and SH-EP cells; PUMA was elevated only in IMR32 cells. Enforced miR-885-5p expression did not alter p53 and p21 waf1 protein expression in HDN33 cells or p21 waf1 and PUMA protein expression in TP53 mutant SK-N-BE(2)c cells, and miR-331-3p expression had no effect on p53, p21 waf1 and PUMA protein levels in any of the cell lines analyzed. Expression of miR-885-5p downregulated E2F1 in SH-EP, IMR32, KELLY and HDN33 cells, while it had no effect on MYCN and p27 protein levels (Figure 3a, and data not shown). Caspase-3 activity was elevated in SK-N-BE(2)c and IMR32 after miR-885-5p introduction although the increases were less than two fold (Supplementary Figure S3). Western blot analysis revealed reduction of procaspase-3 levels in all cell lines (Figure 3a) with no significant appearance of mature caspase-3 (data not shown). Taken together, the western blot data reveal that enforced miR-885-5p expression causes p53 and p21 waf1 induction in TP53 wild-type neuroblastoma, which is in line with the observed G0/G1 arrest after enforced expression of this miRNA.

Querying the miRNA databases at www.microrna.org [START_REF] Betel | The microRNA.org resource: targets and expression[END_REF] , TargetScan 17 (www.targetscan.org) and searching for candidate 3'UTRs using the miRanda algorithm [START_REF] John | Human microRNA targets[END_REF] in the UTResource 3′UTR database (http://www.ba.itb.cnr.it/UTR/) retrieved STAT3, CUL3, CDK2, MCM3, MCM5 and CDK6 as candidate miR-885-5p targets, and p21 waf1 and E2F1 as candidate miR-331-3p targets. Cleavage of STAT3 and MCM3 by apoptosis-executing proteases has been reported, which could have interfered with our expression analysis of these proteins [START_REF] Darnowski | STAT3 cleavage by caspases: impact on full-length STAT3 expression, fragment formation and transcriptional activity[END_REF][START_REF] Schwab | Selective proteolysis of the nuclear replication factor MCM3 in apoptosis[END_REF] . We examined the protein expression of the candidate targets after miR-885-5p and miR-331-3p transfection. CDK2 and MCM5 protein levels decreased after miR-885-5p transfection in all tested cell lines, supporting both as direct miR-885-5p targets (Figure 3b). Enforced miR-331-3p expression reduced E2F1 levels in KELLY, IMR32 and SK-N-BE(2)c and reduced CDK2 expression in IMR32, although to a lesser extent than miR-885-5p. MCM3

cleavage was observed after miR-885-5p transfection of IMR32 and SK-N-BE(2)c cells, which prohibited estimation of direct targeting of MCM3 by miR-885-5p.

To test whether the observed effects of miR-885-5p depend on the manner of overexpressing miRNAs SH-EP and KELLY cell lines stably expressing either miR-885-5p or miR-331-3p were generated (Supplementary Figure S1c). The phenotype of miR-885-5p-SH-EP and miR-885-5p-KELLY cells was highly similar to that of SH-EP and KELLY cells transiently transfected with the miR-885-5p mimic. Most importantly, CDK2 and MCM5 were downregulated and p53 and p21 waf1 were upregulated in only the miR-885-5p-SH-EP cells and miR-885-5p-KELLY cells (Supplementary Figure S4).

Accordingly, our data from in silico prediction and western blotting support the notion that CDK2 and MCM5 are targeted by miR-885-5p.

miR-885-5p directly targets the predicted binding sites in CDK2 and MCM5 3'-UTRs.

To test whether miR-885-5p binding sites in the CDK2 and MCM5 3'UTRs are directly regulated by miR-885-5p we performed luciferase reporter assays. Relative firefly luciferase activities of reporter plasmids containing MCM5 or CDK2 3'-UTR binding sites were inhibited by 20-40% in miR-885-5p-SH-EP cells and premiR-885-5p-transfected SH-EP cells but not parental, vector-or miR-331-3p-SH-EP cells and control miRNA or miR-331-3p-transfected cells. Importantly, mutation of the CDK2 and MCM5 binding sites in the reporter constructs restored luciferase activity in miR-885-5p-SH-EP and premiR-885-5p-transfected cells (Figure 4).

These data show that miR-885-5p directly targets the predicted binding sites in the 3'UTRs of CDK2 and MCM5.

CDK2 and MCM5 knockdown mimic miR-885-5p-induced phenotype

Previous studies showed that CDK2 and MCM5 are connected to the p53 pathway. MCM5 was identified as a negative regulator of p53-induced growth arrest [START_REF] Agarwal | DNA replication licensing factor Minichromosome Maintenance Deficient 5 rescues p53-mediated growth arrest[END_REF] . Moreover, CDK2 inhibition led to p53 accumulation in MYCN-amplified neuroblastoma cells [START_REF] Molenaar | Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells[END_REF] . This suggests a link between phenotypic changes induced by miR-885-5p in neuroblastoma cells and suppression of CDK2 and MCM5. To test this possibility we introduced siRNAs targeting CDK2 and/or MCM5 into the given set of neuroblastoma cells lines and analyzed the phenotype with the same panel of assays used for the characterization of the miR-885-5p-induced phenotype. CDK2 or MCM5 knockdown alone as well as the combined knockdown inhibited cell proliferation and anchorage-independent growth in all tested cell lines (Figure 5ab). Cell cycle analysis at day 3 after transfection of CDK2 or CDK2/MCM5 siRNAs revealed accumulation of cells in G1/G0 phase in most cell lines (SH-EP, KELLY, IMR32 and HDN33, Figure 5a). MCM5 knockdown alone showed an inconsistent cell cycle phenotype with G1/G0 arrest in SH-EP and KELLY and G2/M arrest in the other cell lines. SK-N-BE(2)c cells failed to respond with a G1/G0 arrest after CDK2 and/or MCM5 knockdown, but consistently showed accumulation of cells in G2/M phase. Most SA-ß-Gal positive cells were found after the combined CDK2/MCM5 knockdown in SH-EP and HDN33 cells (Figure 5c), both of which also showed SA-ß-Gal positive cells after miR-885-5p overexpression. In addition, an increase of sub-G1 fraction and Annexin V positive cells were observed in HDN33 after CDK2 and/or MCM5 knockdown (Figure 5c), which were also observed after miR-885-5p overexpression in these cells. Neither CDK2 nor MCM5 inhibition led to significant increases in apoptotic cell death in SH-EP, KELLY, IMR32 and SK-N-BE(2)c.

We found p53 and p21 waf1 upregulation after CDK2 knockdown in SH-EP, KELLY and IMR32 cells and PUMA upregulation after CDK2 knockdown in IMR32 cells (Figure 5d). CDK2 and/or MCM5 knockdown did not affect p53, p21 waf1 and PUMA levels in HDN33 cells. Together, these data suggest that CDK2 and MCM5 inhibition is involved in miR-885-5p-mediated growth arrest and senescence in

miR-885-5p-induced growth arrest is p53 dependent in TP53 wild-type neuroblastoma

To determine whether miR-885-5p-induced growth arrest is indeed p53-dependent in TP53 wild-type neuroblastoma we sequentially transfected TP53 wild-type SH-EP, KELLY and IMR32 cells with siRNA targeting p53 and miRNA mimics and assessed cell proliferation with Alamar Blue assay.

While the treatment of control siRNA transfected cells with premiRs induced the same phenotype as premiR treatment alone, miR-885-5p failed to inhibit proliferation in p53 knockdown SH-EP, KELLY and IMR32 cells. In addition, p53-knockdown abolished miR-885-5p-induced G1/G0 arrest and inhibition of anchorage independent growth by miR-885-5p in these cells (Figure 6ab). Consistently, the levels of p21 waf1 and PUMA remained unaffected in response to miR-885-5p and after p53 knockdown in these cells (Figure 6c).

To further validate that miR-885-p regulates p53, CDK2 and MCM5 we thought to suppress endogenous miR-885-5p in neuroblastoma cells. According to our RT-qPCR and northern blot data (Figure 1d, Supplementary Figure S1) only IMR32 and SK-N-BE(2)c cells express significant levels of miR-885-5p. We did not observe growth upregulation upon antimiR-885-5p as determined by Alamar Blue assay (data not shown). However, a trend towards increased anchorage-independent growth was found in antimiR-885-5p transfected IMR32 and SK-N-BE(2)c cells. Importantly, p53, p21 waf1 and PUMA levels were reduced in antimir-885-5p-transfected SK-N-BE(2)c and IMR32 (Figure 6d).

