Julia Nafz

Johanna De-Castro

Verena Arce

Andrea Fleig

Sybille Patzelt

Mazurek

Dr Frank Rösl

Interference with energy metabolism by 5-aminoimidazole-4carboxamide-1-β-D-ribofuranoside induces HPV suppression in cervical carcinoma cells and apoptosis in the absence of LKB1

Keywords: HPV transcription, AP-1, AMPK, p53, therapy

Carcinogenesis is a dynamic and stepwise process, which is accompanied by a variety of somatic and epigenetic alterations in response to a changing microenvironment. Hypoxic conditions will select for cells, which have adjusted their metabolic profile and maintain proliferation by successfully competing for scarce nutritional and oxygen resources. In the present report we have studied the effects of energy depletion in the context of HPV-induced pathogenesis. We show that cervical carcinoma cell lines are susceptible to undergo either growth arrest or cell death under conditions of metabolic stress induced by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a known activator of the AMP-activated protein kinase (AMPK). Our results revealed that AICAR treatment led to a reduced binding affinity of the transcription factor AP-1 and in turn to a selective suppression of HPV transcription. Moreover, the outcome of AICAR on proliferation and survival was dependent on p53 activation and the presence of LKB1, the major upstream kinases of AMPK. Using non-malignant LKB1 expressing somatic cell hybrids, which lose expression after tumourigenic segregation as well as siRNA LKB1 knock-down approaches, we could further demonstrate that expression of LKB1 protects cells from cytotoxicity induced by agents which modulate the ATP:AMP ratio. Since simulation of low energy status can selectively eradicate LKB1 negative cervical carcinoma cells, AICAR may represent a novel drug in the treatment of cervical cancer.

INTRODUCTION

Cervical cancer is a sexually transmitted disease with an apparent causal relationship between the infection with certain "high-risk" human papillomavirus types (mostly HPV-16, but also HPVs 18,[START_REF] Kyriakis | At the crossroads: AMP-activated kinase and the LKB1 tumor suppressor link cell proliferation to metabolic regulation[END_REF][START_REF] Chan | Cooperative effects of genes controlling the G(2)/M checkpoint[END_REF] and some others) and the onset of tumour formation [START_REF] Zur Hausen | Papillomaviruses and cancer: from basic studies to clinical application[END_REF]. While the multi-step process of virus-induced carcinogenesis can be mimicked in part under tissue culture conditions [START_REF] Münger | Human papillomavirus immortalization and transformation functions[END_REF], accumulation of malignant cells in a patient represents a more complicated constellation, which is determined by the three dimensional environment, depending on external parameters such as blood vessel formation, nutritional supplementation, growth hormones and oxygen [START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF]. To compensate initial poor vascularisation and low nutritional supply, transformed cells have to adapt to their new surroundings through a profound change in their energy metabolism [START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF]. Indeed, many tumours including cancer of the cervix uteri are characterized by an increased anaerobic catabolism of glucose as energy source [START_REF] Warburg | On respiratory impairment in cancer cells[END_REF]. Furthermore, glycolytic intermediates are used as precursors for synthetic processes, debranching from glycolysis such as nucleic acid, phospholipid and amino acid synthesis [5; 6].

Although the glycolytic phenotype seems to be disadvantageous, yielding 18 times less ATP than oxidative phosphorylation [START_REF] Eigenbrodt | Glycolysis -one of the keys to cancer?[END_REF], its maintenance in primary and metastatic malignancies has a quite obvious selective benefit: cells, which shift to anaerobic glycolysis not only survive the hypoxic microenvironment, but also select for a phenotype resistant to acid-induced toxicity, caused by secretion of lactic acid during incomplete glucose metabolism [START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF]. Likewise, chronic acidification can even promote invasion of formerly premalignant cells since adjacent normal populations, which are sensitive to acidosis are destroyed [START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF]. Consequently, fully transformed cells have undergone genetic mutations to an extent that they have partly lost their flexibility to respond to changing conditions such as deprivation of glucose, which makes them susceptible to drugs interfering with energy metabolism [START_REF] Seyfried | Targeting energy metabolism in brain cancer: review and hypothesis[END_REF].

HPV-positive cells, derived from highly invasive cancers depend mainly on glycolysis for energy production [START_REF] Vrbacky | Inhibitory effects of Bcl-2 on mitochondrial respiration[END_REF]. In several studies including animal models, interference with glycolysis by particular inhibitors or glucose withdrawal disturbs the proliferation of tumour cells [9; 10]. In our previous study we have shown that the glucose availability as energy source is not only advantageous for an increased metabolism, but seems to be even indispensable to maintain HPV-18 oncogene transcription in cervical carcinoma cells. This was mainly deduced from the fact that the glucose analogue 2-deoxyglucose (2-DG) was able to suppress viral expression at the level of initiation of transcription. Moreover, 2-DG restored Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Copyright 2007 Biochemical Society the normal half-life of the tumour suppressor p53, in response to viral E6/E7 oncogene suppression [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF].

Besides a general inhibitory effect on protein glycosylation [START_REF] Kang | Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose[END_REF], 2-DG also reduces the intracellular ATP level and in turn increases AMP. Alteration of the intracellular ATP:AMP ratio as a consequence of nutrient starvation or hypoxia usually activates the AMP-activated protein kinase (AMPK), a metabolic stress-sensing protein, which attracted increasing attention in cancer research due to its central role in controlling energy consumption and cell proliferation [START_REF] Hardie | AMP-activated protein kinase: the energy charge hypothesis revisited[END_REF]. AMPK represents a multi-component enzyme complex, which switches off, once activated, many ATP-utilizing processes to sustain energy homeostasis [START_REF] Hardie | AMP-activated protein kinase: the energy charge hypothesis revisited[END_REF]. AMP binding allosterically activates AMPK, facilitating the binding of upstream kinases, which enhance its activity. 5-aminoimidazole-4-carboxamide-1-β-Dribofuranoside (AICAR), an adenosine analogue has been shown to mimic AMP once phosphorylated to ZMP by adenosine kinase [START_REF] Sabina | 5-Amino-4imidazolecarboxamide riboside (Z-riboside) metabolism in eukaryotic cells[END_REF]. Its effect on survival of cells depends strongly on cell type and concentration used and varies from induction of growth arrest, prolonged survival, induction of senescence and apoptosis [START_REF] Luo | AMPK, the metabolic syndrome and cancer[END_REF]. Moreover, AICAR can even induce apoptosis without AMPK activation [START_REF] Lopez | 5-Aminoimidazole-4carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved[END_REF], which is evidently causally linked with the absence of LKB1, a serine/threonine tumour suppressor kinase, which normally phosphorylates AMPK [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF].

In the present report we describe the effects of cellular metabolic stress on HPVpositive cervical carcinoma cells. We show that both viral transcription and cellular growth were strongly affected by AICAR treatment. Notably, the final outcome of the biological response upon AICAR application was determined by the AMPK upstream kinase LKB1 whose expression protects non-malignant HPV-positive cells from apoptosis, while LKB1 negative cells were eliminated. This may open new strategies in the treatment of cervical cancer, using LKB1 expression as predictor for the therapeutic success.

MATERIAL AND METHODS

Cell lines and somatic cell hybrids. The cervical carcinoma cell lines HeLa, SiHa, CaSki, SW756, C33a, the non-tumourigenic somatic cell hybrid (referred to as "444") of HeLa and primary human lung fibroblasts, its tumourigenic segregant (referred to as "CGL3") [START_REF] Stanbridge | Genetic analysis of human malignancy using somatic cell hybrids and monochromosome transfer[END_REF], β-"444" , the lung carcinoma cell line H1299 and the chronic myelogenous leukaemia cell line K562 were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma, München, [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF] (Invitrogen, Karlsruhe, Germany). β-"444" cells were generated by stable transfection of "444" cells with a β-actin promoter driven E6/E7 transcription cassette, representing a viralcellular chimeric cDNA isolated from SW756 cells. AICAR, 2-DG and uridine (all from Sigma, München, Germany) were dissolved in sterile water. Usually, 2 x 10 4 cells per cm 2 of the respective cell lines were seeded, and left 24 h before treatment.

RNA extraction and Northern Blot. Cytoplasmic RNA was extracted according to the guanidinium-thiocyanate method or using RNeasy RNA purification kit (Qiagen, Hilden, Germany). 5 µg of total cellular RNA were separated on 1% agarose gels in the presence of ethidium bromide under non-denaturating conditions and transferred to nylon membranes (GeneScreen, NEN Life Science, Köln, Germany). The filters were subsequently hybridized under stringent conditions with specific 32 P-labeled DNA probes [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF].

DNA hybridization probes. pHPV-16/18 represent the unit-length genome of HPV-16/18 DNA cloned in pBR322. pHF-βA1 harbours an approximately full-length insert of the fibroblast β-actin gene. All probes were labelled by random-priming in the presence of 32 P-dCTP [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF].

Reverse Transcription and PCR. For reverse transcription, 2 µg of DNase purified RNA was mixed with 0.2 µg of random primers (Roche, Mannheim, Germany), heated at 70°C for 10 min, and chilled on ice. After the annealing, the mixture was supplemented with reaction buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 ), 10 mM dithiothreitol, 500 µM deoxynucleoside triphosphate mixture (Invitrogen, Karlsruhe, Germany) and incubated at 25°C for 10 min. 100 Units of reverse transcriptase SuperScript II (Invitrogen, Karlsruhe, Germany) was added, the reaction was incubated at 42°C for 50 min, heated to 70°C for 15 Taq polymerase (Sigma, München, Germany) and 2 µl of reverse-transcribed product. The amplification was performed in an MJ Research PTC-200 thermal cycler in a total volume of 50 µl. The following primers and conditions were used: p21: 5'-ATGTCAGAACCGGCTGGGGATG-3´ and 5'-TTAGGGCTTCCTCTTGGAGAAG-3´ located in exon 3 and 4 (product length: 494), 4 min 94°C, 28 cycles of 30 sec at 94°C, 45 sec at 55°C, and 30 sec at 72°C, final extension 10 min at 72°C; p53: 5'-CTGAGGTTGGCTCTGACTGTACCACCATCC and 5'-CTCATTCAGCTCTCGGAACAT-CTCGAAGCG-3´, located in exons 7 and 10 (product length 371), 4 min 94°C, 30 cycles of 30 sec at 94°C, 45 sec at 57°C, and 30 sec at 72°C, final extension 10 min at 72°C; LKB1: located in exons 9 and 10 (product length 314), 5'-ATGGAGTACTGCGTGTGTGG -3' and 5´-CCAGATGTCCACCTTGAAGC-3', 4 min 94°C, 35 cycles of 30 sec at 94°C, 45 sec at 58°C, and 1 min at 72°C, final extension 10 min at 72°C; GAPDH (internal control):5'-TGGATATTGTTGCCATCAATGACC-3' and 5'-GATGGCATGGACTGTGGTCATG-3' , 4 min 94°C, 25 cycles of 30 sec at 94°C, 45 sec at 65°C, and 30 sec at 72°C, final extension 10 min at 72°C. The PCR products were analyzed in 2% agarose gels containing ethidium bromide.

