Alain Da

Silva Morais

Alain Saliez

Isabelle Leclercq

Yves Horsmans

MD Peter Stärkel
email: peter.starkel@gaen.ucl.ac.

Inhibition of the RAS oncoprotein reduces proliferation of hepatocytes in vitro and in vivo in rats

Keywords: ras, hepatocellular carcinoma, S-Farnesylthiosalicylic acid, cell cycle, MAPkinase, partial hepatectomy

Introduction

There is a general consensus that the Ras oncoprotein may play an important role, not only in the regulation of normal cellular proliferation and differentiation but also in carcinogenesis [START_REF] Lowy | Function and regulation of ras[END_REF][START_REF] Kelloff | Progress in chemoprevention drug development: The promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancer-a plan to move forward[END_REF]. The Ras oncoprotein activates several intra-cellular signalling pathways including the MAPkinase pathway that plays an important role in promoting DNA synthesis and cell proliferation [START_REF] Lavoie | Cyclin D1 expression is regulated positively by the p42/p44 MAPK and negatively by the p38/HOG MAPK pathway[END_REF]. In vitro, studies have shown that expression of the Ras oncoprotein increased between the G1 and S phases of the cell cycle [START_REF] Ikeda | H-ras gene expressed at the G1 phase in primary cultures of hepatocytes[END_REF][START_REF] Kost | Effect of epidermal growth factor on the expression of protooncogenes c-myc and c-Ha-Ras in short-term primary hepatocyte culture[END_REF] and its inhibition resulted in a cell cycle block in G1 phase. [START_REF] Dobrowolski | Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells[END_REF][START_REF] Talarmin | The mitogen-activated protein kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G1 phase progression in proliferating hepatocytes[END_REF]. In vivo in the liver, it remains, however, ill defined whether interfering with ras activation could lead to reduced liver cell proliferation. An interesting model to study the impact of targeted inhibition of intracellular pathways in the liver is liver cell proliferation following partial hepatectomy (PH). A cascade of events moves cells from their resting state into cell cycle progression in order to restore the liver mass [START_REF] Campell | Liver regeneration[END_REF]. Several intra-cellular signalling pathways are involved including activation of the small GTPase Ras in response to mitogens [START_REF] Campell | Liver regeneration[END_REF][START_REF] Marshall | How do small GTPase signal transduction pathways regulate cell cycle entry?[END_REF]. Ras mRNA expression increases in the regenerating liver in close relationship to the peak of DNA synthesis [START_REF] Goyette | Expression of cellular oncogenes during liver regeneration[END_REF] and seems to be specifically related to cellular proliferation and cell cycle progression in various animal models of liver cell proliferation [START_REF] Stärkel | After portal barnch ligation in the rat, cellular proliferation is associated with selective induction of c-Ha-ras, p53, cyclin E and cdk2[END_REF][START_REF] Laurent | Expression of presumed specific early and late factors associated with liver regeneration in different rat surgical models[END_REF]. In addition, modulation of ras expression in the liver influences the course of liver regeneration in mice [START_REF] Lüdde | Ras adenovirus modulate cyclin E protein expression and DNA synthesis after partial hepatectomy[END_REF]. Very recently, it has also been shown that ras and the MAPkinase pathway might play a role in the development of hepatocellular carcinomas in humans [START_REF] Calvisi | Ubiquitous activation of ras and Jak/Stat pathways in human HCC[END_REF]55], a cancer with poor survival and scarce therapeutic options. However, additional and unequivocal data are needed before targeting of the ras oncoprotein as a future approach in preventing cellular transformation and malignant liver cell growth can be recommended.

Farnesylation of the Ras oncoprotein is important for its membrane anchorage and activation [START_REF] Kato | Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity[END_REF] and inhibition of this process arrests cell growth [START_REF] Egozi | Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist[END_REF][START_REF] Haklai | Dislodgement and accelerated degradation of Ras[END_REF]. The Ras antagonist S-Farnesylthiosalycilic acid (FTS) acts over the three isoforms of Ras and seems to achieve inhibition of their membrane anchorage and activation by two distinct mechanisms: the first one involves inhibition of the enzyme prenylated protein methyltransferase (PPMTase) [START_REF] Marciano | Farnesyl derivatives of rigid carboxylic acids -inhibitors of ras-dependent cell growth[END_REF][START_REF] Marom | Selective inhibition of Ras-dependent cell growth by farnesylthiosalicylic acid[END_REF], the second one, the antagonism of the mature form of the Ras oncoprotein [START_REF] Marom | Selective inhibition of Ras-dependent cell growth by farnesylthiosalicylic acid[END_REF][START_REF] Gana-Weisz | The Ras antagonist S-farnesylthiosalicylic acid induces inhibition of MAPK activation[END_REF].

Since FTS exhibits anti-tumoral effects in non liver cell tumours in vivo in nude mice and prevents cirrhosis development in rats [START_REF] Weisz | A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice[END_REF][START_REF] Reif | The Ras antagonist, farnesylthiosalicylic acid (FTS), inhibits experimentally-induced liver cirrhosis in rats[END_REF], we were prompted to evaluate the impact of inhibition of Ras activation through FTS on normal hepatocyte proliferation in rats after partial hepatectomy (PH) in vivo as well as on a hepatic tumour cell line in vitro. The

Materials and Methods:

Cell culture HepG2 cells (2,25x10 5 cells per 100 mm of diameter dish), a human hepatoblastoma derived cell line were cultured in DMEN (Gibco, Harelbeek, Belgium) supplemented with 10% fetal bovine serum, 1% streptomycin and penicillin (Gibco), 1% sodium pyruvate (Gibco) and 1% minimal essential medium (MEM, Gibco) over 7 days. DMSO (solvent for FTS) or FTS at a final concentration of 100 µM were included into to the culture medium from the beginning of the experiment. BrdU was added to the culture medium at a final concentration of 10 µM from 48 hours of culture onwards. Culture medium was changed every other day. Cells were harvested by trypsinisation at the end of the experiment for flow cytometry analysis and cell count using a Burker cell. In a subset of dishes, cells were directly lysed and the supernatant was recovered for Western blot analysis. Cell viability was checked using trypan blue staining. At least, three independent experiments were performed for each analysis.

Animals

Male Wistar rats (body weight 180-250 g) were obtained from the rat breeding facilities of the Catholic University of Louvain Medical School, Brussels, Belgium. Animals were kept in a temperature and humidity-controlled environment in a 12 h light-dark cycle. They were allowed free access to water and standard food pellet diet (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orges, France). The animals were handled according to the guidelines established by the Catholic University of Louvain.

Surgical procedures and experimental design

All operations were carried out under light ether anaesthesia at room atmosphere using a clean but a not sterile technique. Partial hepatectomy (PH) was performed according to Higgins and Anderson [START_REF] Higgins | Experimental pathology of the liver[END_REF]. Rats were injected intra-peritoneally (i.p.) with 50mg/kg b.w. of BrdU 2 hours before sacrifice. Animals were sacrificed at 12, 24 and 48h after PH. Livers were removed, weighed and snap-frozen or conserved in formol.

The initial approach consisted in testing the toxicity of FTS in our animal model by injecting a dose of 75mg/kg b.w. per injection, the inferior limit of the reported LD50 in rats, initially to non-hepatectomised rats and then to rats subjected to two-third PH. Three intra-peritoneal injections were administered to each animal during a 24 hours period respecting an interval of 8 hours between each injection. Death of the animals was considered as the primary end-point for this part of the study. According to the results obtained during assessment of FTS toxicity, the highest dose of FTS that does not produce any death in hepatectomized rats during the first 24 hours after PH was chosen for the final experiments. Consequently, the scheme that was used for the final experimental procedure comprised 3 i.p. injections of 50mg/kg b.w. of FTS at 1, 8 and 16 hours after PH. Controls received a similar volume of DMSO (vehicle for FTS) at similar time points. At least three and maximum six animals per experimental group were used.

