Amalia Fernández-Martínez

Belén Mollá

Rafael Mayoral

Lisardo Boscá

Marta Casado

Paloma Martín-Sanz

Dr Paloma Martín Sanz

Cyclo-oxygenase 2 expression impairs serum-withdrawal-induced apoptosis in liver cells

Keywords: COX, prostaglandins, apoptosis, liver, hepatocyte, hydrodynamic transfection

INTRODUCTION

Cyclooxygenase (COX) enzymes catalyze the first step in the biosynthesis of prostaglandins (PGs) and thromboxanes [1;2]. The COX-1 isoform is constitutively expressed in many tissues [START_REF] Pilbeam | Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells[END_REF], whereas COX-2 is induced by growth factors, tumor promoters and cytokines [2;4;5]. The role of COX-2 in the production and development of neoplasic tumors is well established, especially in colon cancer [START_REF] Kawai | Cyclooxygenases and colon cancer[END_REF]. Several authors have moreover reported that increased tissue COX-2 expression and PG production may contribute to the development of other cancers, including hepatocellular carcinoma (HCC) [START_REF] Kondo | Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma[END_REF].

Expression of COX-2 has been demonstrated in many contexts related to liver cell pathologies: regeneration after partial hepatectomy (PH) [8;9]; animal models of cirrhosis [START_REF] Yamamoto | JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis1[END_REF]; human hepatoma cell lines [11;12]; chronic hepatitis C infection [7;13] and HCC [14;15]. Failure of apoptosis is an important event in tumour progression and resistance to cytotoxic therapy, and a principal mechanism of selective COX-2 inhibitors (COXIBS) is their pro-apoptotic activity. Apoptosis is mediated by activation of caspases, a family of cysteine proteases [START_REF] Talanian | Caspases as targets for anti-inflammatory and anti-apoptotic drug discovery[END_REF]. Caspase activation occurs by at least two mechanisms: the extrinsic or death receptor pathway, initiated by agonists of the tumour necrosis factor superfamily such as TNF-α, Fas and Apo2 ligand/TRAIL [START_REF] Ashkenazi | Death receptors: signaling and modulation[END_REF], and the intrinsic or mitochondrial pathway [START_REF] Green | Mitochondria and apoptosis[END_REF]. In the mitochondrial pathway caspase activation principally occurs as a result of the release of cytochrome c from the organelle, a process closely regulated by the Bcl-2 family of proteins. Cytochrome c in the cytosol associates with Apaf-1 and ATP and pre-caspase-9 in a multi-protein complex called the apoptosome. Once activated in the apoptosome, caspase 9 in turn activates downstream executioner caspases, such as caspase-3 and caspase-7 [START_REF] Jiang | Cytochrome c-mediated apoptosis[END_REF]. In liver, the extrinsic and intrinsic apoptotic mechanisms are both operative. Constitutive expression of Fas is found in mouse and human liver, and this pathway appears to be very important in executing apoptosis in healthy hepatocytes and in the pathogenesis of diseases including liver injury, viral hepatitis and cirrhosis [START_REF] Kondo | Essential roles of the Fas ligand in the development of hepatitis[END_REF]. However, HCC is one of the tumours known to be resistant to Fas-mediated apoptosis, because Fas expression is down regulated and the Fas-activated signalling pathway is altered [START_REF] Lee | Expression of Fas and Fas-related molecules in human hepatocellular carcinoma[END_REF].

We have used two approaches to express COX-2 in liver cells in order to elucidate the mechanisms implicated in PGE 2 -dependent inhibition of apoptosis. In the first, we generated liver cell lines expressing COX-2 protein by stable transfection with a vector containing the human COX-2 cDNA. In the second, we used hydrodynamics-based transient transfection to systemically administer Human full-length COX-2 cDNA [START_REF] Jones | Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines[END_REF] in pcDNA1/Amp was a kind gift of S. Prescott's lab (Huntsman Cancer Institute, Utah). The COX-2 cDNA was subsequently cloned into the pcDNA3 expression vector. COX-2-GFP was obtained by inserting 720 bp of the GFP open reading frame (excluding the initiator methionine and the termination codon) from pEGFP-N1 vector into the BspEI site of the human COX-2 cDNA. This inserts the GFP at the C-terminal end of COX-2 polypeptide, as described previously [START_REF] Trifan | Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism[END_REF]. The orientation and integrity of the constructs were determined by sequencing. Largescale preparation of plasmids was done using EndoFree plasmid mega kit (Qiagen).

Cell cycle distribution and cell counting

Cell counts were performed by initially plating 1.6 x10 5 cells. At the indicated times, cells were trypsinised, stained with trypan blue and the cells that excluded trypan blue were counted under the microscope. For the analysis of cell cycle distribution, cells were resuspended in PBS, and fixed in 70% ethanol. Propidium iodide (PI) staining was performed after incubation of the cells with 0.05% PI and 0.1 mg/ml RNAase for 30 min and analysed in a CyAn MLE-R cytometer (DakoCytomation).