Moreover, MCM5 was moderately induced in antimiR-885-5p-transfected SK-N-BE(2)c and CDK2

was induced in antimiR-885-5p-transfected SK-N-BE(2)c and IMR32 (Figure 6d). According to these data, antimiR-885-5p affects p53, p21 waf1 , CDK2 and MCM5 protein levels altogether, which is consistent with the results from miRNA mimic transfections and strengthens the link between miR-885-5p and p53/CDK2/MCM5.

miR-885-5p activates the p53 transcriptional program

In previous sections we provide evidence that miR-885-5p-mediated growth inhibition requires functional p53 in TP53 wild-type neuroblastoma cells. To further characterize downstream effects of miR-885-5p expression in neuroblastoma cells with wild-type TP53, gene expression profiles 3 days after miR-885-5p mimic transfection in SH-EP and KELLY cells were generated using oligo microarrays. Profiles were compared to SH-EP and KELLY cells transfected with either control miRNA or miR-331-3p mimic, and genes with expression changes of at least 2-fold in both cell lines were identified. There was little overlap between genes regulated in cells expressing either miR-885-5p

or miR-331-3p (8 upregulated and 28 downregulated genes, Supplementary Tables S1 andS2). Several p53 target genes, IGFBP3, ZMAT3, TNFRSF10B, BTG3, PPAP2B, PPM1D, RRM2B, PTPRE and DLC1, were upregulated in miR-885-5p transfectants [START_REF] Wei | A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome[END_REF] (Table 1). Only one p53 target, GDF15, was upregulated in miR-331-3p transfectants. IGFBP3 and ZMAT3 were among the top-5 genes upregulated in KELLY cells after miR-885-5p expression. These data implicate p53-directed transcriptional changes in the downstream effects of miR-885-5p in neuroblastoma cells. We used the expression profiles of miR-885-5p transfectants to assess expression of genes controlling p53 transcription either positively or negatively. Several genes known to positively regulate p53, namely DYRK2, HIPK2, and GAS1, were upregulated after miR-885-5p expression. Enforced miR-331-3p expression altered expression of none of these genes, but did suppress the positive p53 regulator, PML (Supplementary Table S2).

To validate that miR-885-5p upregulated p53 target genes are dependent on functional p53 we additionally profiled Kelly cells 3 days after miR-885-5p or miR-331 transfections in the presence of p53 targeting siRNA or control siRNA. We observed that from nine p53 target genes that emerged from the first profiling series (Table 1), seven validated miR-885-5p-induced genes (IGFBP3, BTG3, ZMAT3, PPAP2B, PPM1D, RRMB2, PTPRE) showed a lack or reduced induction in the presence of p53 targeting siRNA in Kelly cells (Supplementary Table S3), indicating that miR-885-5p induces these genes via increasing p53 transcriptional activity in these cells.

We further tested whether genes downregulated after enforced miR-885-5p expression were enriched for potential miR-885-5p targets compared with upregulated genes, based on the presence of at least one miR-885-5p potential binding site in the 3'UTR detected by in silico prediction. We found 74 downregulated genes (10.7%) as compared to only 3 upregulated genes (0.9%, Supplementary Table S1). CDK2 was among the downregulated genes, identified as a miR-885-5p target here (Figure 3b; Supplementary Figure S4), as was caspase-3 (CASP3), ß-catenin (CTNNB1), citron rho-interacting kinase (CIT), farnesyl transferase (FNTA) and microphthalmia-associated transcription factor (MITF).

Genes downregulated after miR-331-3p expression similarly included putative miR-331-3p targets harboring miR-331-3p binding sites (90 genes, 15.6%, Supplementary Table S3). These included PML, the serine/threonine-protein kinase, PIM1, neurobeachin (NBEA), the nerve growth factor receptor, NGFR and ERBB2, which has also recently been reported to be a miR-331-3p target [START_REF] Epis | mir-331-3p regulates ERBB-2 expression and androgen receptor signaling in prostate cancer[END_REF] . Nineteen (8.2%) of the genes upregulated after miR-331-3p expression also contain miR-331-3p target sites (Supplementary Table S2). This distribution of prospective target genes within the genes regulated after enforced miR-331-3p expression makes less sense that those among the genes regulated after enforced miR-885-5p expression. Together with the lack of apparent phenotype in miR-331-3p-transfected neuroblastoma cells, these results support a less specific role for miR-331-3p in directing changes in the neuroblastoma cell phenotype.

Taken together, these data indicate that miR-885-5p expression activates p53 pathway in neuroblastoma cells, including the induction of p53 and p53 downstream pathway activity, but multiple pathways can be employed by miR-885-5p during regulation of neuroblastoma cell phenotype especially in TP53 mutant background.

Discussion

In this report, we provide evidence that miR-885-5p is a candidate neuroblastoma tumor suppressor and involved in directing the neuroblastoma cell phenotype. Several genomic regions have been characterized that are commonly lost in neuroblastoma, although, the genes responsible for tumor suppression still remain unidentified. Region 3p25.3 is deleted in approximately 14% of neuroblastomas, and is the location of miR-885-5p. We show that miR-885-5p functions as an inhibitor of proliferation and survival, and positively regulates the p53 pathway. We present data for CDK2 and MCM5 being direct targets of miR-885-5p.

Initially, two miRNAs, miR-885-5p and miR-331-3p, were chosen as candidate tumor suppressive miRNAs in neuroblastoma. Our findings show that miR-885-5p is downregulated in neuroblastoma tumors with heterozygous deletion of 3p25.3, and that expression in neuroblastoma cell lines is low or absent also independent of 3p loss. Expression of miR-331-3p correlates inversely with amplified MYCN in neuroblastoma tumors, but no such trend was detected in neuroblastoma cell lines. Only experimentally enforced expression of miR-885-5p, but not miR-331-3p, in neuroblastoma cell lines resulted in growth inhibition, senescence and apoptosis. Clearly, miR-885-5p fulfills the criteria of a tumor suppressor gene in the context of neuroblastoma.

A master gene orchestrating cell cycle arrest, programmed cell death and senescence is TP53. We provide evidence for p53 involvement in miR-885-5p-driven cell cycle arrest, senescence and apoptosis. Neuroblastoma cells having wild-type TP53 displayed G1-arrest coupled with p53 and p21 waf1 induction upon miR-885-5p introduction. What genes are targeted by miR-885-5p to create this phenotype? Among six candidates, CDK2 and MCM5 met the miR-885-5p target criteria: (1) they were downregulated upon miR-885-5p introduction in all cell lines examined (2) predicted miR-885-5p binding sites in their 3'UTRs reduced expression of a reporter construct in the presence of miR-885-5p.

It has been described that CDK2 and MCM5 are linked to negative regulation of p53 pathway activity [START_REF] Agarwal | DNA replication licensing factor Minichromosome Maintenance Deficient 5 rescues p53-mediated growth arrest[END_REF][START_REF] Molenaar | Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells[END_REF] . In our study we found prominent phenotypic similarity between enforced miR-885-5p expression and siRNA-mediated CDK2/MCM5 knockdown in TP53 wild-type neuroblastoma cells (SH-EP, IMR32 and KELLY). Consistently, antimiR targeting endogenous miR-885-5p elevated the levels of CDK2 and MCM5 and reduced p53 and its downstream targets. In addition, siRNA-mediated p53 knockdown reverted phenotypic as well as protein and transcriptome changes induced by miR-885-5p. Together, this indicates that CDK2 and MCM5 inhibition executes miR-885-5p-mediated growth inhibition in TP53 wild-type neuroblastoma cells at least in part via p53 induction. Importantly, miR-885-5p did not affect deregulated MYCN expression in MYCN amplified neuroblastoma cells.

Deregulated MYC proteins modulate p53 function by transcriptionally inhibiting p21 waf1 leading to an impaired G1/G0 arrest [START_REF] Seoane | Myc suppression of the p21 Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage[END_REF] . Our data now indicate that miR-885-5p restores p21 waf1 function leading to G1/ G0 arrest in TP53 wild-type neuroblastoma cells despite high MYCN levels. This miR-885-5pmediated effect is mainly achieved through targeting of CDK2, because primarily CDK2 and not MCM5 knockdown exerts G1/G0 arrest and p21 waf1 induction similar to those induced by miR-885-5p.

An interesting question is why miR-885-5p also triggers apoptosis and senescence in neuroblastoma cells with mutant TP53. Such cells cannot engage p53 in response to CDK2 inhibition, which is the cause of G0/G1-arrest failure in these cells. However, CDK2 may still act as prosurvival signal in a TP53 mutant background, as demonstrated by CDK2 knockdown in HDN33 cells. Alternatively, the other arm of miR-885-5p action, MCM5 inhibition, may prevail in TP53 mutant cells inducing a senescence-like process and G2/M phase arrest, as evidenced by MCM5 knockdown in HDN33 cells.

However, other miR-885-5p targets may influence growth and survival in p53-mutant neuroblastoma, A model of how miR-885-5p function in neuroblastoma cells in the context of targeting CDK2 and MCM5 is proposed in Figure 7.

In addition, our data provide indirect evidence that caspase-3 might be targeted by miR-885-5p. In the presence of elevated p53 protein levels, we found only moderate upregulation of caspase-3 activity, reduction of procaspase-3 protein with no significant presence of its mature form as well as downregulation of CASP3 upon miR-885-5p introduction in neuroblastoma cells. According to miRanda algorithm CASP3 3´UTR contains a miR-885-5p binding site. Intriguingly, caspase-3 can be involved in degradation of p21 waf1 26 . We propose that caspase-3 targeting may further determine the response of miR-885-5p-transfected cells, favoring p21 waf1 accumulation in neuroblastoma cells (Supplementary Figure S5).

Given that, one can speculate about miR-885-5p functions in normal cells; it may withdraw normal cells from cycling via p53 activation but maintain survival. Therefore it would be interesting to determine whether miR-885-5p plays a role in developmental processes, for example, neuronal maturation.

To sum up, our findings lead us to conclude that miR-885-5p engages p53-dependent and independent regulatory layers jointly to interfere with cell cycle progression and survival of neuroblastoma cells. It negatively regulates CDK2 and MCM5 and activates the p53 pathway in TP53 wild-type neuroblastoma and is able to trigger cell death and senescence in TP53-mutant neuroblastoma.