Synthesis of farnesylthiosalicylic acid (FTS)

FTS was prepared as described previously [START_REF] Marciano | Farnesyl derivatives of rigid carboxylic acids -inhibitors of ras-dependent cell growth[END_REF]. Briefly, thiosalicylic acid (2.65 g, 6 mmol), guanidine carbonate (3.82 g, 7 mmol), and trans,-trans-farnesyl bromide (5 g, 6 mmol) were mixed overnight in 220.6 ml of acetone at room temperature. After the acetone had evaporated, chloroform was added together with a few drops of 2 N HCl. The mixture was washed with water, and the organic phase was separated and dried on magnesium sulfate and evaporated. FTS was purified on silica gel with mixtures of chloroform and ethyl acetate. FTS was dissolved in DMSO (20mg FTS / 500µL DMSO) for injection.

Preparation of liver homogenates, cell lysates and nuclear extracts

Livers were homogenized and cells lysed (HepG2) in ice-cold buffer (0,1% Triton X-100, 50mM HEPES, 150mM NaCl, 10% glycerol, 1.5mM MgCl2, 1mM DTT, 1mM NaF, 1mM PMSF, 100mg/ml Leupeptin, 10mg/ml Aprotinin and 0.1mM Na3VO4). Nuclear extracts were prepared from frozen liver using a commercially available nuclear extract kit according to the instructions of the manufacturer (Active Motif, Rixensart, Belgium).

Preparation of rat liver cytosolic and membrane fractions

Liver membrane fractions were prepared as described by Wheeler et al. [START_REF] Wheeler | Impaired Ras membrane association and activation in PPARα knockout mice after partial hepatectomy[END_REF] with minor modifications. Livers were homogenized in a buffer containing 10 mM Tris HCl (pH 7.4), 1 mM EDTA (pH 7.6), 0.25 M sucrose, 2µg/ml PMSF, 2µg/ml Aprotinin, 2µg/ml Leupeptin, 1mM DTT and 1mM Benzamidin and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was centrifuged at 100,000 g for 1 h at 4°C to prepare cleared cytosol. The resulting pellet was solubilized in 1ml of a lysis buffer containing 20mM Tris HCl (pH 7.4), 2.5mM EDTA, 1% Triton X-100, 1% NaDOC, 0.1% SDS, 100mM NaCl, 100mM NaF and 1mM Na3VO4 and centrifuged at 100,000 g for 1 h at 4°C and was used as the membrane fraction.

Western Blotting

Proteins were separated by SDS-PAGE and transferred to PVDF Transfer membranes according to standard techniques. Membranes were incubated with primary and secondary antibodies as specified in table 1. The antigen-antibody reaction was visualized using the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer) detection system. Membranes were stripped using Restore Western blot stripping buffer (Pierce Chemical, Rockford, IL, USA) and reprobed whenever possible following the instructions of the manufacturer. Quantification of immune-reactive proteins was obtained by densitometry using the Gel Doc 2000 device and software (Bio-Rad, Nazareth, Belgium).

Immunoprecipitation and Cdk kinase assays

Immunoprecipitation and Cdk kinase assays were performed as previously described [START_REF] Stärkel | After portal barnch ligation in the rat, cellular proliferation is associated with selective induction of c-Ha-ras, p53, cyclin E and cdk2[END_REF].

The histone H1 kinase assay was used to assess Cdk2 activity and the Rb (769) fusion protein (Santa Cruz Biotechnology) functioned as a substrate for Cdk4.

Histology and immunohistochemistry

Hepatic sections were stained with hematoxylin-eosin for conventional histology or incubated with a mouse anti-BrdU antibody (Dako; 1/100; overnight) and a secondary anti-mouse Envision System Labelled Polymer-HRP antibody (Dako; 1h) to visualise cell proliferation.

Flow cytometry

Incorporation of BrdU into DNA was quantified by flow cytometry essentially as described by Borbath et al. [START_REF] Borbath | Use of 5-[(76)Br]bromo-2'fluoro-2'-deoxyuridine as a ligand for tumour proliferation: validation in an animal tumour model[END_REF].

Reverse transcription and quantitative polymerase chain reaction (PCR)

cDNA was prepared by reverse transcription of total hepatic RNA as previously described by Stärkel et al. [START_REF] Stärkel | Expression and DNA-binding activity of signal transducer and activator of transcription 3 in alcoholic cirrhosis compared to normal liver and primary biliary cirrhosis in humans[END_REF]. Quantitative PCR analysis was performed with the GeneAmp 5700 Sequence Detection System and software (Applied Biosystems, Den Ijssel, Netherlands) according to the standardized thermal profile of the system previously set by the manufacturer with SYBR Green for detection. All primers (table 2) were designed using the Primer Express design software (Applied Biosystems). Ribosomal protein L19 (RPL19) RNA was chosen as an internal standard. Expression of mRNA was quantified using the ∆∆CT method as specified by the manufacturer.

Statistical Analysis

Results are expressed as means ± SEM. The statistical differences between the groups were tested using the Student's t-test. Statistical significance was assumed for P values < 0.05.

Results: FTS inhibits liver tumour cell proliferation in vitro

First, we examined whether FTS is able to inhibit proliferation of the hepatic tumour cell line HepG2 in vitro. After 3 days of culture in the presence of FTS, a slight non significant reduction in cell proliferation was be observed compared to untreated cell cultures (not shown). However after 7 days of culture, FTS treated cultures showed a marked reduction in overall cell density (figure 1a) and a total cell count using a Burker cell confirmed a significant reduction in the number of cells per dish (figure 1b). Trypan blue staining of the cells showed viability of more than 98% of the recovered cells. Furthermore, a significant decrease in BrdU incorporation into the cells (figure 1b) as well as a reduced expression of PCNA by Western blot (figure 2) was found in the FTS treated cultures compared to controls.

These observations clearly show that FTS inhibits proliferation of tumour derived hepatocytes in vitro.

FTS significantly reduces hepatocytes proliferation in vivo after PH

Since FTS effectively inhibited hepatocytes proliferation in vitro, we next examined whether FTS is also able to inhibit hepatocytes proliferation in vivo. To study the in vivo effect of FTS on hepatocytes, the partial hepatectomy model was chosen as it induces a synchronous, vigorous and reproducible proliferation of hepatocytes in rats.

Injection Scheme of FTS and mortality

As a first step we determined the potential toxic/lethal dose of FTS in our experimental setting. Given the lethal dose 50 (LD50) of FTS of 75 to100mg/kg b.w. reported in rats [START_REF] Weisz | A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice[END_REF],

we started with intraperitoneal injections of 75mg/kg of FTS, the inferior limit of the LD50 range. Considering death as the primary end-point, we treated rats, subjected or not to a PH, with upto three consecutive injections of 75mg/kg of FTS at 1, 8 and 16 hours. No mortality was observed in the non-hepatectomised rats (n=6/group) receiving either FTS or DMSO (vehicle for FTS). By contrast, three injections of 75mg/kg of FTS caused death within 24 hours in 57% of the rats subjected to the PH (4 out of 7 rats) but not in rats receiving the vehicle (DMSO) only (n=8). Therefore, we decreased the dose of FTS to 50mg/kg b.w. and repeated the experiment in partial hepatectomised rats. At that time, three consecutive injections of FTS at 1, 8 and 16 hours after PH did not induce any mortality in the animals (n=8). These results suggest that, in our experimental conditions, only the association of three injections of 75mg/kg of FTS with PH elicits death in rats. Consequently, the scheme of 3 injections of the lower dose of FTS (50mg/kg b.w.) at 1, 8, and 16 hours after PH was used for further studies assessing the impact of FTS on ras inhibition, cell proliferation and the cell cycle.