Immunocytochemistry

Cells (5x10 4 per well) were cultured in 24-multiwell plates on glass coverslips and maintained overnight in DMEM/1% FBS. Cells were then fixed for 15 min with 4% paraformaldehyde pH 7.0, washed with PBS and permeabilised with methanol for 10 min at -20ºC. After blocking with 3% bovine serum albumin (BSA) for 1 h at room temperature (RT) the cells were incubated for 2 h at RT with the corresponding monoclonal anti-COX-2 antibodies diluted 1:150 in PBS/1% BSA. After three washes with PBS, the coverslips were incubated with a 1:500 dilution of fluorescent secondary antibody (IgG-Cy3, DakoCytomation SA, Barcelona, Spain), washed x3 with PBS, and treated with Hoechst 33258 for 30 min at RT. Fluorescence was visualized and quantified with a MRC 1024 microscope (Bio-Rad) and Lasersharp software. For tissue immunohistochemistry, liver samples were snap frozen in isopentane immersed in liquid N 2 , and serial 10 µm-thick sections were cut onto gelatinised glass with a Microm HM550 sledge microtome. After fixing and blocking, the sections were incubated overnight with GFP antiserum (diluted 1:50; Molecular Probes) in 5% horse serum at 4ºC. After three washes with PBS, the sections were incubated with a 1:500 dilution of fluorescent secondary antibody (IgG-Alexa Fluor 488), washed, and analysed as above. For the detection and quantification of apoptosis, the TUNEL commercial kit for cell death detection (Roche) was used in accordance with the manufacturer's Copyright 2006 Biochemical Society instructions. Histological examination was performed on 4% paraformaldehyde-fixed mouse liver sections prepared by routine histological staining (hematoxylin/eosin) procedures.

Caspase assays

Cell extracts were prepared by lysing in 10 mM HEPES pH 7.9; 1 mM EGTA, 1 mM EDTA, 120 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mM NaF, 1 mM NaVO 4 , 10 mM Na 2 MoO 4 and 0.5 % Nonidet P-40 (buffer A). After centrifugation of the cell lysate the supernatant was stored at -80ºC (cytosolic extract) and protein content was assayed with the Bio-Rad protein reagent. The activities of caspases 3, 8 and 9 in cytosolic extracts were determined with the fluorogenic substrates N-acetyl-DEVD-7-amino-4-trifluoromethylcoumarin, N-acetyl-IETD-7amino-4-trifluoromethylcoumarin, and N-acetyl-LEHD-7-amino-4-trifluoromethylcoumarin, respectively, and in accordance with the supplier's instructions (Calbiochem). The linearity of caspase assays was determined over a 30 min reaction period [START_REF] Hortelano | Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease[END_REF].

Cell extracts and Western blotting

Cells (2-3x10 6 ) or tissue samples (100mg) were homogenized in a lysis buffer containing 10 mM Tris-HCl pH 7.5, 1 mM MgCl 2 , 1 mM EGTA, 10% glycerol, 0.5% 3-((3cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), 1 mM β-mercaptoethanol, and 0.1 mM PMSF. Extracts were vortexed for 30 min at 4ºC and after centrifuging for 20 min at 13,000g the supernatants were stored at -20ºC (cytosolic extracts). To obtain nuclear extracts the pellets were processed in the same volume with 20% glycerol and 0.4M KCl and mixed for 30 min at 4ºC as previously described [START_REF] Casado | Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy[END_REF]. To determine the release of cytochrome c from the mitochondria to the cytosol, cell extracts were obtained by controlled lysis of the plasma membrane as previously described [START_REF] Hortelano | Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease[END_REF]. For Western blot analysis, whole-cell extracts were boiled for

Determination of metabolites

PGE 2 levels were determined in culture media by specific immunoassay (Amersham). Alanine aminotransferase (ALT) was assayed spectrophotometrically in plasma [START_REF] Martin-Sanz | Effect of a rhodium complex on alterations of hepatic function in thioacetamide-induced hyperplastic noduligenesis in rats[END_REF]. Protein levels were determined with the Bradford reagent.

RNA isolation and RT-PCR

Total RNA was extracted from liver with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. One µg total RNA was reverse transcribed with 50U of expand reverse transcriptase and pd(N) 6 Random Hexamer as primer (Amersham Biosciences). The resulting cDNAs were amplified with the following oligonucleotide sequences: COX-2, 5´CAGAGTTGGAAGCACTCTATGG3´

(sense) and 5´CTGTTTTAATGAGCTCTGGATC3´ (antisense); 18s rRNA, 5´GCAATTATTCCCCATGAACGA3´ (sense) and 5´CAAAGGGCAGGGACTTAATCAA3´

(antisense).