SDS-PAGE and Western blotting.

Total cellular protein extracts were prepared using RIPA buffer (0.5% deoxycholate, 0.5% NP-40, 0.5% SDS, 50 mM Tris pH 7.4 and 100 mM NaCl) supplemented with serine-protease inhibitor phenyl-methyl-sulfonyl-fluoride (PMSF) to a final concentration of 10 µg/ml. Cells were sonified using a Sonifier 250 (Branson Ultrasonics, Geneva, Switzerland) and after removing the cell debris by centrifugation, the supernatant was immediately frozen and kept at -80°C. Nuclear and cytosolic extracts were prepared according to the method of Schreiber et al. [START_REF] Schreiber | Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells[END_REF], adding the protease inhibitors N-N- MAb (p-ACC, Cell Signalling, Frankfurt, Germany). The incubation was carried out overnight at 4°C in Tris-buffered saline (TBS) supplemented with 5% skim milk powder (Sigma, München, Germany), 0.05% Tween 20 (Sigma, München, Germany) and the diluted antibody. The bands were visualized with anti-mouse IgG antibodies conjugated with horseradish peroxidase using the ECL detection system (Amersham-Pharmacia, Freiburg, Germany). For reincubation with additional antibodies, the filters were stripped with 200 mM NaOH for 5 min at room temperature. Equal protein transfer and loading was controlled by reincubation with an actin specific antibody.

Electrophoretic mobility shift assays (EMSA).

Oligonucleotides for an HPV-18-specific AP-1-binding site 5 -CGCACCTGGTATTAGTCATTTTCC-3 within the enhancer region, position 7596-7620, the AP-1 consensus sequence 5'-CGCTTGATGACTCAGCCGGAA-3' from the human collagenase promoter [START_REF] Lee | Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements[END_REF] and Oct-1 consensus sequences 5'-TGTCGAATGCAAATCACTAGAA-3' derived from the immunoglobulin light chain enhancer [START_REF] Scheidereit | A human lymphoid-specific transcription factor that activates immunoglobulin genes is a homoeobox protein[END_REF] were generated in an Applied Biosystems (Foster City, CA, USA) synthesizer and purified by high performance liquid chromatography. The annealed oligonucleotides were labeled with 3000 Ci/mmol [ - 32 P]ATP (Amersham Biosciences, Freiburg, Germany) and T4 polynucleotide kinase (New England Biolabs, Frankfurt a.M., Germany) and gel-purified.

Binding reactions were performed in 20 µl containing 10% glycerol, 12 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 60 mM KCl, 1mM EDTA, dithiothreitol, 0.6 mg/ml bovine serum albumin, 2 µg of poly(dI-dC), and 2 µg of nuclear extract. After 5 min, 10000 cpm of the probe was added and incubation was continued for 30 min at room temperature. For super shifts, 2 µg of either c-Jun (sc-822X), Fra-1 (sc-183X) or c-Fos (sc-52X) are added and incubated for one hour (all antibodies for supershift: Santa Cruz, Heidelberg, Germany). The complexes were resolved with 5.5% non-denaturing polyacrylamide gels (29:1 cross-linking ratio). The gels were dried and exposed to x-ray films (Amersham-Pharmacia, Freiburg,

Germany)

Transfection of a HPV-18 URR reporter construct. To circumvent corrections for transfection efficiencies, a luciferase reporter construct under the control of the complete HPV-18 URR was transfected on 1x10 6 HeLa cells in a semistable fashion. In detail, cells were seeded in 6 cm culture dishes for 24 h. The next day, cells were transfected using Effectene reagent (Qiagen, Hilden, Germany) containing 2 µg of the HPV-18 URR-Luc Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Copyright 2007 Biochemical Society plasmid and 0.2 µg of a plasmid carrying a dominant selection marker for neomycin resistance. The cells were first incubated in the absence of the selective antibiotic G-418 (Geneticin). 24 h post transfection, medium was changed and supplemented with 1 mg/ml G-418. The outgrowing clones were pooled und used for luciferase assay. H1299 cells semistable transfected with HPV-18 URR-Luciferase construct were kindly provide by Daniela Holland (DKFZ, Heidelberg-Germany) and served as a control for HPV-negative cells. All experiments were performed by triplicate. For the luciferase assay, 6 well plates were seeded with 5 x 10 5 cells. The next day, 3 wells were supplemented with 4mM AICAR without changing the medium and the remaining 3 wells were used as controls. 24 h after treatment, cells were collected and lysed in passive lysis buffer (Promega, Mannheim, Germany) and luciferase activity was evaluated in a luminometer (Berthold, Bundoora, Australia) using Luciferase Assay Reagent II (Promega, Mannheim, Germany). siRNA transfection. For knock-down experiments, 75 nM SMARTpool siRNA against LKB1 (Dharmacon, Chicago, USA) was transfected using HiPerFect (Qiagen, Hilden, Germany) according to the instruction of the manual. siRNA directed against the luciferase gene served as negative control. 2 x 10 6 cells were transfected and seeded in 10 cm plates.

The next day, cells were split 1:4 and cultured for additional 24 h. To control the efficiency of the knock-down, cells were collected for RNA preparation using RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. RT-PCR was performed to monitor LKB1 expression.

DNA staining, flow cytometry and quantification of apoptosis. Cells including

supernatants were harvested by trypsination, washed twice with phosphate-buffered saline (PBS) and fixed overnight with 70% ethanol. After fixation and centrifugation, the cell pellets were resuspended in DNA staining solution containing 5 µM DAPI for DNA and 5 µM SR 101 as a protein counter stain following the protocol published by Stoehr et al. [START_REF] Stoehr | Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses[END_REF].

Processing and cell cycle analysis were performed according to Dean and Jett [START_REF] Dean | Mathematical analysis of DNA distributions derived from flow microfluorometry[END_REF] with the

RESULTS

Selective down-regulation of HPV transcription in cervical carcinoma cell lines. 2-DG

treatment was reported to reduce selectively the transcription of viral mRNA encoding HPV-18 specific oncogenes E6/E7 in HeLa cells concomitant with a decrease in intracellular ATP levels [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF]. As subsequently shown, also other conditions of energy deprivation such as hypoxia and low glucose levels have the same effect on HPV gene expression [START_REF] Kang | Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose[END_REF]. 2-DG is however only effective as an ATP depleting agent in high dosage, since glucose and DG compete for uptake [START_REF] Vrbacky | Inhibitory effects of Bcl-2 on mitochondrial respiration[END_REF]. To mimic a decreased ATP:AMP ratio at a constant ATP level, cells were treated with AICAR at different concentrations. AICAR is a cell-permeable drug and its phosphorylated form ZMP structurally resembles AMP, a known activator the AMP-activated kinase (AMPK) [START_REF] Hardie | AMP-activated protein kinase: the energy charge hypothesis revisited[END_REF].

As depicted in Figure 1A, 0.5 mM of AICAR was sufficient to down-regulate E6/E7 viral transcription in HPV-18 positive cervical carcinoma HeLa cells in a time-dependent manner. Strongest reduction was detected with 4 mM AICAR after 24 h treatment. HPV-18 suppression was selective since other reference genes such as the housekeeping gene β-actin were not affected. To demonstrate that the effect of AICAR was not merely a cell line specific phenomenon, additional HPV-16/18 positive cells were examined. CaSki cells contain approximately 500 both full length and truncated copies of HPV 16 integrated at different chromosomal locations, SW756 cells harbour multiple truncated copies of HPV-18 located at a single integration site [START_REF] Mincheva | Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization[END_REF]. As demonstrated in Figure 1B, both cell lines also showed a strong decrease in HPV expression, clearly indicating that AICAR treatment negatively regulates viral transcription independently of the HPV type, the copy number or the viral integration locus (Figure 1B).

Consistent with the down-regulation of HPV-18 seen on RNA level (Figure 1A), 0.5 mM AICAR treatment also leads to a selective and time-dependent reduction of the E7 oncoprotein (Figure 2). Due to the unavailability of appropriate antibodies, we could only indirectly measure the fate of the E6 oncoprotein. Figure 2 shows that in the same way as E7 and viral transcription was decreased (Figure 1A), p53 was stabilised compared to untreated control cells. Cells cultured in the presence of AICAR showed a strong decrease in UTP and CTP levels as a result of 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) depletion, an important precursor for pyrimidine nucleotide synthesis [START_REF] Thomas | Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation[END_REF]. To overcome pyrimidine depletion and to demonstrate that the metabolism of the treated cells was not irreversibly disturbed, HeLa cells were cultured with 0.5 mM AICAR in the presence of 20 µM uridine. Under these conditions, the inhibitory effect on oncogene expression and stabilisation of p53 Copyright 2007 Biochemical Society could be completely prevented (Figure 2). Conversely, at higher AICAR concentrations uridine failed to inhibit the transcriptional attenuation of HPV expression even when uridine was supplemented in concentrations up to 1 mM (data not shown).

The viral control region determines down regulation of HPV transcription. Decrease in oncogene mRNA levels can be either due to a labilization and shortening of the cytoplasmic mRNA half-life or by direct suppression of viral upstream regulatory region (URR)-directed transcriptional activity. To investigate the effect of AICAR treatment on the HPV-URR, we established a clonal subline derived from HeLa cells fused with normal human fibroblasts, which express an additional E6/E7 cDNA transcription cassette under the control of the βactin promoter [START_REF] Bachmann | Disturbance of tumor necrosis factor alpha-mediated beta interferon signaling in cervical carcinoma cells[END_REF]. As depicted in Figure 3A, incubation with 0.5 mM AICAR only resulted in a suppression of the URR-directed E6/E7 transcription (referred to as "endo" for endogenous HPV-18 expression), while the β-actin driven viral cDNA (referred to as "exo") was not affected. Uridine supplementation was able to abrogate the AICAR effect indicating that down-regulation of viral gene expression was regulated at the level of initiation of transcription rather than by post-transcriptional mechanisms. Additionally, to exclude any positional effect due to integration and to unequivocally demonstrate that the URR was indeed a target of the negative regulation upon AICAR treatment, HeLa cells were transfected with a HPV-18-URR luciferase reporter plasmid. H1299 cells, which contain the same reporter construct were used to monitor the effect of AICAR on the HPV-18 URR-directed transcription in HPV-negative cells. As shown in Figure 3B, in both cell lines, 4 mM AICAR treatment lead to a 6-10 times reduced luciferase activity, clearly indicating that the HPV-18-URR was target of the negative regulatory process induced by AICAR.