PCNA Expression

The proliferating Cell Nuclear Antigen (PCNA) is widely used as a cell proliferation marker [START_REF] Wolf | Hepatocyte regeneration in acute fulminant and non-fulminant hepatitis: a study of proliferating cell nuclear antigen expression[END_REF]. Therefore, homogenates from DMSO and FTS-treated rats, sacrificed at 12, 24 and 48 hours after PH, were subjected to Western blot analysis with a PCNA specific antibody. Low PCNA expression was found in DMSO-treated animals at 12h. PCNA peaked at 24 h and decreased at 48h (figure 3a). FTS treatment significantly reduced PCNA expression at 12 and 24h after PH compared to the DMSO group. At 48h, PCNA expression was similar in FTS and DMSO treated animals. However and by contrast to the DMSO group, no prominent peak of PCNA expression was observed during the first 48h after PH in the FTS-treated animals (figure 3b).

BrdU incorporation

To confirm the impact of FTS treatment on DNA synthesis, we determined the incorporation of BrdU into DNA of hepatocytes during S-phase of the cell cycle by immunohistochemistry and quantified this incorporation by flow cytometry analysis (FACS). BrdU incorporation was negligible at 12h after PH. BrdU incorporation peaked in DMSO-treated animals at 24h and decreased thereafter (figure 4a). As with PCNA, FTS strongly reduced BrdU incorporation at 24h after PH predominantly in hepatocytes (figure 4a,b) and returned thereafter to levels observed in DMSO-treated animals (figure 4a). In parallel to PCNA expression, BrdU incorporation was also blunted in FTS-treated animals over the first 48h after PH compared to their DMSO-treated littermates that exhibited a clear peak at 24h. Taken together, the data obtained for PCNA expression and BrdU incorporation strongly suggest that FTS impairs hepatocytes proliferation.

liver to body weight index

The liver to body weight index reflects recovery of the liver mass proportional to the weigth of the animal. In accordance with reduced cellular proliferation in FTS-treated animals at 24 hours after PH, the liver to body mass index was significantly lower in FTS-treated rats compared to DMSO controls (1,5% ±0.08 vs 1,7% ±0.03, respectively; p<0.001). The differences between both groups were attenuated with partial recovery of cellular proliferation at 48h after PH (2.4% ±0.01 vs 2.5% ±0.08 in FTS and DMSO-treated animals, respectively).

FTS dislodges ras from the cell membrane and interferes with downstream signaling through the MAPkinase pathway

Since FTS reduced cell proliferation, we next verified that this effect was indeed due to functional inhibition of the ras protein.

Sub-cellular ras localization

Cytsol and membrane fractions from DMSO and FTS-treated animals were analysed for ras expression by Western Blotting. During the first 48 hours after PH, Ras expression was principally confined to the cell membranes in DMSO animals (figure 5). No ras immunoreactive protein was detected at 12 hours (not shown) and only small amounts of ras protein were found in the cytosol beyond the 12 th hour after PH (figure 5). By contrast, Ras membrane expression decreased progressively in FTS-treated animals and substantial amounts of ras protein could be demonstrated in the cytosol fractions from the 12 th hour onwards after PH (figure 5). In addition, FTS did not affect ras mRNA expression which significantly and similarly increased in both groups after PH (not shown).

These results confirm that FTS principally dislodged the ras oncoprotein from the cell membranes without affecting the induction of its mRNA levels.

expression of membrane bound p-raf

Activation of the ras oncoprotein recruits cytosolic raf to the plasma membrane where it becomes activated by phosphorylation [START_REF] Marshall | Cell signalling. Raf gets it together [news[END_REF]. Therefore, we subjected membrane fractions of DMSO and FTS-treated animals to Western blot analysis for p-raf expression. P-raf was selectively detected at the membranes of DMSO but not of FTS treated animals at 24 hours after PH (figure 6a). No p-raf expression could be detected at any time during the first 48 hours after PH in FTS-treated rats (not shown) suggesting FTS treatment interferes with activation of raf by ras.

activation of the MAPkinase ERK1/2

The activation of Ras leads to down stream activation of the MAPkinase cascade that includes the phosphorylation and activation of the ERK 1/2 proteins [START_REF] Talarmin | The mitogen-activated protein kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G1 phase progression in proliferating hepatocytes[END_REF][START_REF] Heidecker | Mutational activation of c-raf-1 and definition of the minimal transforming sequence[END_REF]. Thus, the presence of phosphorylated ERK1/2 might serve as a marker for ras activation. Therefore, we assessed the expression of phosphorylated ERK1/2 in homogenates of DMSO and FTS-treated rats by Western blotting. Phospho-ERK 1/2 was significantly reduced in FTS treated animals during the 48h of the experiment (figure 6b). The strongest reduction was found during the first 24 hours in the FTS group. With interruption of the treatment 16 hours after PH, phospho-ERK 1/2 levels increased again but did not reach the level of expression observed in the DMSO treated animals (figure 6c).

Taken together, these data confirm the functional inhibition of ras by FTS and suggest that reduced cell proliferation might be mediated by inhibition of down-stream signalling through the MAPkinase pathway.

The effect of FTS is connected to the cell cycle principally through reduced CyclinE-

Cdk2 expression and activity

The cyclinD/Cdk4 and the cyclinE/Cdk2 complexes constitute the principal factors that are involved in controlling the G0/G1 and the G1/S checkpoints of the cell cycle, respectively. It has been proposed that activation of the ras oncoprotein might directly influence both cell cycle checkpoints [START_REF] Marshall | Small GTPases and cell cycle regulation[END_REF][START_REF] Albanese | Transforming p21 ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions[END_REF][START_REF] Mittnacht | Ras signalling is required for inactivation of the tumor suppressor pRb cell-cycle control protein[END_REF]. We therefore examined the expression of both components of these complexes in the nucleus as well as the activity of their kinase sub-units.

Nuclear cyclin D and Cdk4 expression and activity

Cyclin D expression did not change significantly during the first 48 hours after PH regardless whether the animals were administered the solvent (DMSO) or FTS, the active substance (figure 7a). Although, FTS reduced the expression of Cdk4 during the first 24 hours after PH (figure 7a,b), this reduction did not translate into a significant impact on the activity of this kinase (figure 7c).

Nuclear cyclin E and Cdk2 expression and activity

FTS treatment decreased Cyclin E expression during the first 48 hours after PH compared DMSO-administered animals (figure 8a). FTS treatment also reduced Cdk2 expression during the first 24 hours after PH in contrast to DMSO animals in which a steady and progressive increase in Cdk2 levels occurred throughout the experiment (figure 8a,b). As a consequence of reduced Cyclin E and Cdk2 levels, Cdk2 activity remained strikingly lower in FTS treated animals during the 48 hours after PH (figure 8c). Interestingly, Cdk2 activity at 48 hours after PH, i.e. more than 24 hours after the last dose of FTS, did not recover in the FTS group despite an increase of nuclear Cdk2 in both DMSO and FTS-treated animals (figure 8a,b,c).

These observations suggest that FTS exerts an inhibitory effect on the Cdk2 kinase activity even 24 hours beyond drug withdrawal.

p27 and p21 mRNA expression

We next investigated whether reduced Cdk2 activity could be related to increased expression of the Cdk inhibitors p27 and p21. No significant differences in p27 mRNA expression was found at any time point in FTS-treated animals compared to their respective control (DMSO) group (data not shown).

until 48 hours after PH in DMSO-treated animals. Compared to the kinetics of DNA synthesis, the peak in p21 expression followed the up-regulation of Cdk2 activity as well as the first wave of DNA synthesis in DMSO-treated animals. By contrast, p21 levels were significantly higher in FTS-treated animals at 12 hours after PH preceding the increase in Cdk2 activity observed in DMSO-treated animals as well as the first wave of DNA synthesis, and remained elevated until 48 hours after PH (figure 9). In parallel to strong reduction in DNA synthesis in the FTS group, peak levels of p21 expression were also lower in FTStreated animals compared to the DMSO-group. These observations suggest that p21 might contribute to down-regulate Cdk2 activity and subsequently DNA synthesis in FTS-treated animals.