Materials and methods

Patients

Neuroblastoma tumor samples were collected prior to any cytoreductive treatment, snap-frozen and stored at -80°C until RNA or DNA isolation. Written informed consent was obtained from patients' parents for tissue sampling. Genomic MYCN status was assessed in the reference laboratories of the German Neuroblastoma trial in Cologne and Heidelberg.

aCGH of primary neuroblastomas and M-FISH

Patient DNA tumor samples (n=193) were analyzed using either 44k (n=143) or 105k (n=51) aCGH microarrays (Agilent Technologies). 2.5 μg each of genomic tumour and reference DNA were used for each hybridization. Labelling and hybridization was performed as previously described [START_REF] Spitz | Oligonucleotide array-based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome[END_REF] . Arrays were preprocessed using the R/Bioconductor statistical software package. Arrays were segmented using the daglad function after lowess normalization, implementing a modified version of the methodology described in Hupe et al. [START_REF] Hupé | Analysis of array CGH data: from signal ratio to gain and loss of DNA regions[END_REF] . Different array platforms were matched using the chromosomal positions of the probes, and missing values were imputed only if two neighbouring probes had the same segmented values. Multicolor FISH (M-FISH) analysis on neuroblastoma cell lines was performed using the 24

XCyte probe-kit (Meta-Systems, Altlussheim, Germany). Hybridization was performed according to the manufacturer's instructions. Slides were counterstained with 4,6-diamidino-2-phenylindol (DAPI) (Sigma). Images were captured with a Zeiss Axio Imager Z1 microscope and analysed with Isis/mFISH imaging software (MetaSystems).

Cell culture and transfection

SH-EP, KELLY, IMR32, SK-N-BE(2)c and HDN33 were grown in RPMI supplemented with 10% (v/ v) fetal calf serum, 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37°C and 5% CO2. miRNA mimics and antimiRs were purchased from Ambion. siRNAs to p53, CDK2 and MCM5 and control non-targeting siRNA were purchased from Qiagen, Invitrogen and Santa Cruz. Cells were plated 24 hours before transient transfection with 30 nM of miRNA mimic or antimiR or Cy3-labeled control miRNA or antimiR using Lipofectamine 2000. siRNA transfections were performed using Lipofectamine 2000 according to manufacturer's instructions. Unless otherwise stated, cells were assayed 4 days after transfection. For p53 knockdown experiments, cells were retransfected with miRNA mimics 24 hours after transfection with p53 siRNA or control siRNA.

siRNA transfections were performed using Lipofectamine 2000 according to manufacturer's instructions. To construct the miR-885-5p and miR-331-3p expressing vectors the 500 bp fragments surrounding corresponding miRNA genes were amplified and cloned into the Nhe1 and EcoR1 sites in pcDNA3.1 (Invitrogen). SH-EP and KELLY cells were plated in 6-well plates for stable transfection with 400 µg pcDNA3.1-miR-331-3p, pcDNA3.1-miR-885-5p or empty pcDNA3.1 vector using Effectene (Qiagen). Individual clones were selected that grow in the presence of G418. Stable transfectants were maintained in culture no longer than 4 weeks during experiments.

Clonogenic assay, cell cycle, senescence and apoptosis assays

For cell proliferation evaluation, Alamar Blue (ABD Serotec) was used according manufacturer's instructions. Fluorescence was detected using the FluorStar Optima microplate fluorescence reader (BMG Labtech). The cell cycle distribution was determined as previously described [START_REF] Deubzer | Histone deacetylase inhibitor Helminthosporium carbonum (HC)-toxin suppresses the malignant phenotype of neuroblastoma cells[END_REF] . To assay for senescence, cells were fixed and stained using the SA-ß-Gal kit (Cell Signaling Technology) according to the manufacturer's instructions. Three fields containing 100 cells were counted for each experiment and the results of three independent experiments are presented. Apoptosis was evaluated in FACS analysis by the extent of DNA fragmentation and by extent of phosphatidylserine externalization using FITC Annexin V Detection kit (BD Pharmingen) [START_REF] Nicoletti | A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[END_REF][START_REF] Koopman | Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis[END_REF] .

Soft agar assay

Cells were trypsinised 18 hours after miRNA mimic transfection, and 10 5 cells were resuspended in a top agar solution containing 0.35% noble agar, then poured onto a base layer containing 0.5% agar in 6-well plates. Each well was covered with 3 ml of RPMI full medium, and incubated under standard culture conditions for two weeks. Colonies were fixed and stained with 10% methanol, 10% acetic acid, and 0.5% crystal violet. The colony counting was performed using ImageJ software.

Luciferase reporter assay

miRNA-3'UTR binding sites were predicted by miRanda (www.microrna.org) and Targetscan (www.targetscan.org) algorithms. 3'UTR database was obtained from UTResource (http://www.ba.itb.cnr.it/UTR/) [START_REF] Mignone | UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs[END_REF] . pmiRGLO vector (Promega) was used for reporter assay according to manufacturer' instructions. Putative miR-885-5p target sites in the CDK2 and MCM5 3'UTRs were cloned into pmiRGLO vector downstream of the firefly luciferase gene in pmiRGLO vector to create reporter plasmids. The sequences containing miR-885-5p predicted binding sites were used to construct pmiRGLO-CDK2-site are listed in the Supplementary Table S4.

Cells were plated in 96-well plates, and transfected with 20 ng of either pmiRGLO empty, pmiRGLO-CDK2-site, pmiRGLO-CDK2-mut-site, pmiRGLO-MCM5-site or pmiRGLO-MCM5-mut-site using Effectene (Qiagen), or miRNA mimics. Cells were lysed in passive cell lysis buffer (Promega) 24 hours post-transfection, and lysates were processed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's directions. Luciferase activity was detected in a FluorStar Optima microplate fluorescence reader. Firefly luciferase activity was normalized to Renilla luciferase activity as an internal transfection control. Transfections were performed in six independent experiments, and assayed in duplicate.

RT-qPCR

TaqMan miRNA assays (Applied Biosystems) were used to quantify endogenous miR-885-5p and miR-331-3p. RNA was isolated from neuroblastoma cell lines using TRIzol, and 5 ng of total RNA was used to reverse transcribe cDNA for miRNA assessment. Data from cell lines were normalized to U44 RNA expression. miRNA expression in primary tumor samples was analysed as previously described [START_REF] Mestdagh | A novel and universal method for microRNA RT-qPCR data normalization[END_REF] , and normalization was performed by using a set of miRNAs stably expressed in neuroblastomas. Real-time RT-PCR was performed by using the Applied Biosystems 7000 Sequence Detection system (Applied Biosystems).

Northern Blotting

Northern Blotting was performed as previously described [START_REF] Afanasyeva | New miRNAs cloned from neuroblastoma[END_REF] . Total RNA was isolated with TRIZOL reagent (Invitrogen). 30 µg of total RNA was loaded per lane and separated on 15% denaturing polyacrylamide gels, transferred by electroblotting to GeneScreen+ membranes (Perkin Elmer).

Methylene blue staining was performed prior to blocking with ULTRAhyb-Oligo hybridization buffer (Ambion) to assess RNA loading. Blots were incubated with 10 ng/ml 3'-biotinylated DNA probes complementary to either miR-885-5p or miR-331-3p in ULTRAhyb-Oligo buffer then visualized using the North2South Chemiluminescent Hybridization and Detection Kit (Pierce).

Western Blotting

Whole cells were lysed in a buffer containing 7 M urea, 1% Triton X-100, 100 mM DTT, 20 mM Tris-HCl, pH 8.5. Protein concentrations were determined by Bradford assay (Bio-Rad), and 20 µg protein lysate were separated per lane on either 12% or 15% PAGE gels then transferred to nitrocellulose membranes (Protran). Antibody to MCM5 was purchased from Abcam. Antibody to PUMA was from ProSci Inc. Antibodies to p53, MCM3, STAT3, E2F1, MYCN, CUL3 and ß-actin were from Santa Cruz Biotechnologies. Antibodies to p27, CDK2, CDK6 were from Dianova. Antibodies to precursor and mature CASP3 were from BD Transduction Laboratories and Cell Signaling, respectively. HRPconjugated antibodies against mouse or rabbit were obtained from Jackson Immunoresearch. Sheep anti-goat HRP-conjugated antibody was from Santa Cruz Biotechnologies. Membranes were incubated with the appropriate antibodies, and bands were visualized using the ECL system (Pierce). Images were captured with a CCD camera (Vilber Lourmat) and densitometry analysis was performed using ImageJ software.

Gene expression analysis

In the first series of experiments, RNA was isolated from cells 3 days after transient transfection with miR-885-5p, miR-331-3p, or control miRNA. In a second series of experiments, RNA was isolated from cells pretreated with p53 siRNA or control siRNA for 1 day and subsequently transfected with miR-885-5p, miR-331-3p, or control miRNA for 3 days. Gene expression profiles were generated as two one-color replicates using the whole genome oligonucleotide microarray platform from Agilent (Agilent Technologies) as previously described [START_REF] Westermann | Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas[END_REF][START_REF] Oberthuer | Customized oligonucleotide microarray gene[END_REF]

TP53

  wild-type neuroblastoma cells. Also, CDK2 and MCM5 inhibition can play a role in miR-885-5p-triggered apoptosis and senescence in TP53 mutant HDN33 cells. However, although CDK2 and/or MCM5 knockdown in SK-N-BE(2)c shared p53 upregulation with miR-885-5p-transfected SK-N-BE(2)c, they did not undergo apoptosis, suggesting that targets other than CDK2 and MCM5 modify the SK-N-BE(2)c response to miR-885-5p.