Selective reduction of AP-1 after AICAR treatment. HPV transcriptional activity is

strongly determined by a certain set of host specific transcription factors, which bind to their cognate cis-regulatory recognition sites within the URR [START_REF] Butz | Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region[END_REF]. In the case of 2-DG it was shown that the trans-activating potential of the transcription-factor Sp1 was diminished by an increase in O-linked glycosylation [START_REF] Kang | Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose[END_REF]. Since the β-actin promoter contains numerous Sp1like sequences [START_REF] Quitschke | The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor[END_REF], but was not affected upon AICAR incubation (Figure 3), we focused our attention on the transcription factor AP-1, which is known to play a central role in the transcriptional regulation of almost all HPV types investigated so far [START_REF] Rösl | Regulation of E6 and E7 oncogene transcription[END_REF].

To test this notion, we examined the AP-1 binding by electrophoretic mobility shift assays (EMSAs). Incubating of nuclear extracts obtained from cells treated with different Copyright 2007 Biochemical Society concentrations of AICAR with 32 P-labeled AP-1 oligonucleotides, a strong reduction of AP-1 binding could be discerned (Figure 4A). Super-shift analysis confirmed that c-Jun is the main dimerisation partner of the AP-1 complex of HeLa (Figure 4B). Monitoring the c-Jun steady state level by Westernblot, however, no quantitative reduction was detectable under conditions of low AP-1 binding (Figure 4C). These data suggest that decreased AP-1 affinity in EMSA might be the result of a post-translational modification of c-Jun, probably interfering with its affinity to DNA. To demonstrate that suppression of AP-1 binding was a selective process, EMSAs with 32 P-labeled Oct-1 specific oligonucleotides were carried out.

In fact, under conditions where AP-1 binding was reduced (Figure 4A), both Oct-1 and Sp1 binding (data not shown) were not affected. These data clearly demonstrate that an unbalanced energy metabolism triggers a decrease of AP-1, but apparently did not impair the binding of transcription factors in general.

The effect of AICAR on the cell cycle and p53/p21 CIP1 expression. Since viral expression is necessary to maintain a proliferative phenotype of cervical carcinoma cells [30], we examined the fate of HeLa cells at different time points after seeding in the presence of increasing concentrations of AICAR. As shown in Figure 5A, 0.5 mM AICAR completely blocked cellular growth, while at higher amounts of AICAR, the number of cells were even diminished. In fact, as revealed by phase contrast microscopy (Figure 5B), addition of 4 mM AICAR for 24 hours led to the appearance of typical apoptotic figures indicated by condensation of the cytoplasm and nuclear shrinkage (karyorhexis). In order to analyse at which phase of the cell cycle the cells became growth arrested and to quantify the extend of apoptosis, flow-cytometric analyses were performed. As depicted in Figure 5B, lower concentrations of AICAR blocked cells within S-phase, which in turn resulted in a reduced number of cells in G2/M. At 4 mM AICAR, a substantial fraction of cells (approximately 27%) accumulated in a sub-G1 position, confirming that apoptosis was taking place. At 2 mM AICAR, S-phase arrest was partially released and cell morphology changed similar to cells treated with 4 mM but without the appearance of apoptotic figures. In contrast, the HPV negative cervical carcinoma cell line C33a did not undergo apoptosis, but a reduced growth rate was observed (see supplementary data).

To get insight in the dosage effect, we monitored the expression of p53 and p21 CIP1 at different AICAR concentrations. As shown in Figure 2 Copyright 2007 Biochemical Society consequence of its reconstituted half-life due to viral oncogene suppression (Figure 1) rather than by a transcriptional effect. One major target gene involved in cell cycle control is p21 CIP1 , an inhibitor of cyclin-dependent kinases [START_REF] Imamura | Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line[END_REF]. Intriguingly, only when cellular growth arrest was accomplished, p21 CIP1 was up-regulated, both on RNA and on protein level (Figure 6). In contrast, under conditions of apoptosis, p21 CIP1 was not induced transcriptionally despite increased p53 protein levels. Moreover, Western blot analysis revealed that p21 CIP1 was even not expressed, indicating that under these conditions p21 CIP1 induction was not necessary (Figure 6B). These findings suggest that growth of HeLa can be efficiently inhibited by lower concentration of AICAR, while increasing concentrations of AICAR triggered apoptosis.

Expression of LKB1 and its role in induction of apoptosis. As already shown for cells

without any HPV aetiology, AICAR also induces p53 and p21 accumulation [START_REF] Imamura | Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line[END_REF]. Apparently, p53 is a direct target of activated AMPK [START_REF] Jones | AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[END_REF], providing a novel link between cell cycle control and metabolic regulation. AMPK in turn is activated by AMPK upstream kinases (AMPKK), such as LKB1 [START_REF] Kyriakis | At the crossroads: AMP-activated kinase and the LKB1 tumor suppressor link cell proliferation to metabolic regulation[END_REF]. To examine the expression of LKB1 in the context of HPVinduced carcinogenesis, we analysed the presence of the corresponding mRNA in different cervical carcinoma cells (Figure 7A). Consistent with previous results [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF], HeLa cells lacked detectable LKB1 expression, while other cervical carcinoma cell lines, such as CaSki, SW756 and C33a, respectively, were found to be positive for the corresponding mRNA. Exceptions were the HPV-16 positive cervical carcinoma cell line SiHa and the tumourigenic segregant (referred to as "CGL3") of the former non-tumourigenic somatic cell hybrid made of HeLa and primary human fibroblasts ("444"), where the conversion to tumourigenicity was obviously accompanied by a loss of LKB1 expression. The chronic myelogenous leukaemia cell line K562, known to be LKB1 positive [START_REF] Tiainen | Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest[END_REF] was used as a positive control for the RT-PCR reaction (Figure 7A).

As predicted recently, the absence of LKB1 and therefore the lack of AMPK activation committed cells to undergo apoptosis when they were challenged with drugs signalling low cellular energy conditions [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF]. To confirm this notion, various cell lines were treated with 4 mM AICAR and programmed cell death was monitored. In fact, as depicted in Figure 7B, all cells, which lack detectable LKB1 expression showed the appearance of the PARP cleavage after AICAR treatment, characteristic for ongoing apoptosis (see also Figure 8A for HeLa cells). This suggests that LKB1-deficient cervical carcinoma cells undergo apoptosis under conditions that mimic an elevated AMP:ATP ratio. Copyright 2007 Biochemical Society AMPK functionality and apoptosis. In order to elucidate the activity of AMPK, phosphorylation of the downstream target acetyl-CoA-carboxylase (ACC), an enzyme involved in the fatty acid synthesis pathway, was monitored [START_REF] Luo | AMPK, the metabolic syndrome and cancer[END_REF]. For this purpose, we chose LKB1-positive "444" cells and LKB1-negative parental HeLa cells for comparison. As demonstrated in Figure 8A, only "444" cells showed increased ACC phosphorylation, which is consistent with the presence of LKB1 expression and an activation of AMPK upon 4 mM AICAR treatment. In contrast, HeLa cells were completely refractory under the same experimental conditions, while apoptosis, as indicated by a strong PARP cleavage, was induced. To show that there still exist a redundant non-LKB1 dependent pathway activating AMPK, HeLa cells were treated with 10 µM oligomycin, an inhibitor of mitochondrial respiration that depletes, like 2-DG, the intracellular ATP level [START_REF] Hurley | The Ca2+/calmodulin-dependent protein kinase kinases are AMPactivated protein kinase kinases[END_REF]. Indeed, oligomycin activated AMPK and also led to a cleavage of PARP, while AICAR-mediated apoptosis seemed to be entirely independent from AMPK activity. Since LKB1 signalling is involved in the cellular response to low energy, we hypothesised that the presence of LKB1 should protect cells from apoptosis induced by drugs that further elevate intracellular AMP.

Taking advantage of our somatic cell hybrid model system, metabolic stress such as the application of 2-DG can be investigated both in non-malignant cells expressing LKB1 and in tumourigenic segregants derived from the very same hybrid ("CGL3"), but lacking LKB1 (see Figure 7A). As predicted, 2-DG was inducing apoptosis only in LKB1-negative cells (HeLa and "CGL3"), while "444" cells were resistant (Figure 8B).

Knock-down of LKB1 transcription by siRNA.

To study the fate of AICAR treated cells after knocking-down LKB1 expression, both C33a (data not shown) and "444" hybrids were transfected with LKB1 siRNA in comparison with a siRNA directed against the luciferase gene (siLuc) as non-specific control. As shown in Figure 9A

DISCUSSION

A hallmark of many highly invasive tumours, including cervical cancer, is their high glycolytic rate, strongly supporting Warburg´s dictum of a relationship between a glycolytic shift and malignant progression. Furthermore, a glycolytic phenotype, which guarantees constant energy supply even when oxygen levels decreased is evidently also an essential prerequisite to maintain efficient transcriptional activity of the viral oncoproteins E6 and E7 [START_REF] Kang | Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose[END_REF]. Here, not only 2-DG and limited glucose availability selectively reduce HPV expression in cervical carcinoma cells [11; 12; 36], but also AICAR (Figure 1), a known activator of AMPK, which simulates low energy condition without ATP depletion. The suppressive effect was not limited to HPV-18 positive HeLa cells, but could also be visualized in other cell lines (Figure 1), differing in their HPV type, their viral copy number and viral chromosomal localization (Figure 1B). Since both E6 and E7 transcription was down-regulated by AICAR treatment, p53 stabilisation can be explained by reduced expression of E6 (Figure 2).

Remarkably, there seems to be an evolutionary well-designed circuit between sustained HPV transcription and oncoprotein functionality, since E7 itself is capable to reprogram the metabolism of its host cell. E7 physically interacts with the M2 pyruvate kinase (M2-PK) [START_REF] Zwerschke | Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein[END_REF] and the acid α-glucosidase [START_REF] Zwerschke | Allosteric activation of acid alphaglucosidase by the human papillomavirus E7 protein[END_REF], both key regulatory enzymes involved in the intracellular accumulation of glycolytic phosphometabolites and glycogen breakdown [START_REF] Maehama | Selective down-regulation of human papillomavirus transcription by 2deoxyglucose[END_REF].