Discussion

The ras oncoprotein is thought to play an important role in cell proliferation mechanisms including those of hepatocytes. The aim of our study was to examine whether interfering with the activation of the mature ras oncoprotein would affect liver cell proliferation, an approach that could be exploited in liver carcinogenesis. Therefore, we studied the effect of FTS on HepG2 cells, a human hepatoblastoma derived cell line in vitro and on normal hepatocytes in vivo after partial hepatectomy in rats. FTS has been shown to inhibit the activation of the ras oncoprotein in vitro [START_REF] Haklai | Dislodgement and accelerated degradation of Ras[END_REF][START_REF] Marciano | Farnesyl derivatives of rigid carboxylic acids -inhibitors of ras-dependent cell growth[END_REF][START_REF] Marom | Selective inhibition of Ras-dependent cell growth by farnesylthiosalicylic acid[END_REF][START_REF] Gana-Weisz | The Ras antagonist S-farnesylthiosalicylic acid induces inhibition of MAPK activation[END_REF]. This inhibiting effect is either due to inhibition of Ras farnesylation, that is essential for its membrane anchorage [START_REF] Kato | Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity[END_REF], or results from binding of FTS to the specific Ras membrane binding-site acting as a real competitive inhibitor of Ras membrane anchorage [START_REF] Marom | Selective inhibition of Ras-dependent cell growth by farnesylthiosalicylic acid[END_REF]. Our data clearly show that FTS does inhibit proliferation of the hepatic tumour cell line HepG2 in vitro which suggests that the treatment acts on transformed hepatocytes. Similarly, it has been shown that inhibition of Ras by FTS induces inhibition of the human tumor cells Panc-1 proliferation in vitro and in vivo [START_REF] Egozi | Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist[END_REF][START_REF] Weisz | A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice[END_REF]. Low dose chronic administration of FTS to rats did also inhibit stellate cell proliferation thereby preventing the occurrence of cirrhosis [START_REF] Reif | The Ras antagonist, farnesylthiosalicylic acid (FTS), inhibits experimentally-induced liver cirrhosis in rats[END_REF].When FTS was administered to rats during the first 24 hours after PH, a delay in recovery of the liver mass as well as a highly significant reduction in hepatocytes proliferation was also observed that was confirmed by two independent proliferation markers, PCNA expression and BrdU incorporation. Although cell proliferation in FTS-treated rats remained on levels observed in the DMSO group at 48h after PH, a prominent peak of PCNA expression and BrdU incorporation that usually occurs early after PH, was not seen in FTS-treated animals. This observation suggests that a delay in hepatocytes proliferation persisted at 48 hours after PH and this despite withdrawal of the ras inhibitor at 16h after PH. Interestingly, incomplete recovery of cell proliferation at 48 hours after PH occurred in spite of an increase in cell cycle effector proteins like cyclin D, cdk4 and cdk2 to levels observed in untreated animals. However, neither phospho-ERK 1/2 levels nor Cdk2 kinase activity did fully recover after FTS withdrawal which might at least in part explain the prolonged effect of FTS on cell proliferation. In addition, the Cdk inhibitor p21 increased before initiation of DNA synthesis in FTS-treated animals and remained elevated until 48 hours after PH. These findings suggest that the bock in G1/S phase transition and the prolonged down-regulation in Cdk2 activity could be related to persistently elevated p21 levels in FTS-treated animals. Since later time points have not been examined in our study, we cannot exclude that the cells might finally escape from the block leading to complete liver regeneration on the long term. Recent data confirms that FTS acts in a specific manner on the active, GTP-bound forms of ras proteins. It principally competes with ras-GTP for binding to specific binding sites in the plasma membrane preventing active ras from activating intracellular down-stream signalling pathways [START_REF] Zundelevich | Supression of lung cancer tumor growth in a nude Mouse model by the Ras inhibitor salirasib (farnesylthiosalicylic acid)[END_REF]. Although FTS might have some effects on farnesylation of ras through interaction with the PPMTase, the physiological impact of these interactions is rather minor. Therefore, it is unlikely that effects on other farnesylated molecules besides ras account for inhibition of cellular proliferation in our study. The ras-ERK pathway is primarily involved in cell proliferation in the regenerating liver. However, we cannot exclude, that other ras-dependent downstream signalling pathways, e. g. the AKT or SAPkinase pathway, might have influenced to some extent the proliferative response elicited by partial hepatectomy.

As previously shown by our group, Ckd2 kinase activity is essential for transition from G1 to S-phase and blockage or reduction of its activity blocked hepatocytes in late G1 phase [START_REF] Stärkel | Blunted DNA synthesis and delayed s-phase entry following inhibition of Cdk2 activity in the regenerating rat liver[END_REF]. It has been shown that ras expression increases at a critical time point in the regenerating rat liver [START_REF] Goyette | Expression of cellular oncogenes during liver regeneration[END_REF][START_REF] Thompson | Sequential protooncogene expression during rat liver regeneration[END_REF]. In addition, interference with ras gene expression also reduced liver regeneration presumably through reduction of cyclin E/Cdk2 expression [START_REF] Lüdde | Ras adenovirus modulate cyclin E protein expression and DNA synthesis after partial hepatectomy[END_REF]. Our results of strongly reduced liver cell proliferation using the Cdk2 inhibitor roscovitine [START_REF] Stärkel | Blunted DNA synthesis and delayed s-phase entry following inhibition of Cdk2 activity in the regenerating rat liver[END_REF] and the ras inhibitor FTS in this study adds further evidence for a critical role of the ras-cyclin E/Cdk2 axis in hepatocytes proliferation. It has been suggested that ras signalling via the MAPkinase pathway may be important in this process. The observations made in FTS treated animals in our study sustain an important role for the ras-raf-MAPkinase pathway in liver cell proliferation. Reduced ras membrane expression, increased cytosolic ras retention, and decreased activation of raf and Erk 1/2 in FTS treated animals suggests that the antiproliferative effect of FTS is at least partially mediated by inhibition of the ras-raf-MAPkinase pathway. How this pathway is connected to the cell cycle still remains ill understood. It has been shown that the Ras protein activates the transcription of the cyclin D1 gene [START_REF] Mittnacht | Ras signalling is required for inactivation of the tumor suppressor pRb cell-cycle control protein[END_REF]. Our results are not in favour of a major effect on this cell cycle effector since cyclin D1 nuclear protein levels did not change significantly after administration of the ras inhibitor FTS. In addition, the activity of its preferred kinase partner, cdk4, was also not affected in the nucleus by the treatment making it unlikely that the inhibitory effect is a result of a reduced activation of the cyclin D/cdk4 complex. By contrast, FTS treatment reduced cyclin E and cdk2 nuclear expression with a prolonged inhibitory effect on cdk2 kinase activity in the nucleus. These results suggest that the ras-raf-MAPkinase pathway principally modulates the activity of the cyclin E/cdk2 complex in our experimental conditions. A similar observation has been made by Lüdde et al. who showed that the Ras activation, in vivo, has a direct impact on the hepatocytes proliferation by modulating the activity of the cylin E/cdk2 complex [START_REF] Lüdde | Ras adenovirus modulate cyclin E protein expression and DNA synthesis after partial hepatectomy[END_REF].

The partial hepatectomy model has been chosen since it allows to study vigorous and synchronous hepatocyte proliferation under strictly controlled and reproducible conditions.

The observation that the inhibitory effect of FTS on cell proliferation extended beyond drug withdrawal in this model is of particular interest from the oncology point of view. Tumour cells show less vigorous, non-synchronized proliferation patterns over a longer time period.