Table 1 . miR-885-5p activates p53 transcriptional program HGNC symbol Chromosome, Band Description HGNC symbol Chromosome, Band Description P53 targets upregulated in miR-885-5p transfectants P53 targets upregulated in miR-331-3p transfectants

 1 . Raw microarray data were normalized using quantile normalization. Expression ratios are given as the mean of two replicates. All raw and //www.ebi.ac.uk/arrayexpress; Accession: E-TABM-38) or iCHIP (www.ichip.de/).Statistical significance was calculated using the Student t-test. Data are presented as mean±SD from at least three experiments.expression-based classification of neuroblastoma patients outperforms current clinical risk stratification. J Clin Oncol 2006; 24: 5070-5078.

	normalized	microarray	data	are	available	at	the	ArrayExpress	database
	(http:Statistical analysis

Acknowledgements

The authors thank Gabriele Becker and Yvonne Kahlert for excellent technical assistance, Filip Pattyn for critical remarks and Kathy Astrahantseff for manuscript proofreading. This work was supported with grants N2KR-S19T03 and 01GS0895 within NGFN-2 and NGFN Plus from the BMBF, and the EET-pipeline (#037260) from the EU.


Vilma Graupner

Eva Alexander

Tim Overkamp

Oliver Rothfuss

Vincenzo De Laurenzi

Bernhard F Gillissen

Peter T Daniel

Klaus Schulze-Osthoff

Frank Essmann
email: frank.essmann@ifib.uni-tuebingen.de

Transactivate Bak

Differential regulation of the pro-apoptotic multidomain protein Bak by p53 and p73 at the promoter level

Keywords: Apoptosis / Bak / Bax / transcription / p53 / p73 BS, binding site, CDK, cyclin-dependent kinase, EMSA, electrophoretic mobility shift assay, qRT-PCR, quantitative real-time PCR

During apoptosis, Bcl-2 proteins control permeabilization of the mitochondrial outer membrane leading to the release of cytochrome c. Essential gate keepers for the cytochrome c release are the pro-apoptotic multidomain proteins, Bax and/or Bak. The expression of bax is upregulated upon cellular stress by the tumor suppressor p53. Despite the high functional homology of Bax and Bak little is known about how the bak gene is regulated. To investigate its transcriptional regulation in further detail, we have analyzed a region spanning 8200 bp upstream of the bak start codon (within exon 2) for transcription factor binding sites and identified three p53 consensus sites (BS1-3). Reporter gene assays in combination with site-directed mutagenesis revealed that only one putative p53-binding site (BS3) is necessary and sufficient for induction of reporter gene expression by p53. Consistently, p53 induces expression of endogenous bak. At the mRNA level, induction of bak expression is weaker than induction of puma and p21. Interestingly, Bak expression can also be induced by p73 that binds however to each of the three p53 binding sites within the bak promoter region. Our data suggest that expression of bak can be induced by both, p53 and p73 utilizing different binding sites within the bak promoter.

Introduction

In multi-cellular organisms apoptosis is of utmost importance for organ development, tissue homeostasis and cancer prevention, because superfluous and malfunctional cells are eliminated by this genetically encoded suicide program. The two main apoptosis pathways are triggered either by ligation of death receptors or by the release of cytochrome c from the mitochondrial intermembrane space. The mitochondrial pathway can enhance the response to death receptor ligation or can be activated intrinsically e.g. by genotoxic stress such as γ-irradiation or cytostatic drugs. In the intrinsic pathway members of the Bcl-2 family of proteins constitute the central regulators. [START_REF] Bakhshi | Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18[END_REF][START_REF] Danial | Cell death: critical control points[END_REF][START_REF] Tsujimoto | The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining[END_REF] They share homology in at least one of the four identified Bcl-2 homology (BH) domains and have either anti-or pro-apoptotic capacity. Based on the number of their BHdomains the pro-apoptotic proteins are further subdivided into multi-domain proteins (i.e. Bax and Bak) that contain BH1-3 and so-called BH3-only proteins that only share homology in the BH3-domain with the remainder of the family. Both types of pro-apoptotic proteins have been shown to interact with anti-apoptotic family members such as Bcl-2, Bcl-w, Bcl-xL, and Mcl-1either constitutively or after activation. [START_REF] Fletcher | Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax[END_REF][START_REF] Galonek | Upgrading the BCL-2 network[END_REF][START_REF] Kim | Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies[END_REF][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] The BH3-only proteins act as sensors for cellular stress and their activity is regulated at the transcriptional level as well as by post-translational modifications. Upon activation the multidomain proteins Bax and/or Bak oligomerize at the mitochondrial outer membrane, leading to its permeabilization and the subsequent release of cytochrome c into the cytosol. [START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF] Cytosolic cytochrome c associates with Apaf-1, thereby forming the heptameric apoptosome which is the activation platform for the initiator caspase-9. [START_REF] Li | Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade[END_REF] Active caspase-9 then cleaves and activates downstream caspases that dismantle the cell. [START_REF] Slee | Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner[END_REF] Due to the mutual but also selective interaction specificities of pro-and anti-apoptotic proteins the mechanisms underlying Bcl-2 signaling are not fully elucidated yet. Currently, two models of intracellular Bcl-2 family signaling are discussed: the direct activation and the derepression model. The former proposes that the BH3-only proteins Bim, Bid and Puma are necessary to directly activate Bax and/or Bak via transient interaction. The de-repression model suggests BH3-only proteins to inactivate anti-apoptotic counterparts by binding them and thereby indirectly releasing Bax and Bak from their inactivating interaction with anti-apoptotic proteins. [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] Willis | Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak[END_REF] Interestingly, the BH3-only protein Nbk/Bik, which interacts strongly with the Bax antagonizing proteins Bcl-2 and Bcl-x L but only weakly with Mcl-1 that predominantly interferes with Bak, does not induce apoptosis in Bax-deficient cells, arguing for a de-repression mechanism. [START_REF] Gillissen | Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis[END_REF] In addition to the specific interaction pattern of Bcl-2 proteins, their relative expression level influences the cellular response to apoptosis inducing stimuli. The expression of the upstream acting BH3-only proteins Puma and Noxa is induced by p53 in response to DNA damage. [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF][START_REF] Oda | Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis[END_REF] p53 also upregulates the expression of the downstream acting protein Bax [START_REF] Miyashita | Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[END_REF] resulting in a cooperative sensitization of cells to apoptosis. Furthermore, p53 negatively influences the expression of the anti-apoptotic protein Bcl-2 [START_REF] Bourgarel-Rey | Transcriptional down-regulation of Bcl-2 by vinorelbine: identification of a novel binding site of p53 on Bcl-2 promoter[END_REF] which can lead to an enhanced expression of Bcl-2 in p53 deficient cells. [START_REF] Miyashita | Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo[END_REF][START_REF] Sohn | p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation[END_REF] Since bax is a transcriptional target of p53, we wondered whether also expression of Bak, the other main pro-apoptotic multidomain protein, is regulated by p53. We therefore analyzed the human bak1 gene locus for transcription factor binding sites. Indeed, we identified a functional p53 consensus binding site and verified p53-mediated regulation of the bak1 promoter region by luciferase reporter gene assays, qRT-PCR, and Western blot analysis. Upregulation of Bak in response to the DNA-damaging agent etoposide occurred later than p53-mediated induction of p21 expression and cell cycle arrest. Our data suggest a model in which cells in sustained cell cycle arrest are sensitized for apoptosis by enhanced Bak expression that shifts the Bcl-2 rheostat of anti-apoptotic proteins and pro-apoptotic multidomain proteins.

Results

Identification of putative p53-binding sites in the bak1 gene locus. The tumor suppressor protein p53 suppresses the expression of Bcl-2 [START_REF] Miyashita | Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo[END_REF] , whereas it induces expression of several BH3only proteins, such as Puma and Noxa, and the pro-apoptotic multidomain protein Bax. [START_REF] Miyashita | Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[END_REF] This prompted us to analyze the possibility that p53 in addition to Bax also regulates the expression of the homologous pro-apoptotic multidomain protein Bak. We initially analyzed 8200 bp of the bak1 gene locus on chromosome 6p21.31 for potential promoter regions using the "Gene2Promotor" and "ElDorado" software (Genomatix Software GmbH, www.genomatix.de).

These 8200 bp span a region starting 5550 bp upstream of exon 1, include exon 1, intron 1, and the first 16 codons of the coding sequence within exon 2. The software indicated two potential promoter regions, the first starting upstream of and extending into exon 1 (bp -3188 to -2115) and the second starting within intron 1 and extending into the coding region of exon 2 (-508 to 50; Fig. 1a). Based on these findings we analyzed a total of 3350 bp (-3300 to 50) including both promoter regions for transcription factor-binding sites using the "MatInspector" (Genomatix) software. The MatInspector software identified binding sites for different transcription factors, e.g. SP1/2, STAT, NF-κB and also p53 (Fig. 1a). Because p53 transactivates expression of several pro-apoptotic Bcl-2 proteins we analyzed the three potential p53-binding sites in this region -the first one directly upstream of exon 1 and the other two following exon 1. To test whether these putative p53-binding sites were functional, we cloned the region containing all 3 potential p53 binding sites and exon 1 (-2790 to -2020; a total of 770 bp) into the pGL3-basic vector generating pGL3-p53BS and performed luciferase reporter gene assays.

p53 induces target gene expression from bak promoter constructs. In a next step we cotransfected HeLa cells with different amounts of a p53 expression vector together with the reporter plasmid pGL3-Basic-p53BS that contains all three putative p53-binding sites. Cell extracts were prepared 48 h post transfection and luciferase activity measured luminometrically.