Binding of E7 to M2-PK, for instance, changes its catalytic property, which finally leads to an increase of nucleotide precursors required for DNA synthesis and cell growth [START_REF] Zwerschke | Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein[END_REF].

Accordingly, for a successful virus-host interaction it is reasonable to assume that viral transcription, which continuously needs a high metabolic state for keeping up cell proliferation, is down-regulated by a negative feedback loop when the host cell is sensing low energy conditions.

As depicted in Figure 3A, we could show that AICAR exclusively affected URRdirected gene expression, while the transcriptional activity of a β-actin-driven E6/E7 transcription cassette, which contains a 3´-cellular poly-adenylation side from SW756 cells remained unchanged. The same was true, when HPV18-URR driven luciferase reporter constructs were transfected (Figure 3B), clearly supporting the notion that HPV is suppressed at the level of initiation of transcription rather than post-transcriptionally. Recently, it has been demonstrated that 2-DG, hypoglycaemia and hypoxia can affect the binding affinity of the transcription factor Sp1 by changing its glycosylation profile, which is added in a posttranslational manner [START_REF] Kang | Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose[END_REF]. In our experiments AICAR treatment, however, had no effect on Sp1 (data not shown), but a decreased binding affinity of the transcription factor AP-1 could Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Copyright 2007 Biochemical Society be discerned (Figure 4A). In agreement with our finding, evidence has been provided that AICAR also interferes with AP-1 binding in other systems [START_REF] Weigert | AP-1 proteins mediate hyperglycemia-induced activation of the human TGFbeta1 promoter in mesangial cells[END_REF], suggesting a general functional relationship between AP-1 activity and the metabolic state of the cell.

Actually AP-1 is not only involved in various cellular regulatory processes [START_REF] Eferl | AP-1: a double-edged sword in tumorigenesis[END_REF], but also in the maintenance of HPV expression [START_REF] Rösl | Regulation of E6 and E7 oncogene transcription[END_REF]. This notion has been confirmed by sitedirected mutagenesis of the AP-1 sites within the viral enhancer/ promoter region, where in contrast to other cis-regulatory sequences, URR-driven transcription was almost completely abolished [START_REF] Butz | Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region[END_REF]. The finding that Oct-1 binding was not reduced under these conditions clearly demonstrated the selectivity of this process. Decreased AP-1 binding in the EMSA, however, could not be attributed to a reduction of c-Jun, the major dimerisation partner of the Junfamily members in cervical carcinoma cells (Figure 4B) [START_REF] Soto | Conversion of HPV 18 positive non-tumourigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-alpha mediated repression of viral transcription and modification of the AP-1 transcription complex[END_REF], since Western blot analysis of nuclear extracts revealed that similar expression levels were preserved (Figure 4C). In respect of functionality it should be stressed that AICAR also causes activation of the glycogen synthase kinase-3 (GSK3) [START_REF] King | AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3[END_REF], which phosphorylates c-Jun at its C-terminal DNA binding domain thereby preventing DNA binding [START_REF] Boyle | Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity[END_REF]. This may explain the reduced AP-1 signals in the EMSA (Figure 4A) despite unchanged amounts of c-Jun, even after longer AICAR treatment (Figure 4C). Note that GSK3 inhibitor lithium chloride (LiCl) [START_REF] Stambolic | Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells[END_REF] was not relieving the transcriptional block on HPV-18 transcription (data not shown). How AICAR is definitively diminishing AP-1 binding still remains to be elucidated. Although all tested AICAR concentrations reduced HPV-18 transcription in HeLa cells to a similar extent (Figure 1), the effects on proliferation and survival differed significantly (Figure 5A). While 0.5 mM AICAR caused accumulation of cells in S-phase with a depletion of the G2/M fraction, addition of 4 mM AICAR for 24 hours led to the induction of apoptosis, which was indicated by the appearance of the characteristic sub-G1 peak after flow cytometric measurement (Figure 5B). Growth arrest at lower and moderate AICAR concentrations was in line with a post-translational increase of p53 and the induction of its major target gene p21 CIP1 , both on RNA and on protein level (Figure 6). Elevation of p53 and inhibition of oncogene expression could be completely prevented when 0.5 mM AICAR was applied in combination with 20 µM uridine, pointing to an inhibition of phosphoribosyl-pyrophosphate-synthetase and nucleic acid synthesis as a further target of AICAR (Figure 2) [START_REF] Thomas | Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation[END_REF]. p53 is a central protein in cell cycle control that signals either cessation of growth or induction of apoptosis, depending on the severity of cellular damage [START_REF] Jones | AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[END_REF].

Moreover, its activity is also regulated by AMPK, demonstrating that p53 is part of a metabolic checkpoint, which determines the outcome of the biological response after energy Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Copyright 2007 Biochemical Society depletion [START_REF] Jones | AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[END_REF]. In HepG2 cells, induction of p53 and p21 CIP1 after AICAR treatment has been also described [START_REF] Imamura | Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line[END_REF]. However, p53 was induced on transcriptional level, whereas in our case p53 was stabilised as a consequence of viral oncogene suppression (Figure 1 and2). Note that under conditions when cells underwent apoptosis, p21 CIP1 mRNA was not induced and the corresponding protein was not detectable (Figure 6A). This is consistent with previous reports, suggesting that p21 CIP1 has anti-apoptotic activity [START_REF] Chan | Cooperative effects of genes controlling the G(2)/M checkpoint[END_REF] and can be cleaved by a caspase-like mechanism [START_REF] Munoz-Pinedo | Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing[END_REF]. Despite the fact that AICAR is considered to be a specific inducer of AMPK [START_REF] Hardie | AMP-activated protein kinase: the energy charge hypothesis revisited[END_REF], no phosphorylation of its downstream target ACC [START_REF] Luo | AMPK, the metabolic syndrome and cancer[END_REF] can be detected in HeLa cells (Figure 8A). This correlates with the absence of the AMPK upstream kinase LKB1 [START_REF] King | AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3[END_REF] (see also Figure 7), a putative tumour suppressor gene, first described to be mutated in the rare inherited disease, the Peutz-Jeghers-syndrome [START_REF] Alessi | LKB1-Dependent Signaling Pathways[END_REF]. Monitoring the presence of LKB1 mRNA in other cervical carcinoma cells, the picture was not uniform (Figure 7A), suggesting that loss of LKB1 expression is not a general feature of HPV-induced carcinogenesis. There is strong evidence that not AMPK itself but one of the upstream kinases (either LKB1 or the Calmodulin-dependent kinase) determine the biological outcome after AICAR treatment [17; 35]. LKB1 deficient mouse embryonic fibroblasts (MEF), for example, undergo extensive apoptosis, but not wild type cells or their heterozygous controls [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF]. In HeLa cells, LKB1 seems to be not transcribed due to promotor de novo methylation [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF], but is reconstituted upon somatic cell hybridization with normal human fibroblasts ("444"). Tumourigenic segregants derived from the same hybrids ("CGL3") have lost LKB1 expression, suggesting that progression to malignancy is paralleled by LKB1 down-regulation. Remarkably, similar to the aforementioned situation, only cells lacking LKB1 underwent cell death, while others were found to be resistant (Figure 7B and8A). According to the model presented by Shaw et al. [START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMPactivated kinase and regulates apoptosis in response to energy stress[END_REF], lack of LKB1 expression should also sensitise cell to apoptosis when the ATP:AMP ratio is altered. In fact, 2-DG, which activates AMPK via the Calmodulin-dependent kinase [START_REF] Hurley | The Ca2+/calmodulin-dependent protein kinase kinases are AMPactivated protein kinase kinases[END_REF] only induced apoptosis in LKB1-negative HeLa cells and "CGL3" hybrids, but not in their non-malignant, LKB1-positive counterparts (Figure 8B), clearly confirming the relationship between AICAR induced cytotoxicity and the absence of LKB1 expression. However, apoptosis (Figure 7 Copyright 2007 Biochemical Society LKB1 expression during the carcinogenic process (e.g. HeLa, CGL3), a short-term knockdown of LKB1 followed by AICAR treatment lead apparently to a different biological outcome. LKB1 deficient cells underwent apoptosis (Figure 5B and 8B), whereas siRNA mediated knock-down lead to necrotic cell death. Since the activation of AMPK by AICAR in "444" cells (Figure 8A) did correlate with survival, it can be concluded that by short-term LKB1 suppression the cells can not counteract the AICAR induced metabolic stress, resulting in extensive cytotoxicity (Figure 9B).

The susceptibility of cells lacking LKB1 to undergo cell death raises the question whether interference with energy metabolism might be an additional option to eliminate chemoresistant cancer cells by a combinatorial therapy. Several strategies combining ATP depletion and anticancer agents have been tested in vitro and in vivo. Inhibition of glycolysis by either glucose withdrawal or 2-DG application enhanced radiosensitisation [START_REF] Lin | 2-Deoxy-D-glucose-induced cytotoxicity and radiosensitization in tumor cells is mediated via disruptions in thiol metabolism[END_REF], but also death ligand induced apoptosis [START_REF] Munoz-Pinedo | Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing[END_REF]. Furthermore, the combination of the widely used 5-FU with AICAR induced cell death in 5-FU resistant cell lines [START_REF] Hwang | Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways[END_REF]. Preliminary results from our laboratory are in line with these results showing a synergistic effect of AICAR treatment and CD95-ligand addition in the induction of apoptosis. This may open new strategies in the treatment of cervical cancer, using LKB1 expression as predictor for therapeutic success.