Hydrodynamic transient transfection experiments

100 µg of plasmid dissolved in 2 ml isotonic NaCl were injected into the tail veins of 18-22 g adult male Swiss CD1 mice (Charles River) over 8 s (hydrodynamic injection) [START_REF] Liu | Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[END_REF]. Five animals were used for each condition. 24 h after injection, animals were intraperitoneally injected with a single dose of purified hamster anti-mouse FAS monoclonal antibody Jo2 (0.3 µg/g body wt) freshly dissolved in 0.9% NaCl. This dose was chosen on the basis of previous studies [START_REF] Guidotti | Fas/CD95 pathway induces mouse liver regeneration and allows for highly efficient retrovirus-mediated gene transfer[END_REF]. Animals were killed five hours later, and livers were rapidly removed and freeze-clamped in liquid N 2 . Animals were treated in accordance with Institutional Care Instructions.

Data analysis

Data are expressed as means + standard deviation (SD). Statistical significance of differences between the CHL-C and CHL-V groups was evaluated by the U of Mann-Whitney test. All tests have been calculated two-tail, and the significance has been considered at P< 0.05.

Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780

Copyright 2006 Biochemical Society

RESULTS

Characterization of CHL liver cells expressing COX-2

The human Chang liver cell line (CHL) was transfected with the COX-2 expression vector pcDNA3hCOX-2 or the control vector pcDNA3, and selected with 500 µg/ml G418. Among 20 clones analysed, we selected two clones: a control clone CHL-V, transfected with the empty vector; and a COX-2 clone, CHL-C, transfected with pcDNA3hCOX-2 and selected on the basis of its high COX-2 expression. These clones were used for all subsequent experiments. As shown in Fig. 1A, COX-2 protein was significantly expressed in CHL-C cells, as determined by Western blot, but was not expressed in WT or CHL-V cells. COX-1 protein was expressed at low levels and without significant differences in CHL and in the transfected cells. Immunocytochemistry revealed clear staining for COX-2 in the endoplasmic reticulum and perinuclear membranes (Fig. 1B). RT-PCR of COX-2 mRNA confirmed increased COX-2 mRNA expression in CHL-C cells (Fig. 1C). COX-2 expression in liver cells was functional; Fig. 1D shows that CHL-C cells released significantly greater quantities of PGE 2 into the culture medium than did CHL-V (3.7-fold increase). This PGE 2 synthesis was markedly increased after incubation of the cells for 30 min with 30 µM arachidonic acid (AA). The contribution of COX-2 to this process was confirmed by inhibition (>90%) of AA-inducible PGE 2 synthesis after treatment with the COX-2 selective inhibitor DFU (5 µM). When the cells were incubated in the presence of indomethacin (50 µM), PGE 2 levels returned to the control (or even lower) values. These data indicate that COX-2 is the major contributor to PGE 2 synthesis, whereas the contribution of COX-1 could be evaluated in less than 5% of the PGE 2 levels in CHL-C cells. Fig. 1E shows the clear morphological differences between CHL-V and CHL-C cells, with CHL-C being larger and exhibiting a less rounded shape than the CHL-V control cells.

COX-2 expression in liver cells promotes an increase in S-phase cells and inhibits apoptosis

The cell number of CHL-C after 72 h of culture in 10% FBS was greater (129%) than that of CHL-V (Fig. 2A). To assess the role of COX-2 in cell proliferation we analysed the dependence of CHL-V and CHL-C cell number on FBS concentration. As Fig. 2B shows, COX-2-dependent proliferation was evident in the absence and presence of serum (229%, 129%). Analysis of cell cycle distribution by propidium iodide (PI) staining and FACS revealed that COX-2-expression is associated with an increase in the number of cells in S-phase both at 0 and 10% FBS (133%, Fig. 2C), with no significant changes in the G 0 /G 1 and G 2 -M populations observed, when considering nonapoptotic cells (Fig. 2D). Figure 2E shows the distribution of apoptotic and nonapoptotic cells in the different steps of the cell cycle.