Clearly, co-transfection of p53 induced luciferase expression in HeLa cells (Fig. 1b, left panel).

Induction of luciferase expression from pGL3-Basic-p53BS was higher when cells were cotransfected with 1.0 µg of the p53 expression plasmid as compared to transfection with 0.5 µg, indicating specific target gene expression from the bak-promoter construct in a concentrationdependent manner. To further confirm these data we also performed similar experiments in HCT116/p53 -/-cells. Again, cells were co-transfected with either 0.5 µg or 1.0 µg of the p53 expression construct together with pGL3-Basic-p53BS. Also in the p53-negative cellular background exogenous p53 specifically induced luciferase activity (Fig. 1b, right panel). These results show that p53 can transactivate gene expression from the cloned region of the bak1 gene locus.

We next set out to investigate the specific role of each of the putative p53-binding sites within the 770 bp region and therefore generated 4 more luciferase expression vectors containing either mutations in one of each putative p53 consensus sequence or mutations in all three putative p53-binding sites as described in materials and methods. The p53 consensus binding site consists of two half sites of the sequence 5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' separated by 0 -13 bp. [START_REF] El-Deiry | Definition of a consensus binding site for p53[END_REF] We introduced mutations in each of the p53 binding sites separately by exchanging the essential cytosine and guanine residues for adenine and thymine (Suppl. Table 1). Additionally, we created a luciferase reporter gene vector in which all three p53-binding sites were mutated simultaneously. These four additional reporter gene plasmids, pGL3-Basic-mutBS1, -mutBS2, -mutBS3, and -mutBS1-3 were then co-transfected together with the p53 expression construct into HeLa cells and luciferase activity in cellular extracts was assessed 48 h post transfection (Fig. 2a). Unexpectedly, mutation of BS1 or BS2 resulted in negligible changes of luciferase activity compared to the non-mutated construct, whereas mutation of BS3 caused a significant reduction of luciferase activity by about 60% (23-fold compared to 61.7-fold; Fig. 2a). The mutation of BS3 had a similar effect when assays were conducted in HCT116/p53 -/-cells. Again, mutation of BS1 or BS2 did not significantly influence reporter gene expression, whereas luciferase activity induced from pGL3-Basic-mutBS3 was reduced by about 60% (6.2-fold compared to 15.4-fold) (Fig. 2b). The simultaneous mutation of all three putative p53-binding sites did not result in a further reduction of luciferase activity compared to the single mutation of BS3 in either cell line.

In agreement with the previous results, overall induction of luciferase expression was higher in HeLa than HCT116/p53 -/-cells. These data do not only demonstrate that p53 induces target gene expression from the bak1 promoter region but, moreover, show that exclusively BS3 is necessary and sufficient for p53-driven gene expression, whereas BS1 and BS2 are dispensable. p53 directly binds to BS3. Because mutation of BS3 largely reduced reporter gene activity, we wanted to confirm p53 binding to the newly identified BS3. To this end, we performed electrophoretic mobility shift assays (EMSAs) using radiolabeled ds-oligonucleotides containing the sequence of each putative binding site. We also included the mutated BS3 and, as a positive control, the p53 consensus binding site in these assays. The radiolabeled probes were incubated with nuclear extracts from HCT116/wt cells that had been treated for 48 h with the DNA-damaging agent etoposide. To further analyze specificity of the DNA/protein complex, an anti-p53 antibody was added to the samples. In samples containing the p53 consensus binding site a protein/DNA complex was readily detected and addition of the p53 antibody produced a supershift proving the identity of the p53/DNA complex (Fig. 2c, left lane). Although each of the binding site oligonucleotides was unexpectedly shifted in EMSAs, exclusively the BS3 oligonucleotide was supershifted by the p53 antibody. Mutation of BS3 abolished protein binding in the presence or absence of the p53 antibody. Hence, in line with our results from luciferase reporter gene assays, p53 directly binds to BS3 and mutation of the essential cytosine and guanine residues in the BS3 sequence disrupts p53 binding. In further mobility shift assays that were performed with extracts from etoposide treated HCT116/p53 -/-cells we also detected protein/DNA complexes of each of the binding sites but, as expected, no supershift was produced by addition of the p53 antibody. These results confirm direct binding of p53 to BS3, but also show that BS1-3 are bound by additional proteins. Lastly, we performed chromatin immunoprecipitation (ChIP) assays to analyze binding of p53 to BS1, BS2 and BS3 within the bak promoter in a cellular context. In anti-p53 ChIPs from samples of etoposide-treated HCT116/wt cells we found enrichment of BS3, but not BS1 or BS2 (Fig. 2d), thus confirming binding of endogenous p53 specifically to BS3 within the bak promoter region.

p53 upregulates expression of endogenous bak. The results obtained so far clearly demonstrate that p53 can induce target gene expression from constructs containing the identified promoter region by binding to BS3. Consequently, we next investigated endogenous bak gene expression. In initial experiments we transduced HCT116/p53 -/-cells with different MOIs of an adenovirus encoding for p53 and analyzed Bak mRNA expression three days later. Bak mRNA levels were elevated (about 6-fold) in transduced cells (Fig. 2e, left bars). Because adenoviral transduction induces high levels of p53, we also aimed to achieve p53 upregulation in a more physiological manner. To this end, we transduced HCT116/wt and HCT116/p53 -/-cells with an adenovirus for the expression of p14 ARF . p14 ARF inhibits MDM2-mediated p53 degradation and induces elevated levels of p53. Enhancing the level of endogenous p53 clearly resulted in elevated levels (more than 10-fold) of Bak mRNA (Fig. 2e, middle bars) in HCT116/wt cells.

The specificity of these results was underlined by transduction of HCT116/p53 -/-cells with Ad-p14 ARF . In the absence of p53 p14 ARF did not induce bak mRNA expression (Fig. 2e, right bars).

We then investigated whether activation of the endogenous p53 signaling pathway in response to DNA damage also drives Bak mRNA expression. To this end, HCT116/wt cells were treated with the topoisomerase II inhibitor etoposide and Bak mRNA expression was analyzed by quantitative real-time PCR (qRT-PCR). As a control we also investigated Bak mRNA expression in response to etoposide treatment in the isogenic HCT116/p53 -/-cell line. The results of qRT-PCR were normalized to internal GAPDH mRNA expression. Bak mRNA was upregulated over time in HCT116/wt cells following DNA damage due to etoposide treatment (Fig. 3a, left panel).

Bak mRNA expression was 1.75-fold after 24 h, 2-fold after 48 h and increased up to 4.5-fold after 72 h. In HCT116/p53 -/-cells treated with etoposide induction of Bak mRNA expression was less pronounced (1.5-fold after 24 h and 48 h and 2.7-fold after 72 h; Fig. 3a, right panel).

The results from EMSA experiments indicated binding of proteins different from p53 to BS1, 2 and 3, which is reflected by Bak mRNA induction in p53-negative cells. To test whether upregulation in the absence of p53 is specific for Bak or does also occur for other p53 target genes, we investigated the induction of several other p53 target genes in HCT116/wt and HCT116/p53 -/-cells. We chose to analyze mRNA expression of the cyclin-dependent kinase (CDK) inhibitor p21 [START_REF] El-Deiry | WAF1, a potential mediator of p53 tumor suppression[END_REF][START_REF] Xiong | p21 is a universal inhibitor of cyclin kinases[END_REF] , the pro-apoptotic multidomain protein Bax [START_REF] Miyashita | Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[END_REF] and the BH3-only protein Puma [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF][START_REF] Yu | PUMA induces the rapid apoptosis of colorectal cancer cells[END_REF] , which all are well established target genes of p53. In line with data from the literature, qRT-PCR analysis showed induction of all these target genes in response to etoposide treatment of HCT116/wt cells. Target gene induction was time-dependent and most pronounced after 72 h. Induction was strongest for p21 reaching a 55-fold expression level after 72 h (Fig. 3b). Induction of Puma was below that of p21 (25-fold after 72 h, Fig. 3c) and that for Bax was weakest (7-fold after 72 h, Fig. 3d). Comparable to the results obtained for Bak, all these established p53 target genes were also induced in p53-deficient HCT116/p53 -/-cells, although to a lesser extent compared to wild type HCT116. We therefore conclude that bak can be considered a bona fide p53 target gene, although a weaker expression is also detectable in the absence of p53, similarly to the established p53 targets p21, puma and bax.

We also tested whether the p53-mediated induction of target genes correlates with the strength of DNA damage. To this end we treated HCT116/wt and HCT116/p53 -/-cells with 10 µM, 50 µM, and 100 µM etoposide for 48 h and performed qRT-PCR analysis. These experiments showed that Bak mRNA induction is concentration-dependent. Whereas the Bak mRNA level was about 2.5-fold in cells treated with 10 µM and 50 µM etoposide, it was 3-fold in response to treatment with 100 µM etoposide in HCT116/wt cells (Fig. 4a). Again, etoposide treatment affected Bak mRNA expression in HCT116/p53 -/-only marginally (Fig. 4a). For comparison we also analyzed induction of Bax, p21, and Puma mRNAs in similarly treated HCT116/wt and HCT116/p53 -/-cells. In HCT116/wt cells induction of Bax mRNA was comparable to that of Bak (Fig. 4b) and induction of Puma mRNA expression was higher than that of Bax and Bak (up to 4.5-fold) (Fig. 4c). Notably, p21 was induced about 16-fold regardless of the etoposide concentration (Fig. 4d). The mRNA expression of Bax and Bak was significantly reduced in HCT116/p53 -/-cells compared to that in HCT116/wt cells (Fig. 4a-b). Expression of p21 and Puma mRNA after etoposide treatment was slightly elevated in HCT116/p53 -/-cells (Fig. 4c-d).