Given the prolonged effect of FTS, these cells might therefore remain susceptible to the antiproliferative effect of the compound. With our demonstration that FTS does inhibit proliferation of hepatic tumour cells in vitro as well as proliferation of normal hepatocytes in vivo, it is tempting to speculate that FTS might have anti-proliferative effects in hepatocellular carcinoma in vivo. Indeed, the ras-raf-MAPkinase signalling pathway seems to be important in liver tumour development and ras mutations or activation of the MAPkinase pathway have been demonstrated in hepatocellular carcinoma [START_REF] Yoshida | Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors[END_REF][START_REF] Calvisi | Ubiquitous activation of Ras and Jak/Stat pathways in human HCC[END_REF].

Taken together, our results show that interfering with the activation of the ras oncoprotein in hepatic tumour cells in vitro as well as in normal hepatocytes in vivo that have acquired a high proliferative competence dramatically reduces their ability to proliferate. This effect lasted for at least 48 hours after partial hepatectomy even though the inhibitor was withdrawn 16 hours after PH. Mutations of Ras seem to be involved in the development of many cancers including the liver [START_REF] Lowy | Function and regulation of ras[END_REF][START_REF] Kelloff | Progress in chemoprevention drug development: The promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancer-a plan to move forward[END_REF]. Knowing from our data that the ras inhibitor FTS is capable to inhibit normal and malignant hepatocytes proliferation with minimal toxic manifestations, it seems worthwhile to test the preventive and/or therapeutic effects of this compound in adequate animal models of hepatocellular carcinomas. Such an approach may potentially lead to the development of new therapeutic strategies in humans. The figure is representative of 3 independent experiments. FTS treatment decreased nuclear cyclin E expression after PH. In addition, nuclear Cdk2 levels were significantly reduced 24 hours after PH but strongly increased thereafter. However and despite an increase in Cdk2 levels at 48 hours after PH, Cdk2 kinase activity remained depressed throughout the 48 hours after PH in FTS treated animals.

Legends to figures

Figure 1 .

 1 Figure 1. A. Representative HepG2 cell density after 7 days of culture in cultures without treatment (control), exposed to the solvent DMSO only or treated with the ras inhibitor FTS (DMSO+FTS). In contrast to control and DMSO cell cultures that nearly reached confluence, a strongly reduced cell density was observed in FTS-treated cell cultures. B. Cell count and BrdU incorporation per culture dish in HEPG2 cells after 7 days of culture without treatment (CTL), exposed to the solvent DMSO only or treated with the ras inhibitor FTS (DMSO+FTS). FTS treatment induced a significant decrease in the number of cells per dish as well as in BrdU incorporation into cells. Represented is the mean value ±SD of 3 independent experiments. P values refer to the control (a) and DMSO (b) cultures, respectively.

Figure 2 .

 2 Figure 2. Western blot of PCNA and densitometry analysis of the blots of HepG2 cells after 7 days of culture in cultures without treatment (control, CTL), exposed to the solvent DMSO only or treated with the ras inhibitor FTS (DMSO+FTS). FTS treatment induced a significant reduction in PCNA expression compared to control or DMSO cultures. The mean value ±SD of 3 independent experiments is represented in the densitometry analysis. P values refer to the control (a) and DMSO (b) cultures, respectively.

Figure 3 .

 3 Figure 3. PCNA expression in DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). A. Representative Western blot of PCNA expression in liver homogenates. B. Quantification of PCNA expression on Western blots by densitometry. FTS administration leads to a significant reduction of PCNA expression at 12 and 24 hours after PH.

Figure 4 .

 4 Figure 4. BrdU incorporation into DNA in DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). A. Quantification of BrdU positive hepatocytes by flow cytometry. B. Representative immunohistochemical staining of BrdU positive nuclei in hepatocytes 24 hours after PH. BrdU incorporation into hepatocytes nuclei was hardly detectable 12 hours after PH and significantly reduced at 24 hours in FTS treated animals.

Figure 5 .

 5 Figure 5. Representative Western blot of Ras expression in the cytosol (upper panel) and in membrane fractions (lower panel) of DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). FTS treatment leads to increased cytosolic localisation of ras and reduces the membrane bound fraction of the oncoprotein at all investigated time points after PH.

Figure 6 .

 6 Figure 6. Activation of the MAPkinase signalling pathway downstream of ras in DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). A. Representative Western blot of p-raf expression in membrane fractions of DMSO and FTS treated animals 24 hours after partial hepatectomy (PH). The membrane bound phosphorylated form of raf was only detected in DMSO but not in FTS treated animals at the 24 hours time point after PH. B. Representative Western blot of phospo-Erk 1/2 (pErk) and total Erk (t-Erk) expression in liver homogenates (D=DMSO, F=FTS). C. Quantification of phospo-Erk 1/2 normalized to total Erk expression (pErk/tErk) on Western blots by densitometry. FTS administration leads to a reduction of phospo-Erk 1/2 expression at all investigated time points after PH.

Figure 7 .

 7 Figure 7. Cyclin D1 and Cdk4 nuclear expression and activity in DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). A. Representative Western blot of cyclin D1 and Cdk4 expression in liver nuclear extracts (D=DMSO, F=FTS, C=untreated control before PH). B. Quantification of Cdk4 expression on Western blots by densitometry. C. Immunoprecipitation of Cdk4 nuclear protein followed by a Cdk4 kinase assay using the Rb (769) fusion protein as a substrate (C=untreated control before PH). The figure is representative of 3 independent experiments. FTS treatment did not affect nuclear cyclin D1 expression at any time point of the experiment but significantly reduced nuclear Cdk4 expression 24 hours after PH. However, reduced Cdk4 levels had no impact on Cdk4 kinase activity that remained almost constant throughout the 48 hours after PH.

Figure 8 .

 8 Figure 8. Cyclin E and Cdk2 nuclear expression and activity in DMSO (vector) and FTS (active compound) treated animals at various time points after partial hepatectomy (PH). A. Representative Western blot of cyclin E and Cdk2 expression in liver nuclear extracts (D=DMSO, F=FTS, C=untreated control before PH). B. Quantification of Cdk2 expression on Western blots by densitometry. C. Immunoprecipitation of Cdk2 nuclear protein followed by the histone H1 kinase assay to assess Cdk2 kinase activity (C=untreated control before PH).

Figure 9 . 22 Clinical

 922 Figure 9. Quantitative PCR of p21 mRNA expression normalized to their respective Rpl19 values (internal standard) in DMSO and FTS-treated animals at various time point after partial

Table 1 .

 1 Antibodies and working conditions used for Western blotting

	Antibody	Reaction conditions	Source
	mouse monoclonal anti-	1/750; overnight	Dako, Glostrup, Denmark
	PCNA
	mouse monoclonal anti-Ras 1/1000; 2h	Upstate, Lake Placid, NY, USA
	rabbit polyclonal anti-	1/500; 2h	Santa Cruz, Santa Cruz, CA, USA
	Phospho-Raf-1
	Rabbit polyclonal anti	1/500; overnight	Cell Signaling, Beverly, MA, USA
	Phospho-p44/42 Map kinase
	Mouse monoclonal anti-	1/500; overnight	BD Biosciences, Erembodegem,
	ERK1		Belgium
	mouse monoclonal anti-b-	1/80.000; 1h	Sigma, Saint Louis, MI, USA
	actine
	secondary peroxidase-	1/20.000 to 1/40000; 1h	Jackson ImmunoResearch, West
	conjugated goat anti-mouse		Grove, PA, USA
	secondary peroxidase-	1/20.000 to 1/100000; 1h Jackson ImmunoResearch
	conjugated goat anti-rabbit

Clinical Science Immediate Publication. Published on 3 Aug 2007 as manuscript CS20070152 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 .