Figure legends

  min, and refrigerated at -20°C until used. PCRs were performed in a solution containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 200 µM deoxynucleoside triphosphate mix (Invitrogen, Karlsruhe, Germany ), 40 pM of upstream and downstream primers, 1 Unit of Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society

(L- 3 -

 3 trans-carboxyoxirane-2-carbonyl)-L-leucyl-agmatine (E64) and 4-(2-aminoethyl)benzolsulfonylflouride (''Pefabloc SC'') in concentrations suggested by the manufacturer (Roche, Mannheim, Germany). Concentrations of RIPA and nuclear extracts were determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, München, Germany). 20 µg cellular and 50 µg total protein extracts were separated in 8-12% SDS PAGE, electrotransferred to Immobilon-P membranes (PVDF, Millipore, Bedford, USA) and probed with the following antibodies: human specific murine monoclonal antibody (MAb) directed against p53 (DO1, Santa Cruz, Heidelberg); p21 MAb (Anti Cip/Waf1, BD Transduction Laboratories, Heidelberg); poly (ADP-ribose) polymerase (PARP) MAb (Sc-8007, Santa Cruz, Heidelberg); actin MAb (clone 4, ICN Biomedicals, Meckenheim, Germany); human specific rabbit Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society antibodies c-Jun (H-79, Santa Cruz 1694, Heidelberg) and phospho-acetyl-CoA carboxylase

  cytofluorograph 30-L (Ortho, Raritan, NJ, USA). Cells were additionally analysed by fluorescence microscopy (Leica DMRD, Leitz, Wetzlar, Germany). Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society

  , treatment with AICAR resulted in a strong increase of the p53 protein. By RT-PCR, no differences at the p53 mRNA level could be revealed (Figure 6A, upper panel), indicating that the quantitative increase of p53 was the Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

  , LKB1 siRNA delivery resulted in an almost complete reduction of the corresponding mRNA, while siLuc had no effect. When cells were incubated with 4 mM AICAR for 24 hour, 100% of LKB1 knock-down cells were eradicated in contrast to siRNALuc transfected controls. To monitor the mode of cell death, incubation with AICAR was reduced to 8h, cells were stained with DAPI and SR 101 as a protein counter stain and analysed by fluorescence microscopy. As depicted in Figure 9B, LKB1 knock-down cells underwent extensive cell death (panel 10-12) in contrast to the siRNALuc controls, which showed no morphological changes (panel 4-6). Panel 12 revealed the presence of necrotic (loss of plasma integrity without morphological changes of the nuclei) rather then apoptotic cells, indicated by loss of cell integrity. This suggests that Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society AICAR or ATP-depleting agents may represent potential therapeutic drugs that selectively eradicate LKB1-deficient cervical carcinoma cells via apoptosis or necrosis. Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society

  and 8) is apparently not the only way to eliminate LKB1 negative cells. Knocking-down LKB1 via transient delivery of siRNA resulted in a complete eradication 24 hours after AICAR treatment. Morphological changes typical for apoptosis were absent and cells seemed to be eliminated via disruption of the cellular membrane and release of DNA (Figure 9, panel 12). In comparison to cells, which already acquired a loss of Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Figure 1 :

 1 Figure 1: Selective down-regulation of HPV-transcription in cervical carcinoma cells after treatment with AICAR. Northern blots of different cervical cancer cell lines treated with AICAR. (A) HPV-18 positive HeLa cells were incubated for the times indicated with different concentrations of AICAR. (B) RNA obtained from HPV 16-positive CaSki cells (left) and HPV 18-positive SW756 cells (right) after treatment with 0.5 mM AICAR for different periods of time. For Northern blot, 5 µg/lane of total RNA was separated under nondenaturing conditions in 1 % agarose gels. After transfer, the membranes were consecutively hybridized with HPV-16/-18 and β-actin specific DNA probes. The positions of the 28S and 18S rRNA in the ethidium stained agarose gels are indicated. ( -): untreated control.

Figure 2 :

 2 Figure 2: Reciprocal regulation of E7 and p53 after AICAR treatment -abrogation of the AICAR effect by uridine. HeLa cells were treated with 0.5 mM AICAR for different periods of time with and without supplementation of 20 µM uridine. Immunoblots were performed using antibodies raised against HPV-18 E7 and p53. Equal loading and protein transfer were confirmed by incubating the filter with an anti-actin-specific antibody. ( -): untreated control.

Figure 3 :

 3 Figure 3: Upstream regulatory region (URR)-mediated down-regulation of viral transcription after AICAR treatment. (A) "444" cells expressing an additional E6/E7 transcription cassette under the control of the β-actin promoter were treated with 0.5 mM AICAR with and without supplementation of 20 µM uridine for the times indicated. 5µg/lane of total RNA was separated under non-denaturing conditions in a 1 % agarose gel and the same filters were consecutively hybridised with HPV-18 and β-actin specific DNA probes. The positions of the 28S and 18S rRNA are indicated in the ethidium stained agarose gels. ( -): untreated control. (B) Luciferase reporter assay with HeLa and H1299 cells carrying the luciferase reporter construct under the control of HPV-18 URR. Semi-stably transfected cells were treated with 4 mM AICAR for 24 h and luciferase activity was compared with untreated controls. Ordinate: renilla luciferase units per µg total cellular protein (RLU/µg).

Figure 4 :

 4 Figure 4: Selective reduction of AP-1 binding after AICAR treatment. (A) Electromobility shift assay (EMSA): nuclear extracts obtained from treated and non-treated cells were incubated with 32 P-labelled oligonucleotides specific for AP-1 or Oct-1. The incubation

Figure 5 :

 5 Figure 5: Growth curves and cell cycle analysis after AICAR treatment. (A) Follow-up of the cellular growth of 1 x 10 4 HeLa cells 1-4 days in the presence of AICAR. Ordinate: number of cells (x 10 5 ), abscissa: different concentrations of AICAR. (B) Representative flow cytometric profiles and the corresponding morphology of HeLa cells after incubation with AICAR. The percentage of cells present in the different phases of the cell cycle (G1, S, G2/M) as well as the percentage of apoptotic cells (reflected by the sub-G1 peak) are given in the table below and represent the mean of three different experiments. Magnification of the phase-contrast micrographs: 200 x.

Figure 6 :

 6 Figure 6: Dosage effect of AICAR on p53 and p21 CIP1 expression. (A) RNAs of AICAR treated cells were reverse transcribed. RT-PCR products of p53, p21 CIP1 and GAPDH after separation in a 1% agarose gel. (Contr.): untreated cells. (B) Immunoblot of total protein extracts with antibodies against p53 and p21 CIP1 . Equal loading and protein transfer were confirmed by incubating the same filter with an anti-actin-specific antibody.

Figure 7 :

 7 Figure 7: LKB1 expression and apoptosis. (A) RNAs from different cervical carcinoma cell lines and derived somatic cell hybrids were reverse transcribed and the RT-PCR products of LKB1 and GAPDH were separated in a 1% agarose gel. (B) The indicated cell lines were treated for 24 h with 4 mM AICAR. For immunoblot, total protein extracts were incubated with antibodies against PARP and actin. The arrow indicates the PARP cleavage product.

Figure 8 :

 8 Figure 8: Absence of LKB1 activity in HeLa cells, but not in non-malignant somatic cell hybrids -selective induction of apoptosis by 2-deoxyglucose in LKB-1 negative cells. (A) HeLa and "444" cells were treated for 24 h with 0.5 and 4 mM AICAR or 10 µM oligomycin, respectively. Total protein extracts were used for Western blot analysis to monitor the phosphorylation of the AMPK downstream substrate ACC (P-ACC) and cleavage of PARP. Actin was again used as a loading and transfer control. (B) Indicated cell lines were treated with 45 mM 2-DG for different periods of time and total protein extracts were used for

Figure 1 Figure

 1 Figure 1

Figure 3 Figure 4 HPV18

 34 Figure 3

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053

ACKNOWLEDGEMENTS

We thank Daniela Holland for the H1299 transfected cells, Karin Butz for the Luciferase reporter plasmid (both DKFZ, Heidelberg, Germany), Eric Stanbridge (University of Irvine, Irvine CA, U.S.A.) for the hybrid cell lines, Monika Frank-Stoehr for cell cycle analysis, Rainer Schmidt and Thomas Hofmann (all DKFZ, Heidelberg) for helpful advices and discussions. This paper is dedicated to Erich Eigenbrodt who deceased in 2004. Biochemical Journal Immediate Publication. Published on 10 Jan 2007 as manuscript BJ20061053 Copyright 2007 Biochemical Society

immunoblots. Filters were incubated with antibodies against PARP and actin, the latter for loading control.


Alexander Arlt

Jörg Minkenberg

Marie-Luise Kruse

Frauke Grohmann

Ulrich R Fölsch

Ph.D Heiner Schäfer

Immediate early gene-X1 interferes with 26S proteasome activity by attenuating expression of the 19S proteasomal components Rpn10 and Rpn2

Keywords: regulated protein degradation, poly-ubiquitination, gene transcription, proteasome assembly, apoptosis AMC, amido-4-methylcumarin, IκB, Inhibitor of κB, IEX-

The stress response gene IEX-1 is involved in the regulation of cell growth and cellular viability. To some extent, these effects include an interference with the proteasomal turnover of certain regulatory proteins. Here, we show that IEX-1 directly attenuates the activity and formation of the 26S-proteasome in 293-cells. We further demonstrate that IEX-1 reduces the overall expression levels of certain protein components of the 19S-proteasomal subunit such as S5a/Rpn10 and S1/Rpn2, whereas the expression of other proteasomal proteins was less or not affected. In contrast to direct apoptotic stimuli, such as the anti-cancer drug etoposide, leading to caspase dependent degradation of Rpn10 and Rpn2, the effect of IEX-1 is independent of proteolytic cleavage of these proteins. Furthermore, the decreasing effect of IEX-1 on Rpn10 and Rpn2 expression is still seen in the presence of cycloheximide, but not in the presence of actinomycinD, and quantitative real time PCR revealed lower mRNA levels of Rpn10 and Rpn2 in IEX-1 overexpressing cells, suggesting an interference of IEX-1 with the gene transcription of Rpn10 and Rpn2. Additionally, luciferase assays confirmed an interference of IEX-1 with the activity of the Rpn10 promoter. These findings indicate a role of IEX-1 in the maintenance and assembly of the 26S-proteasome, obviously involving an altered gene expression of certain proteasomal proteins. Thereby, IEX-1 may essentially modulate signaling pathways related to 26S-proteasome activity and involved in cellular growth control and apoptosis.

Introduction

The ubiquitin-proteasome pathway plays a pivotal role in signal transduction [1,2]. By mediating the tightly regulated turnover of a great variety of regulatory proteins, including cyclins, kinases, phosphatases or transcription factors, the ubiquitin-proteasome pathway essentially contributes to the orchestration of multiple cellular processes such as differentiation, proliferation and apoptosis [1][2][3][4][5][6]. One hallmark of this pathway is the polyubiquitination of the substrate protein which targets it to the proteolytic degradation complex -the 26S proteasome. This high moleceular weight multiprotein-complex comprises a central catalytic part, the 20S proteasome, and two axially positioned regulatory parts, the 19S regulatory caps.