P53-dependent upregulation of Bak protein level in response to DNA damage. We clearly demonstrated p53 specific regulation of bak gene expression and finally wanted to confirm Bak upregulation at the protein level. Therefore, we analyzed Bak protein expression by Western blot analysis of HCT116/wt and HCT116/p53 -/-cells that had been treated with 50 µM etoposide for 24 h, 48 h, and 72 h or had been left untreated. Confirming our qRT-PCR results Bak was significantly upregulated at the protein level in response to etoposide treatment in HCT116/wt cells, whereas induction of Bak expression was less pronounced in HCT116/p53 -/-(Fig. 4e). We also analyzed the expression of the CDK inhibitor p21 and Bax by Western blotting. Again, confirming qRT-PCR data expression of p21 was highly induced already 24 h after etoposide treatment. However, the protein level of p21 appeared to slightly decline over time. Western blot analysis also confirmed p53-dependent induction of Bax (Fig. 4e). Interestingly, the basal level of Bak was reduced in HCT116/p53 -/-cells compared to the wild type cell line. This finding might be indicative of the basal p53 protein expression in HCT116/wt cells upregulating Bak expression on the one hand, and of Bak regulation by other transcription factors than p53 in HCT116/p53 -/- cells on the other. The results obtained from Western blots of dose-response experiments showed that expression of p21 and Bax was already induced by low doses of etoposide, whereas Bak expression increased in a dose-dependent manner (Fig. 4f). p73 transactivates bak. Since we had conclusively established p53-dependent upregulation of the pro-apoptotic protein Bak in response to DNA damage, we were left with the interesting finding that Bak expression, albeit to a weaker extent, is also upregulated in the absence of p53.

Bak induction in the absence of p53 could be due to redundancy of the p53 family proteins regarding their consensus binding sites. Because p73 has been shown to induce expression of Puma in response to DNA-damage by γ-irradiation [START_REF] Melino | p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation[END_REF] , we hypothesized that regulation of Bak expression might also be affected by p73. To test this we co-transfected HCT116/p53 -/-cells with expression plasmids encoding for different p73-isoforms together with the bak promoter construct. All p73 isoforms (α, β, γ, δ) induced luciferase expression to various degrees (data not shown). p73δ showed the highest induction and was used as p73 in subsequent experiments. We next co-transfected the different luciferase reporter gene plasmids containing the bak promoter region and mutations in the p53 consensus sequences together with the p73 expression vector.

Then, 48 h post transfection, lysates were analyzed for luciferase activity. Indeed, p73 induced luciferase expression from the bak promoter region (Fig. 5a). Interestingly, the mutation of each of the putative p53-binding sites reduced p73-mediated reporter gene induction by 20-30%.

Simultaneous mutation of all three binding sites had an additive effect and reduced reporter gene expression by about 60%. These results show that (i) bak gene expression is induced by p73 and (ii) this induction is regulated by all three binding sites and not exclusively by BS3 as is the case for p53. In EMSAs we analyzed whether p73 can bind to all three putative p53 consensus sequences supporting the luciferase data. Indeed, addition of a p73 antibody to the samples produced a supershift of all unmutated ds-oligonucleotides used, showing that p73 binds to BS1, BS2 and BS3 (Fig. 5b).

Both, p53 and p73, can also be expressed as N-terminal truncated isoforms that lack the transactivation domain and these ΔN-isoforms act dominant negative on their respective transactivation competent TA-isoforms. [START_REF] Harms | The functional domains in p53 family proteins exhibit both common and distinct properties[END_REF] We investigated the impact of ΔN-isoforms on p73-mediated reporter gene induction by co-transfection of expression plasmids for Δ133p53 and DNp73, respectively. As expected, p73-induced reporter gene expression from the bak promoter was reduced by co-expression of DNp73 in both, HeLa (Fig. 5c, left panel) and HCT116/p53 -/- cells (Fig. 5c, right panel) to basal level. Interestingly, p73-mediated reporter gene induction was not reduced by co-expression of Δ133p53. In complementary experiments we investigated the influence of DNp73 and Δ133p53 on p53 induced reporter gene expression. In HeLa cells reporter gene induction by p53 was reduced by 70% when DNp73 was co-expressed, and by 30% in case of Δ133p53 co-expression (Fig. 5d, left panel). Likewise, in HCT116/p53 -/-cells coexpression of DNp73 diminished p53-mediated luciferase expression from 12.5-fold to 2.2-fold and Δ133p53 again showed less reduction of luciferase activity (12.5-fold vs. 7.6-fold) (Fig. 5d, right panel).

We finally tested whether p73 induces expression of endogenous Bak. To this end HCT116/p53 -/-cells were transduced with 50 MOI of an adenovirus encoding for p73α and 72 h post transduction cells were harvested and analyzed for Bak mRNA. Quantitative real time PCR showed a 2.5-fold increase of Bak mRNA expression in p73-expressing HCT116/p53 -/-cells as compared to control cells (Fig. 5e). p73-mediated upregulation of Bak mRNA expression in response to etoposide treatment for 72 h was furthermore analyzed in HCT116/wt cells tranfected with an p73-shRNA expression vector and control cells. Expression of p73 was upregulated 4fold in etoposide treated control cells and only 1.5-fold in shp73 transfected cells (data not shown). In line, the 8-fold induction of Bak mRNA in etoposide treated cells was reduced to about 4-fold in p73-shRNA-expressing cells (Fig. 5f, left panel), once more verifying p73 mediated-upregulation of endogenous Bak. To further investigate whether p73 regulates Bak mRNA expression we also incubated HCT116/p53 -/-cells that had been transfected with the p73-shRNA construct in the absence or presence of 50 µM etoposide for three days. Subsequently, Bak mRNA expression was analyzed by qRT-PCR. Upregulation of Bak mRNA expression in response to etoposide treatment was reduced from 2.3-fold in etoposide-treated control cells to 1.7-fold in p73-shRNA expressing cells (Fig. 5f, right panel). These results not only confirm that p73 modulates Bak mRNA expression but, moreover, indicate that p73 modulates expression of Bak mRNA independently of p53.

To investigate the relevance of Bak expression for DNA damage-induced apoptosis, we compared sub-G1 populations in different etoposide-treated HCT116 cell lines that were either wild type, devoid of Bax or Bak, or both Bax and Bak expression, as verified by Western Blot analysis (Fig. 6a). The various cell lines were incubated in the presence of 50 µM etoposide for 24 h, 48 h and 72 h and the percentage of sub-G1 (referred to as apoptotic) cells was assessed by flow cytometry. As expected, Bax knock-out as well as Bak knock-down reduced the number of apoptotic cells at each time point (Fig. 6b). Interestingly, after 72 h of treatment, combined knock-out of Bax and knock-down of Bak had an additive effect that was not evident after 24 h and 48 h. At this late time point the number of sub-G1 cells was reduced by about 50% in HCT116/Bax -/-/Bak kd and only about 25% in either HCT116/Bax -/-or HCT116/Bak kd compared to HCT116/wt (Fig. 6b).

Discussion

In this study we have identified the pro-apoptotic multidomain protein Bak as a transcriptional target of the tumor suppressor p53 and we have furthermore for the first time characterized the bak1 promoter region with respect to p53 binding sites. We found that p53-mediated reporter gene upregulation exclusively depends on the p53-BS3 spanning bp -2085 to -2066. Binding of p53 to this region was verified by a supershift in EMSAs. Induction of endogenous Bak mRNA and protein expression by p53 was confirmed by qRT-PCR and Western blot analyses in HCT116/wt cells and the isogenic p53 knock-out cell line HCT116/p53 -/-. Hence, like other Bcl-2 family proteins, e.g. Puma, Noxa, Bax and Bcl-2, also Bak expression is transcriptionally regulated by p53. The location of the p53 response element downstream of exon 1 within the first intron is reportedly a common feature of some p53 target genes, such as GADD45α, KILLER/DR5 and the p53 negative regulator MDM2. [START_REF] El-Deiry | Definition of a consensus binding site for p53[END_REF][START_REF] Kastan | A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia[END_REF][START_REF] Zauberman | A functional p53-responsive intronic promoter is contained within the human mdm2 gene[END_REF][START_REF] Takimoto | Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site[END_REF] Recent analyses of apoptosis regulation by Bcl-2 proteins have revealed specific interactions of BH3-only proteins with anti-apoptotic Bcl-2 proteins. Likewise, anti-apoptotic Bcl-2 proteins specifically interact with the pro-apoptotic multidomain proteins Bax and Bak. In the de-repression model interaction of BH3-only proteins with anti-apoptotic Bcl-2 proteins results in their inactivation. Therefore, the anti-apoptotic proteins no longer counteract Bax and Bak, which then can oligomerize at the mitochondrial outer membrane and induce its permeabilization. Although further complexity is added to the Bcl-2 network by posttranslational modifications of Bcl-2 proteins that modulates their activity [START_REF] Kutuk | Regulation of Bcl-2 family proteins by posttranslational modifications[END_REF] , transcriptional upregulation of Puma and Noxa by p53 is paramount for apoptosis induction. Also the downstream acting pro-apoptotic multidomain protein Bax is a transcriptional target of p53 [START_REF] Miyashita | Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[END_REF] , whereas the expression of the Bax-antagonizing protein Bcl-2 is reduced in the presence of p53. [START_REF] Miyashita | Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo[END_REF][START_REF] Sohn | p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation[END_REF] Our qRT-PCR data show that Puma induction by p53 is stronger than that of either Bak or Bax but still does not reach the level of p53-mediated p21 induction. This reflects that cell cycleregulatory genes contain more robust p53REs than do pro-apoptotic genes. [START_REF] Weinberg | Comparative binding of p53 to its promoter and DNA recognition elements[END_REF] The strength of p53 mediated induction of the pro-apoptotic genes puma, bax and bak1 correlates with the distance of their respective p53REs from the transcriptional start site, as is the case for many p53 target genes. [START_REF] Riley | Transcriptional control of human p53-regulated genes[END_REF] The puma p53RE is located in the promoter region (-145/-126), those for bax (354/373) and bak1 (596/617, corresponding to BS3) are located in the first intron. Although several other mechanisms might regulate p53 target gene expression, the relative induction of these proteins (p21, Puma, Bax and Bak) reflects the cellular response that is established by p53. The first event to take place is cell cycle arrest via strong induction of p21. Under sustained cell cycle arrest and as long as Puma expression is not sufficient to induce apoptosis, expression of the pro-apoptotic multidomain proteins Bax and Bak rises and both accumulate. Finally, cells cross the borderline to apoptosis due to the overall shift in the Bcl-2 rheostat that drives them into cell death rather than cell cycle arrest.