 2 Primers used for quantitative PCR analysis

	Gene	forward	reverse
	p21	C CTGTTCCACACAGGAGCAA	C ATGAGCGCATCGCAATC
	p27	C TTC CGCCTGCAGA AAC	C TTCTCCAAGT CCCGGGTTAG
	ras	AGCATAAACTGCGGAAACTGAAC CATGAGCTGCAAGTGTGTGCT
	RPL19 CAAGCGGATTCTCATGGAACA	TGGTCAGCCAGGAGCTTCTT

Clinical Science Immediate Publication. Published on 3 Aug 2007 as manuscript CS20070152 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 Aug 2007 as manuscript CS20070152 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 Aug 2007 as manuscript CS20070152 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 Aug 2007 as manuscript CS20070152 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgments

This work has been supported by a research grant from Astra Zeneca, Belgium


Dr Amal Muthumala
email: a.muthumala@ucl.ac.uk

David R Gable

Jutta Palmen

Jackie A Cooper

Jeffrey W Stephens

George J Miller

Steve E Humphries

Is the influence of variation in the ACE gene on the prospective risk of type 2 diabetes in middle aged men modified by obesity?

Keywords: ACE gene, Type 2 diabetes, Obesity, BMI Clinical Science Immediate

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

Introduction

It is likely that angiotensin converting enzyme (ACE) plays a role in the pathogenesis of type 2 diabetes (T2DM) as a renin-angiotensin system exists in the relevant metabolically active tissues, namely adipose tissue, skeletal muscle and pancreas [START_REF] Karlsson | Human adipose tissue expresses angiotensinogen and enzymes required for its conversion to angiotensin II[END_REF].

Angiotensin II, the product of ACE's action on Angiotensin I, is known to be proinflammatory and cause insulin resistance [START_REF] Ogihara | Angiotensin II-Induced Insulin Resistance Is Associated With Enhanced Insulin Signaling[END_REF]. Intervention to disrupt the reninangiotensin system, by the administration of ACE-inhibitors or Angiotensin II Receptor blockers (ARB), has been shown to prevent the development of T2DM in high-risk individuals in a recent meta-analysis [START_REF] Scheen | Renin-angiotensin system inhibition prevents type 2 diabetes mellitus -Part 1. A meta-analysis of randomised clinical trials[END_REF]. This raises the possibility that these individuals with genetically lower levels of ACE may be protected from the development of T2DM.

Serum and tissue ACE activity is strongly associated with a common variant in the ACE gene, with the presence of an insertion (I) of a 287 bp fragment in intron [START_REF] Florez | Effects of the type 2 diabetes-associated PPARG P12A polymorphism on progression to diabetes and response to troglitazone[END_REF], associated with lower ACE activity, and the deletion (D) associated with higher ACE activity [4;5]. The impact of the ACE I/D polymorphism on T2DM risk has been widely studied with varying results. There are however a number of studies in which no association has been found between this genotype and T2DM [6;7]. The D allele was associated with a higher T2DM risk [OR=1.52,p=0.02] in a case-control study [START_REF] Stephens | The D allele of the ACE I/D common gene variant is associated with Type 2 diabetes mellitus in Caucasian subjects[END_REF].

In those with T2DM, D allele carriers have poorer outcomes, with a higher risk of myocardial infarction and renal disease [START_REF] Kennon | Angiotensinconverting enzyme gene and diabetes mellitus[END_REF], and a worse response when starting on an intensive hypoglycaemic medication regime [START_REF] Salonen | Association between angiotensin-converting enzyme insertion/deletion polymorphism and treatment response in type 2 diabetic patients[END_REF]. Paradoxically the D allele has been associated with a higher insulin sensitivity [START_REF] Panahloo | The Insertion Allele of the ACE Gene I/D Polymorphism : A Candidate Gene for Insulin Resistance?[END_REF]. The majority of these studies are limited by being cross-sectional and we therefore set out to examine the hypothesis that men with the low ACE II genotype would be protected from developing T2DM in a prospective 15 year follow up study.

Recently studies have suggested that the ACE gene modulates the development of disease phenotypes through interaction with key environmental factors. Interaction between smoking and the ACE polymorphism has been associated with a higher cardiovascular disease mortality at a younger age [START_REF] Sayed-Tabatabaei | Angiotensin converting enzyme gene polymorphism and cardiovascular morbidity and mortality: the Rotterdam Study[END_REF]. Interaction between the ACE D allele and hypertension leading to a higher risk of heart failure has also been reported [START_REF] Schut | Angiotensin converting enzyme insertion/deletion polymorphism and the risk of heart failure in hypertensive subjects[END_REF]. We are not aware of any reported ACE gene-environment interaction influencing T2DM, however combined SNP interactions or haplotype effects have been reported in diabetic nephropathy [14;15]. Only a few gene-environment interactions have been reported so far in T2DM, with an interaction between PPARgamma Pro12Ala and obesity on T2DM risk [START_REF] Florez | Effects of the type 2 diabetes-associated PPARG P12A polymorphism on progression to diabetes and response to troglitazone[END_REF], while an interaction between lifestyle intervention and a TCF7L2 SNP on T2DM risk has been reported [START_REF] Florez | TCF7L2 polymorphisms and progression to diabetes in the Diabetes Prevention Program[END_REF]. For T2DM, the major environmental determinant of risk is obesity [START_REF] Gable | Variation in the UCP2-UCP3 gene cluster predicts the development of type 2 diabetes in healthy middle-aged men[END_REF]. In view of the raised inflammatory state in obesity, and Angiotensin II being pro-inflammatory, we looked for interaction between obesity and ACE genotype in determining prospective T2DM risk.

Methods:

Subjects were recruited from the prospective Second Northwick Park Heart Study (NPHSII), detailed elsewhere [START_REF] Miller | The Effects of Quality and Timing of Venipuncture on Markers of Blood-Coagulation in Healthy Middle-Aged Men[END_REF]. The study was approved by the institutional ethics committees and performed in accordance with the declaration of Helsinki. Briefly, 3012 unrelated healthy Caucasian middle aged male subjects were enlisted from nine United Kingdom general medical practices throughout the UK and prospectively followed for development of CHD from 1989. Self report by questionnaire identified those with T2DM at baseline (n=76) who were excluded from the prospective analysis.

Subjects requiring insulin, oral hypoglycaemics or any other cardiovascular medication were excluded from entry into NPHSII [START_REF] Miller | The Effects of Quality and Timing of Venipuncture on Markers of Blood-Coagulation in Healthy Middle-Aged Men[END_REF]. New cases were identified by practice note search for physician-diagnosed and treated T2DM according to current national guidelines. The reporting of T2DM was done thoroughly and systematically.

Out of the initial 3012 recruits, 76 with T2DM were excluded, and in 230, DNA could not be extracted. DNA was therefore available for 2706 eligible men and genotype obtained in 2642 subjects (97.5 %). There were only significant differences in SBP and fibrinogen between the group of 230 and 2706 eligible samples but the absolute differences were small and unlikely to have made an impact (supplementary information -Table 1). There were no difference in baseline characteristics between the 64 drop-outs and the 2642 subjects with ACE genotype (not shown). Two of those individuals without T2DM and one with T2DM did not have height recorded and so there are missing data on BMI in these three individuals. Genotypes were determined by leukocyte DNA polymerase chain reaction amplification (PCR) using published primers and conditions for ACE [START_REF] O'dell | Rapid methods for population-scale analysis for gene polymorphisms -the ace gene as an example[END_REF] and resolved using Microarray Diagonal Gel Electrophoresis (MADGE) for approximately 87% of sample and using Taqman, primers and probes for rs4341 (in complete LD with ACE I/D polymorphism) as described in [START_REF] Tanaka | An alternative fast and convenient genotyping method for the screening of angiotensin converting enzyme gene polymorphisms[END_REF] for the remaining samples. Genotype was determined by two independent technicians blind to subject outcome. One DNA array (94 samples) was genotyped for both ACE I/D and rs4341 polymorphisms with 100% concordance.

Obesity was defined as BMI>30 kg/m 2 . Results are presented as hazard ratios (HR) obtained with their corresponding 95% CI. A dominant genetic model was assumed based on the results from our previous cross-sectional study and other studies [8;10;13].