The 20S proteasome presents itself as a cylindrical structure composed of two pairs of ring shaped subunits each consisting of 7 non-identical α-(noncatalytic) and β-(catalytic) subunits, respectively, and the 19S subunit consists of about twenty nonidentical proteins exhibiting ATPase (Rpt proteins) and non-ATPase (Rpn proteins) activity [7,8]. It is meanwhile known that the synthesis of certain proteasomal proteins as well as the formation and maturation of the 20S and 19S subunits and the assembly of the 26S proteasome itself is also a tightly regulated process [9,[START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF]. In yeast, it has been shown that the expression of various proteasomal proteins is under control of a proteasome sensitive transcription factor, RPN4, that accounts for an adequate level of proteasome activity [START_REF] Xie | RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit[END_REF][START_REF] Ju | Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit[END_REF][START_REF] London | Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome[END_REF]. Any deregulation in proteasome activity leads to a loss in cellular homeostasis and to severe cellular dysfunctions.

Accordingly, an inadequate proteasome activity is involved in various pathological conditions including cancer, cachexia and neurodegenerative or autoimmune diseases [START_REF] Devo | The ubiquitin-proteasome system and cancer[END_REF][15][16][17][18][START_REF] Mountz | Significance of increased circulating proteasome in autoimmune disease[END_REF][20][START_REF] Hayashi | Implications of altered apoptosis in diabetes mellitus and autoimmune disease[END_REF].

One of the most prominent signal transduction pathways which are under control of the ubiquitin-proteasome module is the IκBα/NF-κB pathway. NF-κB represents a homo-or heterodimeric transcription factor of the Rel-/NF-κB protein families and plays a fundamental role in various cellular processes, including protection from apoptosis. NF-κB activity is regulated by the κB-inhibitor IκBα which prevents its nuclear translocation and the site specific transactivation of NF-κB target genes. In response to NF-κB inducing conditions, polyubiquitination and subsequent proteasomal degradation of IκBα releases NF-κB from its sequestration in the cytoplasm and promotes its transcriptional activity in the nucleus. As recently shown, the induced IκBα degradation is under control of the stress responsive early response gene , also designated IER3. This widely expressed and inducible protein has been shown to block NF-κB activation by attenuating the signal induced turnover of phospho-IκBα, an effect due to the inhibition of phospho-IκBα degradation by the 26S proteasome [25]. Consequently, IEX-1 prevents the NF-κB dependent protection from apoptosis. Furthermore, since IEX-1 itself is a target gene of NF-κB it might be part of the NF-κB self termination process by preventing inappropriate IκBα degradation in the presence of stimuli like IL-1beta or TNF-alpha [25]. Such an exaggerated NF-κB induction gives rise to various severe pathological conditions, i.e. inflammation and cancer [START_REF] Karin | NF-kappaB: linking inflammation and immunity to cancer development and progression[END_REF][27][28][29]. In accordance with the role of anti-apoptotic protection, the activity of the 26S proteasome is subject of downregulation by proapoptotic triggers, i.e. anti cancer drug treatment. It has been recently

shown that the major excecutor caspase, caspase-3, cleaves certain regulatory components of the 19S proteasomal subunits [START_REF] Sun | Caspase activation inhibits proteasome function during apoptosis[END_REF], thereby efficiently consolidating the initiation and excecution of apoptosis. So, it is tempting to speculate that IEX-1 exerts its proapoptotic potential by affecting the proteasomal activity. We could show, that IEX-1 affects the activity of the 26S proteasome by downregulating the expression of certain regulatory proteins, mainly those forming the 19S subunit. Moreover, this effect of IEX-1 is caspase independent but obviously dependent on altered gene transcription.

MATERIALS AND METHODS

Materials. MG132 and Suc-LLVY-AMC are from Biomol (München, FRG). Etoposide (Vepesid) was from Bistol-Myers-Squibb (München, FRG). Z-VADmk was from R&D-Sytems (Wiesbaden, FRG). Cycloheximide and Actinomycin-D were obtained from SIGMA (Munich, FRG).

Cell lines and their handling. Retrovirally transduced human embryonic kidney 293-cells stably expressing tetrameric and trimeric anti-IEX-1 hammerhead ribozymes or not -designated 293-Ri2/10, 293-Ri2/9 and 293-Babe, respectively [31], were cultured in DMEM low glucose medium (PAA-Laboratories; Cölbe, FRG) supplemented with 1 % (w/v) glutamine (Life Technologies; Eggenstein, FRG), 10 % (v/v) fetal calf serum (Biochrom KG; Berlin, FRG) and 1 µg/ml Puromycine (BD-Clontech; Heidelberg, FRG). Stably mock/pTO-LacZ or pTO-IEX-1 transfected TRex-293 cells (Invitrogen; Schelp, Netherlands) -designated Tet-293-Mock and Tet-293-IEX-1, respectively, were kept in culture using DMEM low glucose medium supplemented with 1 % (w/v) glutamine, 10 % (v/v) fetal calf serum, 5 µg/ml Blasticidine and 400 µg/ml Zeocine (both Invitrogen). All cells were kept at 37°C in a 5 % CO 2 atmosphere at 85 % humidity. Prior to the experiments, cells were kept at least 24 h without selection antibiotics.

Western blotting. Cells were lysed in 500 µl lysis buffer (50 mM Tris/HCl pH 7.4, 1% (v/v) TritonX100, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, and 0.1 mM Na 3 VO 4 ) supplemented with protease inhibitor cocktail (Complete-Mini/EDTA free, Roche) and further treated three times with sonication for 10s. After adjusting to equal protein concentrations (using the D c -Protein assay, Bio-Rad; München, FRG), the samples were supplemented with a 0.5 volume of 3 x SDS-PAGE sample buffer, heated (95°C) for 5 min and submitted to 8-20 % PAA-gradient electrophoresis. Upon semidry transfer onto PVDF membrane, blots were blocked for 2h with 5 % non-fat milk powder, 0.05 % Tween20 in PBS (blocking solution) at room temperature. Afterwards, blots were exposed to the primary 6 antibodies against S5a/Rpn10, S1/Rpn2, S6'/Rpt-5, α6/C2, or hUMP1/POMP (Affiniti/ Biomol; Hamburg, FRG), against caspase-3 (Cell Signalling, Frankfurt FRG), α-tubulin (Sigma, Deisenhofen, FRG) and against HSP90 (Santa-Cruz; Heidelberg, FRG) at a 200-to 1000-fold dilution in primary antibody diluent (5 % bovine serum albumine in 0.05 % Tween20 in TBS) overnight at 4°C. After extensive washing with 0.05 % Tween20 in TBS blots were exposed to the appropriate HRP-conjugated secondary antibody (Cell Signalling) diluted (1:1000) in blocking buffer and were developed using the LumiGlo peroxidase detection kit (Cell Signalling).

Fluorometric Proteasome assay. For the determination of proteasomal activity in 293 cell derived clones, a flurometric assay protocol was performed, as recently described [START_REF] Masdehors | Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic resp onsein B-CLL lymphocytes[END_REF]. In brief, cells were harvested by mild trypsinization. Then, 5 x 10 5 cells were resuspended in 500 µl medium and incubated with the proteasome inhibitor MG132 (5 µg/ml), or not. After 30 min, 10 µl of N-succinyl-L-leucyl-L-leucyl-L-leucyl-7-amido-4-methylcumarin (Suc-LLVY-AMC, 10 mM dissolved in DMSO) were added, and at various time points the formation of the fluorescent dye 7-amido-4-methylcumarin (AMC) was measured at λ=460 nm using a spectralfluorometer (Perkin Elmer). Specific chymotryptic proteasomal activity was determined by subtraction of the mostly proteasome independent fluorescence, measured in the presence of MG132.

Sucrose Gradient centrifugation. Identical amounts (2-3 mg protein) of cellular extracts (in 25 mM Hepes/HCl pH 7.4, 40 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 0.2 mM PMSF, 0.5 mM Na 3 VO 4 , 250 mM sucrose and 0.5% (v/v) TritonX100 supplemented with protease inhibitor cocktail) were loaded on top of 10-30% linear sucrose gradients (in lysis buffer) and the material was centrifuged using a SW28 rotor (Beckman) for 30 h at 28,000 rpm at 4 °C.

After centrifugation, 0.5-ml fractions were collected and 20 µl of each fraction was run on an 8-20% gradient gel, and immunoblotted.

RNA preparation and real time PCR. Total RNA was isolated using the RNeasy Kit from Qiagen (Hilden, FRG) and reverse-transcribed into single-stranded cDNA, as described previously [33]. Two µl of cDNA were subjected to semiquantitative real time PCR (iCycler, Biorad) using the SYBR-green assay with gene-specific primers at a final concentration of 0.2 µM. The primer sequences and the PCR conditions for the detection of the proteasomal subunits were: 5´-gaaggtttccggagtcggcagtt-3` (S1/Rpn2, sense) and 5`-ctccagagcaatgccaatagcct-3` (S1/Rpn2, antisense); 5´-aggcactgtcaccagatgag aac-3` S5a/Rpn10, sense) and 5`-aggcac tgtcaccagatgagaac-3` (S5a/Rpn10, antisense); 5`-gtcaagagtcgggatttgtggg-3` (S6'/Rpt5, sense) and 5´-ttggagctcatgggtgactctc-3` (S6'/Rpt5, antisense); 5`-ggcttactgctgatgctaga CTG-3` (α6/C2, sense) and 5´-agtcaggtcctgttctgcaggaa-3` (α6/C2, antisense); 94°C for 5 min; 35 cylces 94°C for 30 sec, 60°C for 30 sec, 72°C for 90 sec; 72°C for 10 min. For control, βactin was amplified in parallel using primers from BD Biosciences Clontech. The Real-time PCR was performed with a MyiQ Single Color Real-time PCR Detection System (BioRad).

Data were collected during annealing steps and were further analysed by using the i-Cycler iQ Optical system software (BioRad). All samples were analysed in duplicates and data are expressed as amount of mRNA in arbitrary units. The quality of PCR-products was checked by melting curve analysis following the amplification as well as by analytical RT-PCR and subsequent gel electrophoresis.

Cloning of the Rpn10/S5a (psmd4) promoter and luciferase reporter gene assay. Using the 5'primers tccgggctctgtactgggctta (pos. 147722) or ccaactgagaactgcaatggc (pos. 148630) and the 3' primer caagcttcaccaatcacagacctc (pos. 148812) containing a HindIII resctriction site, 1090 bp and 186 bp fragments of the 5`flanking region of the Rpn10 gene (psmd4, Gene bank Acc. No. AL592424; reported transcription start pos. 148806) were amplified and cloned into the pGL3basic luciferase vector (Promega) via KpnI/HindIII sites. Both constructs were verfied by automated BigDye (Perkin Elmer) DNA cyclce sequencing using a ABI 3770 instrument (Perkin Elmer). These psmd4 vectors psmd4 [-186] and psmd4[-1090] or the empty pGL3 vector (0.6 µg each) were co-transfected with 0.2 µg pTK-renilla (pTKRL) used as transfection control, as described recently [25]. After treatment, transfected cells were lysed with dual lysis buffer (Promega) and firefly or renilla luciferase activities were determined using the dual-luciferase reporter kit (Promega) on a luminometer (Bertholdt).