Our data furthermore show that, in addition to p53, another member of the p53 family of transcription factors, namely p73, can drive gene expression from the bak promoter. We found that p73 induces reporter gene expression from the bak promoter construct and that transgenic expression of p73 and also etoposide treatment induce Bak expression in p53-negative HCT116/p53 -/-cells. With regard to the reporter gene assays, it is most interesting to note that all three investigated binding sites equally contribute to p73-mediated reporter gene induction, whereas p53-mediated reporter gene induction exclusively depends on the presence of BS3. These findings were verified by supershift assays that showed that p53 exclusively binds to BS3, whereas p73 binds each of the binding sites. An overlapping response element affinity by p53 and p73 has already been shown by Osada et al. [START_REF] Osada | A novel response element confers p63-and p73-specific activation of the WNT4 promoter[END_REF] However, in addition to binding to p53 consensus sites, p73 has also its own specific consensus sequence. [START_REF] Osada | A novel response element confers p63-and p73-specific activation of the WNT4 promoter[END_REF] The presence of additional specific p73 consensus sites in the bak promoter region is presumable, as mutation of all three putative p53 binding sites does not completely abrogate p73-driven reporter gene activity.

Transactivation by p73 in addition to p53 has also been shown for the BH3-only protein Puma. [START_REF] Melino | p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation[END_REF] We have also detected upregulation of Puma mRNA in etoposide-treated p53-negative HCT116/p53 -/-cells, which might be due to a regulation by p73. [START_REF] Melino | p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation[END_REF] We furthermore found that ΔN-isoforms differentially influence p53-and p73-induced reporter gene expression. Whereas DNp73 suppresses reporter gene induction by both, p53 and p73, the ΔN133p53-isoform specifically reduces reporter gene induction by p53 but not p73.

However, the impact of ΔN133p53 and DNp73 on transactivation by p53 and p73 not only depends on binding specificity and binding affinity, but is highly dependent on the relative expression level of transcription factors. Therefore, transactivation of the bak promoter by endogenous p53-and p73-isoforms might differ to some degree from results of reporter gene assays.

The expression of several Bcl-2 family proteins has been shown to be induced by transcription factors that do not belong to the p53 family. Expression of Puma for instance can also be induced by FoxO3 [START_REF] You | FOXO3adependent regulation of Puma in response to cytokine/growth factor withdrawal[END_REF] and SP1. [START_REF] Ming | Sp1 and p73 activate PUMA following serum starvation[END_REF] Interestingly, Chirakkal et al. identified a SP1-binding site within the promoter region of bak1 34 that was also indicated in our MathInspector analysis (Fig. 1). This SP1 binding site mediates upregulation of Bak in response to butyrate as well as basal Bak expression. The presence of this SP1 binding site in our reporter gene constructs might therefore explain that mutation of p53 binding sites does not completely abrogate reporter gene induction. Most recently NF-κB was shown to induce expression of Puma, Noxa, Bad, and Bim, thereby contributing to apoptosis induction by TNF-α [START_REF] Wang | PUMA is directly activated by NF-kappaB and contributes to TNF-alpha-induced apoptosis[END_REF] . As our software analysis also indicated the presence of several NF-κB binding sites in the Bak promoter region, it is likely that apart from p53 other transcription factors influence expression of Bak under certain conditions. For example, in embryogenesis and tissue development the transcription factor WT1 (Wilm's tumor suppressor protein 1) has been implicated in regulation of Bak expression. [START_REF] Morrison | WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak[END_REF] Mutations in WT1 cause Wilm's tumors and knock-out of WT1 results in agenesis of kidneys associated with embryonic lethality. Transactivation of the bak promoter during differentiation appears to be negatively regulated by brain acid soluble protein 1 (BASP1). [START_REF] Green | Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation[END_REF] In summary, all these findings reflect that, despite the ever increasing advances in our understanding of signaling by Bcl-2 proteins, the regulation of their expression by numerous transcription factors is still poorly investigated. A comprehensive model for the complex mechanisms that underlie control of apoptosis by Bcl-2 proteins would need to incorporate both, transcriptional and posttranscriptional regulation.

Materials and methods

Cell lines, reagents and antibodies. HCT116/wt, HCT116/p53 -/-, HCT116/bax -/-(kindly provided by B. Vogelstein) [START_REF] Zhang | Role of BAX in the apoptotic response to anticancer agents[END_REF] and HCT116 cells stably expressing Bak siRNA (HCT116/bak kd and HCT116/bax -/-/bak kd ) [START_REF] Hemmati | Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells[END_REF] were maintained in McCoy's 5A medium supplemented with 10% heat-inactivated fetal calf serum, 10 mM glutamine and antibiotics (100 units/ml penicillin, 0.1 mg/ml streptomycin) (PAA Laboratories, Linz, Austria). HeLa cells were cultured in RPMI-1640 (PAA) with identical supplements. All chemicals were purchased from Sigma (Munich, Germany). The following antibodies were used: monoclonal mouse anti-Bax (6A7, Trevigen, Gaithersburg, MD), polyclonal rabbit anti-Bak (Upstate, Temecula, CA), monoclonal mouse anti-p53 (Ab-6, Calbiochem/EMD Biosciences, Darmstadt, Germany), monococlonal mouse anti-p73 (ER-13, Sigma), monoclonal mouse anti-p21 (Becton Dickinson, Heidelberg, Germany) and monoclonal mouse anti-β-actin (Sigma). Secondary antibodies to mouse and rabbit IgG were from Promega (Mannheim, Germany).

Cloning and mutagenesis. The bak1 promoter region was amplified by PCR from clone RPCIP704J10291Q (RZPD, Berlin, Germany) using the primer pair BS-for/BS-rev (Suppl. Table 1) that contained the XhoI and HindIII restriction sites. After restriction with XhoI and HindIII the PCR product was ligated into the pGL3-Basic vector (Promega) generating pGL3-Basic-p53BS. Vectors containing mutations in the putative p53 consensus binding sites 1 and 3 were generated by PCR amplification using the primer pair mutBS1-for/BS-rev and BS-for/mutBS3rev (Suppl. Table 1), respectively. Subsequent ligation of the PCR products into pGL3-Basic resulted in pGL3-Basic-mutBS1 and pGL3-Basic-mutBS3. Binding site 2 was mutated using the Quick Change Mutagenesis kit (Stratagene, La Jolla, CA). The vector pGL3-Basic-p53BS served as template and the two complementary primers mutBS2-for and mutBS2-rev (Suppl. Table 1) introduced the desired mutations resulting in pGL3-Basic-mutBS2. To generate the vector pGL3-Basic-mutp53BS containing mutations in all three binding sites simultaneously the template pGL3-Basic-mutBS2 was PCR amplified using the primers mutBS1-for and mutBS3-rev. All vector sequences were confirmed by DNA sequencing.

Western blot analysis. Cells were harvested by scraping, washed in ice-cold PBS and lysed in buffer containing 1% Nonidet P-40, 20 mM HEPES (pH 7.9), 2 mM PMSF, 350 mM NaCl, 1 mM MgCl 2 , 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using the BCA assay kit (Thermo Fischer Scientific, Bonn, Germany) and 15 µg of protein per lane were loaded onto standard SDS-PAGE gels and separated at 200 V. The proteins were transferred onto a polyvinylidene difluoride membrane (Amersham Bioscience GmbH, Freiburg, Germany) by tank blotting. The membranes were blocked in PBS containing 4% BSA and 0.05% Tween-20 for 1 h followed by an overnight incubation with the primary antibody diluted in blocking buffer at 4 °C. After washing the membranes thrice in blocking buffer the appropriate secondary antibodies (1:5000) were applied for 1 h. Finally, membranes were washed for an additional 2 h in PBS, 0.05% Tween-20, before proteins were visualized using ECL reagents (Amersham Biosciences).