Statistical analysis

Statistical Analysis was performed using 'Intercooled STATA' (version 8.2, STATA Corporation, Texas). Baseline characteristics were transformed to a normal distribution as appropriate. Hazard ratios (HR) were obtained from Cox proportional hazard models with their corresponding 95% CI. All models included age as a covariate and were stratified by practice in order to take into account modest differences in the baseline hazard by recruitment site. Interactions were tested as deviations from multiplicative effects in the Cox model using the likelihood ratio test.

Scaled Schoenfeld residuals were used to verify that relative hazards were constant over time. Frequencies were compared by Chi-squared test.

Results:

ACE genotype data was obtained in 2642 men. After 15 years of follow-up, 153 men with ACE genotype developed T2DM. The remaining 2489 men were alive and had not developed T2DM. Baseline BMI, obesity, C-reactive protein (CRP), triglyceride, cholesterol and blood pressure were all associated with increased risk of development of T2DM (Table 1). Alcohol consumption was not different between those who developed T2DM and those who did not. The highest risk was that associated with obesity (HR 3.74[2.66-5.26] p<0.0001). When a stepwise model was used to determine which of these variables were independently associated, BMI remained the most significant predictor, with an age and practice-adjusted HR of 1.91 ([1.64-2.24] p<0.0001). There was no significant difference between those who developed T2DM and those that did not, in ACE genotype distribution (p=0.48) or D allele frequency 6 (p=0.87) (Table 1). Distribution of genotypes was as expected for Hardy-Weinberg equilibrium. HR (adjusted for age and practice) for developing T2DM for DD+ID vs II was 0.91 [0.63-1.32] p=0.63. To eliminate the possibility of confounding we confirmed that there were no significant differences in baseline BMI, blood pressure, lipid parameters, age, CRP or alcohol consumption between the different genotype groups.

Since obesity was the strongest risk factor for development of T2DM and in view of the potential for escalating the inflammatory burden in obese individuals, the interaction between ACE genotype and obesity on 15 year risk of development of T2DM (adjusted for age and practice) was examined. In non-obese men, D allele carriers had a non-significantly lower risk of T2DM -HR ID+DD v II 0.69 [0.45-1.06] while in obese men the association was reversed, HR ID+DD v II 2.11 [0.95-4.69] with a significant genotype-obesity interaction (p=0.02). Figure 1 demonstrates that when adjusting for triglycerides, CRP and SBP (three strongest predictors of T2DM (table 1) which may also be causal) the risk of T2DM in obese men was similarly higher in D allele carriers compared to II homozygotes (HR=4.26 [1.30-13.93]), while in lean men there was no genotype difference in risk (HR=0.75[0.46-1.22]), with an overall interaction p=0.01. A Kaplan-Meier plot (Figure 2) demonstrates that development of T2DM in obese subjects occurs much earlier than in non-obese men, with the curve for obese D carriers separating from obese II homozygotes between 7-10 years. It also shows that after 15 years those subjects with the lowest T2DM risk are the non-obese D allele carriers. When analysis was carried out using BMI as a continuous variable the HR for a one standard deviation increase in BMI was found to be higher, but not significantly so with ID/DD vs II HR 2.18 [1.83-2.61] vs 1.73 [1.27-2.36] p=0.20. However there was clear divergence of risk after BMI>30kg/m2 (supplementary information). Analysis using BMI in four categories using cut-offs previously reported [START_REF] Humphries | Common variants in the TCF7L2 gene and predisposition to type 2 diabetes in UK European Whites, Indian Asians and Afro-Caribbean men and women[END_REF] showed a non-linear pattern of interaction with a threshold effect evident, with the D allele being associated with risk of T2DM (adjusted for age, practice, triglycerides, CRP, SBP) only at high BMI (genotype-BMI interaction p=0.02) as demonstrated in Figure 3.

On the basis of these findings the results in our previous cross-sectional study [START_REF] Stephens | The D allele of the ACE I/D common gene variant is associated with Type 2 diabetes mellitus in Caucasian subjects[END_REF] were re-examined, where those with T2DM in the UDACS study (University College London Diabetes and Cardiovascular disease Study) [START_REF] Dhamrait | Cardiovascular risk in healthy men and markers of oxidative stress in diabetic men are associated with common variation in the gene for uncoupling protein 2[END_REF] were compared to those without T2DM in NPHSII (controls). Analysis was confined to Caucasian subjects with T2DM (605, of whom 574 were successfully genotyped for the ACE I/D gene variant). T2DM was defined as those subjects who fulfilled WHO criteria and not requiring insulin within twelve months of diagnosis. In non-obese patients there was no significant difference in risk of T2DM between D allele carriers and II homozygotes, but in obese men the risk of T2DM was significantly higher in D allele carriers (Odds Ratio 1.79 [1.17-2.72] p=0.007), although the genotype-obesity interaction was not significant (p=0. [START_REF] Humphries | Common variants in the TCF7L2 gene and predisposition to type 2 diabetes in UK European Whites, Indian Asians and Afro-Caribbean men and women[END_REF]. This is demonstrated in Figure 4. A similar pattern of association was present when analysis was confined to only male cases in UDACS, with no genotype difference in risk in non-obese men (D allele carrier 1.28 [0.89-1.85] vs II) but in obese men, OR of T2DM for D allele carriers vs II subjects was 2.26 [1.31-3.91] with genotype-obesity interaction p=0.09.

Discussion:

Based on the protective effect of ACE inhibitors and ARBs on the development of T2DM in a meta-analysis of large clinical trials [START_REF] Scheen | Renin-angiotensin system inhibition prevents type 2 diabetes mellitus -Part 1. A meta-analysis of randomised clinical trials[END_REF], we tested the hypothesis that ACE II subjects, with genetically determined lower ACE levels, would have a lower rate of development of T2DM. This effect was confined to obese men where D allele carriers had a significantly greater risk than their II counterparts, but in non-obese men there was no difference in genotype risk. Possession of the D allele, therefore, may be increasingly harmful when individuals are obese. Further examination suggested that this was not a linear effect of BMI on risk, but rather the effect only became significant once the individual had a BMI>30 kg/m 2 . In a previously published crosssectional study of T2DM [START_REF] Stephens | The D allele of the ACE I/D common gene variant is associated with Type 2 diabetes mellitus in Caucasian subjects[END_REF], re-analysis of the data demonstrated a trend where D allele carriage was associated with risk of T2DM in only obese subjects.

The mechanism of the ACE gene variant and its relationship with obesity in the pathogenesis of T2DM is currently unclear, but is likely to be due to the impact of the different levels of plasma and tissue ACE that will be present constitutively, with the II subjects having roughly 40% lower levels than DD subjects [4;5]. The higher serum and tissue ACE associated with the D allele would lead to higher Angiotensin II (AngII) levels. Renin angiotensin systems are present in the circulation and several tissues in which glucose metabolism are controlled including skeletal muscle, pancreas and adipose tissue. There is now considerable evidence for AngII promoting the development of T2DM through a number of mechanisms, including interfering with insulin signalling, pro-inflammatory effect on tissue beds causing endothelial dysfunction, inhibiting adipocyte differentiation, and causing beta cell dysfunction via oxidative stress [START_REF] Hattori | Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats[END_REF][START_REF] Leung | Pancreatic islet renin angiotensin system -Its novel roles in islet function and in diabetes mellitus[END_REF][START_REF] Engeli | The adipose-tissue reninangiotensin-aldosterone system: role in the metabolic syndrome?[END_REF]. It is likely that the higher AngII levels in D allele carriers would have a bigger impact in the obese state where there is already a greater inflammatory and diabetogenic burden. It is however important to note the wide 95% CI limits for D allele carriers (adjusted for age, practice, Triglycerides, CRP, SBP).

This would be in keeping with a small effect of the ACE (I/D) polymorphism on risk of development of T2DM which ties in with conclusions from the DREAM Trial [START_REF] The | Effect of Ramipril on the Incidence of Diabetes[END_REF] where Ramipril did not reduce incidence of T2DM in high-risk individuals but suggested a small benefit in glucose metabolism.