Optical units determined for firefly luciferase were normalized for renilla luciferase chemoluminescence.

Statistics Data are presented as mean ± SD and analyzed by Student´s t-test. A p-value < 0.05 (indicated as * in the figures) or < 0.02 (indicated ** in the figures) was considered as statistically significant.

Results

IEX-1 attenuates the activity of the 26S proteasome in 293 cells.

For the determination of the activity of the 26S proteasome in IEX-1 overexpressing cells, the formation of the fluorescent AMC product from cleavage of the proteasomal Suc-LLVY-AMC substrate was measured in mock-or IEX-1-transfected Tet-293 cells treated with tetracyclin, or not. As shown in figure 1a, in Tet-293-mock cells, the activity of the proteasome was not altered by tetracycline treatment, whereas in Tet-293-IEX-1 cells, here represented by the two independent clones Tet-293-IEX-1(a3) and Tet-293-IEX-1(a10), the formation of the AMC substrate was greatly reduced if IEX-1 was overexpressed in response to tetracyline administration. To verify the specificity of this effect, the proteasome inhibitor MG132 was added prior to tetracycline stimulation. Under these conditions, the residual activity of AMC formation that is independent of the proteasome was not affected by IEX-1 overexpression.

In contrast, an increase of AMC formation was observed in ribozyme transduced 293 cells with disrupted IEX-1 expression (Figure 1b). In 293-Ri2/10 cells expressing a tetrameric anti-IEX-1 hammerhead ribozyme and exhibiting the most efficient knock-down of IEX-1 expression [31], the cleavage of Suc-LLVY-AMC was more pronounced as compared to con- Copyright 2006 Biochemical Society trol cells (293-Babe). A similar, but slightly lower effect was seen in 239-Ri2/9 cells expressing a trimeric anti-IEX-1 hammerhead ribozyme and exhibiting a slightly less efficient IEX-1 knock-down [31]. Again, these IEX-1 related effects in 293-Ri2/10 and 293-Ri2/9 cells were not seen in the presence of MG132, hence indicating proteasome specificity.

Altered protein levels of Rpn10 and Rpn2 by IEX-1 in 293 cells. In order to elucidate whether the decrease of proteasomal activity could be attributed to an altered expression of certain proteasomal protein components [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF], total lysates from IEX-1 overexpressing or IEX-1 deficient 293 cells were submitted to Western blot analysis detecting the 19S subunit components S5a/Rpn10, S1/Rpn2 und S6'/Rpt-5 as well as of the 20S subunit component α6/C2.

In comparison with mock-transfected Tet-293 cells, the protein levels of Rpn10 and Rpn2 were strongly reduced in the two clones (a3 and a10) of Tet-293-IEX-1 cells if IEX-1 is overexpressed in a tetracycline dependent fashion (Figure 2a). By contrast, the expression of Rpt-5 and α6/C2 was not significantly affected. Conversely, the knock-down of IEX-1 expression in ribozyme transduced 293 cells, as seen in 293-Ri2-9 and 293-Ri2-10 cells, siginificantly increased the expression levels of Rpn10 and Rpn2, but not of Rpt-5 and α6/ C2 (Figure 2b).

To further address the impact of IEX-1 expression on proteasome formation, sucrose density gradient centrifugation was performed and fractions hereof were analysed by Western blot. As compared with tetracycline treated mock-Tet-293 cells, IEX-1 overexpressing 293 cells -here shown for the clone Tet-293-IEX-1(a3) -again exhibited much lower levels of the S5a/Rpn10 protein which is sedimented in higher density fractions corresponding to the 26S proteasome (Figure 3). In contrast, the C2/α6 protein detected over a broader range in the density gradient was not affected in its overall amount by IEX-1 overexpression. However, C2-positive sediments in higher density fractions (containing material of the 26S and 20S proteasome) 10 were less pronounced, whereas those in lower density fractions (containing material of the 20S proteasome precursors) were enriched in Tet-293-IEX-1 cells. Furthermore, expression of the proteasomal maturation factor hUMP1/POMP was strongly increased in IEX-1 overexpressing cells, probably reflecting accumulation of undigested hUMP1 protein due to an impaired proteasome assembly (Figure 3, bottom). Copyright 2006 Biochemical Society higher protein levels of Rpn10 and Rpn2 were still seen in the presence of Z-VADfmk (Figure 4d). These findings demonstrate that the effect of IEX-1 on Rpn10 and Rpn2 expression itself is independent of caspase activity, but significantly enhances the caspase dependent decrease of Rpn2 and Rpn10 protein levels during etoposide induced apoptosis.

The attenuating effect of IEX-1 on

Along with these additive actions of etoposide and IEX-1, apoptotic cell death increases with IEX-1 overexpression as indicated by the stronger caspase-3 activation seen in figure 4c, but decreases with IEX-1 knock down, as indicated by the reduced caspase-3 activation seen in figure 4d.

IEX-1 interferes with Rpn10 and Rpn2 gene transcription. It was therefore tempting to speculate that IEX-1 may interfere with gene transcription of Rpn10 and Rpn2, particularly since these two proteins are subject to tight transcriptional regulation [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF]. For this purpose, quantitative real time PCR was conducted in order to determine the mRNA levels of Rpn10 and Rpn2 as well as of Rpt5 and C2 for comparison. As shown in figure 5a, after normalization to the amount of β-actin mRNA used as internal control, the levels of Rpn10 and Rpn2 mRNA were significantly reduced in tetracycline treated Tet-293 cells overexpressing IEX-1. Compared to Tet-293-mock cells, the mRNA levels of Rpn10 and Rpn2 decreased by 32 % and 47 %, respectively, in Tet-293-IEX-1(a10) cells, and by 35% and 53 %, respectively, in Tet-293-IEX-1(a3) cells. In contrast, mRNA levels of Rpt5 and C2

were not significantly affected by tetracycline treatment in mock-or IEX-1 transfected Tet-293 cells. When analysing 293 cells with disrupted IEX-1 expression, it turned out that in these cells (293-Ri2/9 and 293-Ri2/10) the amount of Rpn10 mRNA (increase by 80 % and 120 %, respectively) and Rpn2 mRNA (increase by 70 % and 90 %, respectively) were significantly higher than in 293 babe control transfectants (Figure 5b), whereas the mRNA levels of Rpt5 and C2 did not differ in this way.

The negative interference of IEX-1 with Rpn2 and Rpn10 is abrogated during transcriptional but not during translational inhibition. In order to show whether the attenuating effect of IEX-1 on Rpn2 and Rpn10 expression depends on de novo mRNA synthesis, the transcriptional inhibitor actinomycinD (0.2 µg/ml, 24 h) was added 16 h after induction of IEX-1 overexpression. As shown by real time PCR (Figure 6a), mRNA levels of Rpn2 and Rpn10 were significantly suppressed in the presence of actinomycinD reflecting blockade of RNA de novo synthesis. Under these conditions, the overexpression of IEX-1 did not further decrease the expression levels of Rpn2 or Rpn10 mRNA, thus indicating a non-additive action of IEX-1 and actinomycinD. In contrast, cycloheximide (0.5 µg/ml, 24h) that blocks protein de novo synthesis did not interfere with the levels of Rpn2 or Rpn10 mRNA and IEX-1 overexpression still decreased the amount of both mRNAs in the presence of cycloheximide, indicating that neither the Rpn2 or Rpn10 gene transcription under these conditions nor the negative effect of IEX-1 involve de novo protein synthesis.

When analysing the effects of these inhibitors also on protein levels (Figure 6b) it turned out that both actinomycinD and cycloheximide decrease the expression of Rpn2 and Rpn10. But whilst no additive effect was seen with IEX-1 and actinomycinD, the decrease of Rpn2 and Rpn10 protein levels seen with cycloheximide was much more pronounced in IEX-1 overexpressing cells indicating an additive action. These data suggest that the supressing effect of IEX-1 on Rpn2 and Rpn10 expression is due to an interference with de novo mRNA synthesis of these genes. This effect does not involve posttranscriptional or cotranslational events that control mRNA stability because this would be additive to the effect of actinomycinD.

Suppression of S5a/Rpn10 promoter activity by IEX-1. Since S5a/Rpn10 appears to be the most affected 19S subunit-gene by IEX-1 (see above) we chose the promoter of this gene for analysing the effect of IEX-1 on promoter activity of proteasomal subunit genes. For this purpose, parts of the Rpn10 promoter, which has not been characterized so far, were fused to the firefly luciferase reporter gene and used for luciferase assays in Tet-293 cells. From a data base search we generated two promoter fragments (Figure 7 When overexpressing IEX-1 by the addition of tetracycline in Tet-293-IEX-1 cells (clone a3), but not in Tet-293-mock cells, the psmd4[-1090] driven luciferase expression declined significantly (Figure 7), whereas the residual promoter activity seen with psmd4[-186] and pGL3 were not affected. These findings strongly support our conclusions drawn from the preceding experiments that IEX-1 specifically interferes with the expression of the 19S subunit genes on the transcriptional level, obviously by exerting an inhibitory effect on the promoter activity, what is shown here in example for the Rpn10 promoter.

Discussion

Under certain cellular conditions (i.e. serum stravation, DNA-damging insults), the stress response gene IEX-1 exerts a strong pro-apoptotic effect [31,[33][START_REF] Segev | Mullerian-inhibiting substance regulates NF-kappa B signaling in the prostate in vitro and in vivo[END_REF][35], that to some extent relates to its attenuating effect on NF-κB activation [25]. This involves an interference of IEX-1 with the signal induced degradation of IκBα by the 26S proteasome. Whereas it is known that certain pro-apoptotic triggers, as shown above for the anti-cancer drug etoposide, decrease the levels of certain proteasomal protein components by caspase mediated cleavage and thereby consolidate the cell death comitting apoptotic program [START_REF] Sun | Caspase activation inhibits proteasome function during apoptosis[END_REF], we could show that the effect of IEX-1 does not involve this process. In fact, IEX-1 affects the expression of these proteins independently of caspase activity and, thereby, acts additive to the decreasing effect of apoptotic stimuli on protein levels of proteasomal components which is caspase dependent.