Transfection and viral transduction. Cells were seeded 24 h prior to transfection in 6-well plates or culture dishes at a density of 7.5x10 4 cells per cm 2 . Transfection was performed using the Fugene 6 (Roche) or jetPEI (PEQLAB, Erlangen, Germany) reagent according to the manufacturer's protocol. Transfected cells were grown for various time periods before harvesting. Preparation of p14 ARF , p53 and p73 adenoviruses [START_REF] Hemmati | Adenovirusmediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis[END_REF][START_REF] Zender | VP22-mediated intercellular transport of p53 in hepatoma cells in vitro and in vivo[END_REF] and transduction of cells were performed as described. [START_REF] Gillissen | Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway[END_REF] Luciferase reporter gene assays. Cells were transfected with the respective pGL3-Basic reporter plasmid together with expression vectors for p53, Δ133p53, DNp73, p73δ or empty vector. [START_REF] Graupner | Functional characterization of p53beta and p53gamma, two isoforms of the tumor suppressor p53[END_REF][START_REF] Grob | Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[END_REF] 48 h post transfection cells were harvested by scraping and lysed in LLB [25 mM glycylglycine (pH 7.8), 15 mM MgSO 4 , 4 mM EGTA (pH 8.0), 1 mM DTT, 1% Triton X-100] for 5 min on ice. After centrifugation (5 min, 10000 x g) 10 µl of the supernatants were assayed in 100 µl luciferase assay buffer [15 mM potassium phosphate (pH 7.8), 25 mM glycylglycine, 15 mM MgSO 4 , 4 mM EGTA, 1 mM DTT, 2 mM ATP] using a microplate luminometer (Berthold Technologies, Bad Wildbad, Germany). Light emission was measured after injection of 100 µl of luciferin (0.3 mg/ml).

Electrophoretic mobility shift assays. The p53-consensus oligonucleotide was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the oligonucleotides representing BS1, BS2, BS3 and mutBS3 (Suppl. Table 2) were from Sigma. Hybridized oligonucleotides were radiolabelled by Klenow-reaction in the presence of [γ]-32 P-ATP. 10 µg of nuclear extracts from etoposide (50 µM, 48 h) treated HCT116/wt or HCT116/p53 -/-cells were incubated with radiolabeled oligonucleotides for 30 min at room temperature. For supershift assays 1 µg of the respective antibody was added to the samples followed by an additional incubation for 30 min on ice. Samples were separated on an 8% 0.5 x TBE polyacrylamide gel at 160 V, dryed and autoradiagraphs were taken by exposure to an x-ray film for 3-5 h.

Quantitative real-time PCR. Cells were seeded at a density of 1x10 5 cells per cm 2 . 24 h later the culture medium was changed for fresh medium with or without etoposide (50 µM). After the indicated periods of time cells were harvested by scraping and total RNA was isolated using the RNeasy mini kit from Qiagen (Hilden, Germany) according to the manufacturer's protocol. cDNA was reverse transcribed from 1 µg total RNA using a 2-step protocol: first RNA was incubated with 50 µM random hexamer primers (Fermentas GmbH, Leon-Rot, Germany) and 40 units RNase-Inhibitor (RiboLock RNase Inhibitor, Fermentas) at 65°C for 10 min. Then, 200 units of reverse transcriptase (Revert Aid H Minus M-MuLV Reverse, Fermentas), 10 mM DTT, 400 µM dNTPs and water were added to a final volume of 25 µl and samples were incubated at 42°C for 50 min followed by final heat inactivation at 72°C for 10 min. qPCR was performed in an ABI7300 instrument (Applied Biosystems, Darmstadt, Germany) using 1x Maxima SYBR Green qPCR Mastermix (Fermentas), 300 nM sense-/antisense primers and 80 ng cDNA. The qPCR programme started with an initial denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Melting curve analysis was performed by heating the samples twice to 95°C for 15 s and 60°C for 1 min. Negative control reactions contained water instead of cDNA and were included in each run to ensure absence of contamination. Primers used for analysis of p21 (Hs_CDKN1A_1_SG), puma (Hs_BBC3_1_SG), and bak (Hs_BAK1_1_SG) expression were purchased from Qiagen and primers for bax expression (forw: 5'-CACGGCAGAGAATGCCTATGA-3', reverse: 5'-CCCAATTGATGCCACTCTCAA-3') were kindly provided by Dr. K. Lauber (University Hospital Tübingen). Results were normalized to GAPDH and analyzed by the ΔΔCt method to give fold-induction as compared to untreated control samples.

Chromatin immunoprecipitation (ChIP) assay. Cells were seeded at a density of 1x10 5 /cm 2 . 24 h later the culture medium was changed for fresh medium with or without 50 µM etoposide.

After three days, cells were harvested by scraping, crosslinked and ChIPs were performed from 5x10 7 cells using an anti-p53 antibody (Ab-6) as described previously. [START_REF] Rothfuss | Parkin protects mitochondrial genome integrity and supports mitochondrial DNA repair[END_REF] Quantitative real-time PCR was performed on a LightCycler480 instrument using the SYBR Green I Master kit (Roche) according to the manufacturer's protocol. Primers specific for amyloid precursor protein (APP), BS1, BS2 and BS3 are indicated in Suppl. Table 3. Values were normalized to APP and fold enrichment over input chromatin was calculated using the 2exp{-ΔΔCt} method.

Lauber for valuable materials and helpful discussion. We also wish to thank Mohamed Hassan for support in EMSA experiments. This work was supported by the Deutsche Forschungsgemeinschaft (SFB773, SFB685, GRK 1302), the BMBF (AID-Net) and the Comprehensive Cancer Center Tübingen.

Legends to figures

Figure 1 :

 1 Figure 1: Representation of the bak1 gene locus and reporter gene assays. (a) Schematic representation of the analyzed region of the bak1 gene locus on 6p21.31. The promoter regions predicted by the Matinspector software, positions of exon 1, intron 1 and exon 2 and positions of the three putative p53-binding sites are depicted. Also shown is the sequence of the cloned bak promoter region. The putative p53binding sites are printed in bold and highlighted by grey boxes, the position of exon 1 is highlighted in grey, potential binding sites for SP1 and STAT are underlined. (b) Luciferase reporter gene assay using the reporter vector containing the illustrated region (bp -2790 to -2020) of the bak1 gene locus. HeLa and HCT116/p53 -/-cells were co-transfected with pGL3-Basic-p53BS together with different amounts of a p53 expression vector. p53 induces luciferase expression in a concentration-dependent manner in HeLa and HCT116/p53 -/-cells. Values are means ± S.D. from 6 independent experiments.

Figure 2 :

 2 Figure 2: Identification of BS3 as the functional p53-binding site. HeLa (a) and HCT116/p53 -/-(b) cells were transfected with the indicated reporter gene constructs together with a vector for the expression of p53. Luciferase reporter gene induction by p53 is not affected by mutation of BS1 or BS2, whereas mutation of BS3 reduces luciferase activity significantly. (c) EMSA using the p53 consensus sequence (p53con), BS1, BS2, BS3 or mutated BS3 (mutBS3) and extracts from etoposidetreated HCT116/wt and HCT116/p53 -/-cells. Addition of a p53 antibody produces a supershift (indicated by *) of p53con and BS3 in samples from wild type but not from p53-deficient cells. No shift of BS1, BS2, or mutBS3 is detected. (d) Anti-p53 ChIP assay from HCT116/wt cells incubated in the absence or presence of 50 µM etoposide for 72 h. The graph shows enrichment of BS3 but not BS1 and BS2 over input DNA. Values are averages of two independent experiments. (e) HCT116 cells were transduced with 10 or 50 MOI of adenoviruses for the expression of p53 or p14 ARF .

Figure 3 :

 3 Figure 3: Time course of p53-dependent gene induction in response to DNA damage. HCT116/wt and HCT116/p53 -/-cells were treated with 50 µM etoposide for 24 h, 48 h and 72 h and expression of Bak (a), p21 (b), Puma (c) and Bax (d) mRNA was analyzed by qRT-PCR. Bak, Bax, Puma and p21 mRNA expression rises over time in response to treatment with etoposide in HCT116/wt and to a lesser extent in HCT116/p53 -/-cells. Values are means ± S.D. from three independent experiments.

Figure 4 :

 4 Figure 4: Analysis of mRNA and protein expression of p53 target genes in response to etoposide. HCT116/wt and HCT116/p53 -/-cells were treated with 10 µM, 50 µM and 100 µM etoposide for 48 h and expression of Bak (a), Bax (b), Puma (c) and p21 (d) mRNA was analyzed by qRT-PCR. Expression of Bak and Puma mRNA increases with concentration of etoposide, whereas induction of Bax and p21 mRNA expression is independent of the concentration of etoposide. (e) HCT116/wt and HCT116/p53 -/-cells were incubated with 50 µM etoposide for 24 h, 48 h and 72 h and expression of p53, Bak, Bax and p21 was analyzed by Western blotting. (f) HCT116/wt and HCT116/p53 -/-cells were incubated with the indicated concentrations etoposide for 48 h and expression of p53, Bak, Bax and p21 protein was analyzed likewise. In HCT116/wt and to a lesser extent also in HCT116/p53 -/-cells the level of Bak expression rises in a time-(e) and concentration-(f) dependent manner. Values are means ± S.D. from three independent experiments.

Figure 5 :

 5 Figure 5: p73-mediated induction of Bak reporter gene expression, DNA binding, and repression by Δ133p53, DNp73, and p73 knock-down. (a)

Figure 6 :

 6 Figure 6: Apoptosis induction in Bak-or Bax-deficient and proficient HCT116 cells. (a) Western blot analyses show the status of Bax and Bak expression in the

Acknowledgements

The authors wish to thank Bert Vogelstein, Martina Müller-Schilling, Stefan Kubicka and Kirsten