Adjustments of the ACE risk effects were made for SBP, triglycerides and CRP because with BMI, these were the strongest predictors of T2DM development in this cohort. Such adjustments did not materially alter the genotype risk pattern seen. All three factors may be involved in the same causal pathway in T2DM pathogenesis. SBP can directly activate the renin angiotensin system [START_REF] Reja | Upregulation of Angiotensin AT1 Receptor and Intracellular Kinase Gene Expression in Hypertensive Rats[END_REF]. Non-esterified fatty acids (from triglyceride hydrolysis) directly inhibits insulin signalling [START_REF] Wang | Free Fatty Acids Inhibit Insulin Signaling-Stimulated Endothelial Nitric Oxide Synthase Activation Through Upregulating PTEN or Inhibiting Akt Kinase[END_REF] and excess intracellular triglycerides promotes increased oxidative stress and inflammation which can cause insulin resistance [START_REF] Bakker | Cytosolic triglycerides and oxidative stress in central obesity: the missing link between excessive atherosclerosis, endothelial dysfunction, and [beta]-cell failure?[END_REF]. AngII's properties of affecting insulin signalling and causing oxidative stress would directly affect this. CRP may act together with AngII in promoting insulin resistance again through direct and indirect effects on insulin signalling and oxidative stress [START_REF] Xu | C-Reactive Protein Suppresses Insulin Signaling in Endothelial Cells: Role of Spleen Tyrosine Kinase[END_REF].

There are several limitations to this study. The method of identification of NPHSII men with T2DM, by the medical record search, is unlikely to include any false positive diagnosis, but, in the absence of a full recall for fasting glucose testing, some T2DM subjects may be misclassified as healthy. This would result in an underestimate of the 15 year incidence of T2DM, reducing the ability to detect an effect of the polymorphism, and would not confound the genetic association seen.

The lack of plasma glucose and serum insulin data prevents an exploration of whether these parameters are affected by ACE (I/D) genotype, in light of the potential detrimental impact of AngII on insulin signalling and glucose metabolism.

Background diet and physical activity data would have also been useful, as they could affect development of T2DM. Measurement of plasma AngII (or ACE) levels would have been useful in light of the proposed importance of AngII in accentuating the T2DM risk in obese men but these measurements have not been made. However, there are strong and consistent published data which demonstrate that ACE levels (and therefore AngII) in subjects with the ACE D allele are significantly higher than those with the I allele [4;5] and this effect of ACE I/D polymorphism remains the most likely direct mechanism.

The incidence of T2DM could possibly have been under-estimated in the non-obese subjects as a whole, in view of general practioners being more alert to the presence of T2DM in obese rather than non-obese patients. However, this is unlikely in this sample, in view of the thorough, systematic review of patients' notes and it is worth noting that greater than 75% of cases with T2DM were from the non-obese group over the 15 years of follow-up. Even if this did take place, it is unlikely it would affect the major conclusions of the studies, because it is implausible that any such underestimation of T2DM would occur by ACE (I/D) genotype. All of the 2489 subjects without T2DM are still alive since they are 'flagged' with the Office of National Statistics and we receive all death certificates. At recruitment no subjects were taking ACE inhibitors, ARBs and Beta receptor blockers, and although a proportion of those who developed T2DM over follow-up may have been prescribed such medications this is unlikely to have confounded the genetic effect on risk observed here, but would rather have the effect of diluting it.

A further limitation is that the study is underpowered to detect associations in which alleles have a small effect. Multiple testing (in this case for interaction and adjustments) may raise the probability that 'chance' is the explanation for the observed associations and these results may therefore be false positives. A similar prospective study of T2DM was not available to us to try and replicate the findings.

Re-analysis of a previously published case control study showed a higher risk of T2DM in obese D allele carriers compared to II subjects but the genotype-obesity Clinical Science Immediate Publication. Published on 12 Jul 2007 as manuscript CS20070158 interaction is not significant. There was however limited power (only 60% to detect the previously observed effect at the 5% significance level) to demonstrate such an effect size. Studies in which there are much larger numbers of cases and controls are necessary for firm conclusions. This is the first prospective study to show that variation in ACE gene may interact with BMI to increase the risk of T2DM. Prospective gene-association studies are better at teasing out gene-environment interactions than the case-control design since the environment can be measured at baseline, and results are not confounded by survivor bias and retrospective patient recall of lifestyle factors [START_REF] Humphries | In search of genetic precision[END_REF]. Further replication is certainly required to confirm these findings. The combination of the proinflammatory D allele and an already 'at risk' obese state, may well underlie an impaired metabolic profile and a possible increased propensity to T2DM. Categories of BMI were as used in a previous analysis [START_REF] Humphries | Common variants in the TCF7L2 gene and predisposition to type 2 diabetes in UK European Whites, Indian Asians and Afro-Caribbean men and women[END_REF].

Figure 2 :

 2 Figure 2: Kaplan-Meier plot for T2DM by ACE genotype and obesity.

Figure 3 :

 3 Figure 3: Categorical Model for BMI Interaction with ACE genotype on prospective risk of T2DM. ID+DD [Number of subjects/Number developing T2DM] v II [Number of subjects/Number developing T2DM]. Reference group is specific for each BMI category -it is the ratio of number of subjects with II genotype who develop T2DM after 15 years to the total number of II subjects within the same BMI category.

Figure 4 :

 4 Figure 4: Odds Ratio for T2DM in NPHSII controls vs UDACS cases by ACE I/D genotype stratified by obesity. [Number of NPHSII controls/Number of UDACS cases]No significant interaction with BMI fitted as a continuous or categorical variable.

  Figure 1

Clinical Science Immediate Publication. Published on 12 Jul 2007 as manuscript CS20070158

Table 1 :

 1 Baseline characteristics by diabetes in NPHSII men with ACE genotyping Hazard Ratio (adjusted for age, practice, triglycerides, CRP and SBP) for T2DM in non-obese and obese NPHSII men by ACE I/D genotype. [Number of subjects/Number developing T2DM]

		No diabetes	With diabetes
		N=2489	N=153	P value
	Age(years)	56.0 (3.4)	56.2 (3.3)	0.53
	BMI a (kg/m 2 )	26.1 (3.3)	28.6 (3.6)	<0.0001
	Obesity[%(N)]	12.3 (307)	33.6 (51)	<0.0001
	SBP a (mmHg)	136.6 (18.7)	142.2 (19.1)	0.0004
	DBP(mmHg)	84.5 (11.4)	86.5 (11.3)	0.03
	Smoking[%(N)]	28.2 (702)	35.3 (54)	0.06
	Cholesterol(mmol/L)	5.72 (1.01)	5.92 (0.98)	0.02
	Triglyceride a (mmol/L)	1.76 (0.92)	2.27 (1.05)	<0.0001
	Fibrinogen a (g/l)	2.70 (0.51)	2.77 (0.54)	0.12
	CRP a (mg/l)	2.41 (2.46)	3.38 (3.24)	0.0004
	Alcohol
	% drinkers	80.7	77.8	0.37
	Median units/wk	6 [1-16]	5 [1-18]	0.84
	[IQR]
	Genotype distribution	584/1232/673	39/68/46	0.48
	II/ID/DD
	D Allele frequency	0.518	0.523	0.87
	(95% CI)	(0.504-0.532)	(0.465-0.580)

a geometric mean (approx sd); [IQR] -Interquartile Range Figure 1:

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 12 Jul 2007 as manuscript CS20070158 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgments

NPHS-II was supported by the UK Medical Research Council, the US National Institutes of Health (grant NHLBI 33014) and Du Pont Pharma, Wilmington, USA. AM, DRG, JAP, JAC and SEH are supported by the British Heart Foundation (SS/CH92025;FS/04/012;RG2005/014).