Intriguingly, IEX-1 directly attenuates the expression of certain proteasomal components, particularly those being part of the 19S regulatory subunit of the 26S proteasome [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF] and conferring specificity for polyubiquitinated substrates such as IκBα [36,37]. This decreased expression of S5a/Rpn2 or S1/Rpn10 in response to IEX-1 is not due to a direct protein interaction as recently described for alpha-synuclein [38], but obviously results from a suppressed gene transcription as shown by the declined mRNA levels of both genes in 293 cells if IEX-1 is overexpressed. In support of this assumption, conditions of transcriptional blockade by ac-tinomycinD abrogated this negative interference of IEX-1 with Rpn2 and Rpn10 expression, suggesting that the effect of IEX-1 does not depend on post-transcriptional events, i.e. a decreased mRNA stabilization or an increased mRNA turn-over, but rather relates to the transcription process and RNA de novo synthesis. Furthermore, treatment with the translational inhibitor cycloheximide did not alter the effect of IEX-1 on Rpn2 and Rpn10 expression, also indicating that IEX-1 expression does not affect a post-transcriptional or a co-translational event. In further support of this assumption, the activity of the putative s5a/Rpn10 promoter as determined by luciferase reporter gene assay was attenuated by IEX-1.

Still, it could be just speculated how IEX-1 interferes with gene transcription of Rpn2 and Rpn10. In yeast, the transcription of proteasomal proteins is under control of the transcription factor Rpn4 which is mainly regulated by its proteasomal degradation [START_REF] Xie | RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit[END_REF][START_REF] Ju | Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit[END_REF][START_REF] London | Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome[END_REF]. Accordingly, during conditions with high proteasomal activity the de novo synthesis of protein components is restrained, whereas the lack of proteasome activity favours the increased expression of particularly those proteins compensating for the declined proteasomal activity [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF]. Whilst the human counterpart of Rpn4 has not been identified yet, recent reports indicated that in mammalian cells the antioxidant related transcription factor Nrf2 (NF-E2 related factor 2) which is known to be involved into cytoprotection during oxidative stress may control the expression Copyright 2006 Biochemical Society of these proteins [START_REF] Kwak | Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway[END_REF][START_REF] Kwak | Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway[END_REF]. Kwak et al. recently described a crucial role of Nrf2 in the induced expression of the proteasomal subunit protein β5/PSMB5 in response to oxidative stress [START_REF] Kwak | Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway[END_REF],

and other conditions are known -including proteasome inhibition, PI3K/Akt or ERK activation -leading to increased Nrf2 activity and, thereby, stimulating expression of proteasomal proteins [41,[START_REF] Numazawa | Nrf2-dependent gene expressions: a moleculartoxicological aspect[END_REF]. However, it still has to be elucidated whether or not the effect of IEX-1 on the gene expression of certain proteasomal proteins is indeed related to the action of this transcription factor as could be expected from Rpn10 promoter analysis and luciferase assays, and whether other transcription factors, i.e. or NF-κB, play also a role.

A major consequence of the blocking effect of IEX-1 on the expression of certain subcomponents of the 26S proteasome is a marked impairment of its assembly (Figure 3) that is already blocked on the level of the precursor particles [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF]44,45]. Moreover, along with the decreased formation of the 26S proteasome, the maturation of the 20S proteasome seems also to be affected as indicated by the accumulation of the maturation factor hUMP1 and by the altered distribution of sedimented 20S protein -i.e. α6/C2 -in the sucrose density gradients (Figure 3). It is not clear yet how the reduced de novo expression of 19S proteins and consequently the 19S subunit depletion influences the assembly of the 20S proteasome. Due to the reduced levels of certain protein components, the immature 19S precursors may disintegrate and assemble improperly with 20S precursors, thereby blocking further 20S subunit maturation. Another explanation could be that the inappropriate assembly of the 19S subunit excessively consumes certain proteasome related chaperons [45] that are then unavailable for the completion of the 20S subunit.

Irrespective of the exact mechanisms, proteasome activity targeted to poly-ubiquitinated proteins (i.e. IκBα) is significantly compromized by IEX-1 giving rise, at least in part, to an increased sensitivity to apoptotic triggers. This is underlined by the finding that many conditions affecting proteasome activity enhance apoptosis. In this way, proteasome inhibitors and anti-cancer drugs and, as can be assumed, IEX-1 expression sensitizes various cell types to diverse apoptotic stimuli [46,47]. Thus, it can be speculated that IEX-1 is part of a cellular regulation process that dampens the exaggerated processing and turn over of regulatory proteins. Thereby, IEX-1 may contribute to the maintenance of cellular homeostasis and integrity which, for example, is a prerequisite in the elimination of pre-malignant cells.

Accordingly, IEX-1 expression has been found to be reduced in certain types of tumours indicating a potential role of IEX-1 in tumour suppression [48][49][50]. Transcription Element Search Software (www.cbil.upenn.edu/tess). After transfection, cells were incubated with 1 µM tetracycline or not for 24 h. Then, cells were lysed and submitted to dual luciferase assay using renilla luciferase activity (pTKRL) for normalization of optical units obtained with firefly luciferase and expressed as mean ± SD (n = 4), * indicates statistically significant (p < 0.05).

Legends to figures

  Rpn10 and Rpn2 expression enhances the reduction of Rpn10 and Rpn2 protein levels resulting from etoposide induced apoptosis and is caspase independent. Next we looked whether downregulation of Rpn10 and Rpn2 is related to apoptosis dependent and caspase-3 mediated degradation of these proteins. Upon administration of the anti-cancer drug etoposide (20 µg/ml) and in the absence of tetracycline, the protein levels of Rpn10 and Rpn2 quite similarly declined in mock-and IEX-1 transfected Tet-293 cells within 24 hours in comparison with untreated cells. If tetracycline was added 16 hours prior to etoposide administration the drug induced decrease of Rpn10 and Rpn2 protein levels was much more pronounced in Tet-293-IEX-1 cells, here the clone Tet-293-IEX-1(a3), as compared to Tet-293-mock cells (Figure 4a). Conversely, in 293 cells subject to ribozyme mediated knock-down of IEX-1 expression (293-Ri2-10 and 293-Ri2-9), the etoposide dependent decrease of Rpn10 and Rpn2 protein levels was much less pronounced compared to 293-babe control transfectants (Figure 4b). In the presence of the Caspase inhibitor Z-VADfmk (Figure 4c,d), the etoposide dependent decrease of Rpn10 and Rpn2 protein levels in all transfected 293 cells was completely blocked, indicating that this decreasing effect by etoposide was indeed due to caspase mediated degradation of Rpn10 and Rpn2. Interestingly, caspase inhibition by Z-VADfmk did not affect the tetracycline induced decrease of Rpn10 and Rpn2 protein levels in IEX-1 overexpressing 293-Tet cells (Figure 4c), neither in the absence nor in the presence of etoposide. Moreover, in ribozyme transduced 293 cells with disrupted IEX-1 expression, the Biochemical Journal Immediate Publication. Published on 3 Nov 2006 as manuscript BJ20061072

  , lower panel): one (psd4[-1090]) containing a potential antioxidant response element (ARE) and binding site for Nrf2 as well as potential binding sites for AP1, PPARα, Sp1, Ets-1 and, most notably, NF-κB, and one lacking these sites (psd4[-186]). As shown in figure 7, significant promoter activity as measured by firefly luciferase optical units (normalized to renilla luciferase) was detected in Tet-293 cells transfected with psmd4[-1090] compared to cells transfected with promoterless pGL3, but only marginal promoter activity was seen in cells transfected with psmd4[-186].

Figure 1 .

 1 Figure 1. IEX-1 inhibits the activity of the 26S proteasome. a) Tet-293-mock and Tet-293-IEX-1 cells (clone a3 and a10) treated with tetracycline (16h, 1 µM) or without, or b) 293babe, 293-Ri2/10 and 293-Ri2/9 cells were submitted to a fluorometric 26S proteasome assay.

Figure 2 .

 2 Figure 2. IEX-1 decreases protein levels of Rpn2 and Rpn10 in 293 cells. Total lysates from a) Tet-293-mock and Tet-293-IEX-1 cells (clone a3 and a10) treated with tetracycline (16 h, 1 µM) or without, or from b) 293-babe, 293-Ri2/10 and 293-Ri2/9 cells were submitted to Western blot analysis detecting the proteasomal proteins a6/C2 (20S proteasome

Figure 3 .

 3 Figure 3. Impaired protein assembly of the 26S proteasome in IEX-1 expressing 293 cells. Cellular lysates from Tet-293-mock and Tet-293-IEX-1 cells (clone a3) treated with tetracycline (16 h, 1 µM) were submitted to sucrose density gradient(10-30%)

Figure 4 .

 4 Figure 4. The decreasing effect of IEX-1, but not of etoposide, on the protein levels of Rpn2 and Rpn10 is caspase independent. a) Tet-293-mock and Tet-293-IEX-1 cells (clone a3) pre-treated with tetracycline (8 h, 1 µM) or without, or b) 293-babe, 293-Ri2/10 and 293-Ri2/9 cells were subject of treatment with etoposide (10 µM) or without for 24h. Then, cell

Figure 5 .

 5 Figure 5. IEX-1 suppresses gene expression of Rpn2 and Rpn10 on the transcriptional level. Total RNA from a), Tet-293-mock and Tet-293-IEX-1 cells (clone a3 and a10) pretreated with tetracycline (16 h, 1 µM) or from b), 293-babe, 293-Ri2/9 and 293-Ri2/10 cells was reversed transcribed and then submitted to semi-quantitative real-time PCR using specific primers for Rpn2, Rpn10, Rpt-5, S1, C2 and, as control, βactin. The amount of mRNA normalized to βactin is shown and expressed as mean value ± SD (n = 5); * indicates

Figure 6 .

 6 Figure 6. The decreasing effect of IEX-1 on Rpn2 and Rpn10 expression is blocked by transcriptional, but not by translational inhibition. Tet-293-mock and Tet-293-IEX-1 cells (clone a3), pre-treated with tetracycline (16 h, 1 µM) or not, were further incubated with the

Figure 7 .

 7 Figure 7. IEX-1 overexpression attenuates Rpn10 promoter activity. Tet-293-IEX-1 (clone a3) or Tet-293-mock cells were cotransfected with pTKRL (for normalization) and

Fig. 1 Suc

 1 Fig.1

  Fig. 5

Biochemical Journal Immediate Publication. Published on 3 Nov 2006 as manuscript BJ20061072

Biochemical Journal Immediate Publication. Published on 3 Nov 2006 as manuscript BJ20061072 Copyright 2006 Biochemical Society

Acknowledgements

This work was supported by a grant of the German Research Society DFG (SFB415/A13 to H.S.).