Flow cytometry of PI-stained cells revealed an inhibition of apoptosis in CHL-C (COX-2-expressing) cells cultured in the absence of FBS (Fig. 3A-C). After 72 h of culture only 4% of CHL-C cells were apoptotic, compared with 11% of CHL-V cells (Fig. 3B). In order to correlate the number of PI positive cells induced after FBS removal with COX-2 expression, the levels of PGE 2 were measured at various times, and as shown in Fig. 3B (inset), a correlation exists between an increase in PGE 2 synthesis and a decrease in the number of PI positive cells. The inhibition of apoptosis could be mimicked in CHL-V control cells by treating with exogenous PGE 2 (Fig. 3D), and the action of COX-2 metabolites in the reduced apoptosis in CHL-C cells was confirmed by treatment with increasing concentrations of DFU, which progressively increased the numbers of apoptotic cells (Fig. 3E). COX-2 expressing CHL were also resistant to the induction of apoptosis by 10 nM staurosporine, camptothecin or a combination of TNF-α and actinomycin D, with 30% fewer cells in apoptosis compared to CHL-V controls (data not shown). We also overexpressed COX-2 in HepG2, as a different cell line with a basal constitutive COX-2 expression (Fig. 3F) obtaining the same results than in CHL cells. COX-2 overexpression, but not the constitutive level, caused a decrease in the number of apoptotic cells after serum deprivation (Fig. 3G).

COX-2 expression leads to altered expression and activity of apoptosis markers

The data presented so far provide convincing evidence that PGE 2 synthesized by COX-2 suppresses apoptotic signals in CHL-C liver cells. To investigate the mechanisms through which this occurs we first analysed the activities of caspases 3, 8 and 9 by fluorimetry (measured as DEVDase, IETDase and LEHDase activities, respectively). As shown in Fig. 4, the activities of caspases 3 and 9

were markedly lower in CHL-C cells than in control CHL-V cells. Caspase 8 activity was also inhibited in CHL-C cells, but to a lesser extent and with a delay with respect to caspases 3 and 9. These results are in agreement with data obtained in gastric mucosal cells [29], where COX-2 expression also suppresses the activation of caspases 8 and 9 in addition to caspase 3. Copyright 2006 Biochemical Society without FBS, but was not observed even after 72 h in CHL-C cells (Fig. 5D). The protein levels of Bcl-2, xIAP and Mcl-1 did not vary between CHL-V or CHL-C cells; however, Bcl-xL decreased in CHL-V cells from 6 to 48 h of culture in medium without FBS, whereas it was maintained in CHL-C cells (Fig. 5). Release of cytochrome c to the cytosol with the corresponding decrease in the mitochondria and cleavage of poly (ADP-ribose) polymerase (PARP-1) were also observed only in CHL-V cells (Fig. 6A,B).

The PI3K/Akt pathway plays a central role in integrating diverse survival signals. The levels of phospho-Akt (Ser 473 ) increased in cells expressing COX-2. Moreover, when the CHL-C cells were incubated with DFU to inhibit COX-2 the increase in P-Akt was not observed (Fig. 6E), suggesting a role for COX-2 in liver cell survival. The same mechanism occurs in HepG2-COX-2 expressing cells (not shown).

These results indicate that the anti-apoptotic effect induced by COX-2/PGE 2 is mediated by the inhibition of the mitochondrial intrinsic pathway. But the inhibition of caspase 8 (Fig. 4C) suggested the possible involvement of death receptor signalling, known to be important in hepatocyte apoptosis and in the pathogenesis of liver diseases. As shown in Fig. 5 A the levels of Fas protein were scarcely detected in either cell line. The above mentioned failure of FAS antibody to induce apoptosis (data not shown)

indicates that CHL-V and CHL-C cells are resistant to FAS-mediated apoptosis, as occurs in many liver cell lines and in HCC [START_REF] Lee | Expression of Fas and Fas-related molecules in human hepatocellular carcinoma[END_REF].

Transient expression of COX-2 inhibits liver apoptosis in vivo.

To examine whether COX-2 expression inhibits liver-cell apoptosis in living animals, we developed an in vivo model to deliver the COX-2 gene to the liver via hydrodynamics-based transfection. COX-2 was expressed in approximately 35% of the liver cells of animals transfected with a construct encoding GFP-COX-2, as indicated by the associated GFP fluorescence (Fig. 7A). Under these conditions, the levels of ALT determined 24 h after transfection were similar between animals transfected with GFP or with GFP-COX-2, and were in the range of the animals receiving saline (Fig. 7B). The transfected COX-2 was active, since intrahepatic levels of PGE 2 increased in the GFP-COX-2 mice but not in the GFP animals (Fig. 7C). As Fig. 7D-F show, transfection of liver with GFP-COX-2

impaired Fas-dependent apoptosis as indicated by the inhibition of caspase-3 activity and reduced staining in TUNEL analysis. Moreover, the analysis of apoptosis markers in the liver of GFP-COX-2 mice demonstrates an increase in the levels of P-Akt and in the Bcl-2/Bax ratio (Fig. 7G).

Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780

Copyright 2006 Biochemical Society

DISCUSSION

The important role of COX-2 as inhibitor of apoptotic pathways has been demonstrated in vivo in transgenic mouse models of COX-2 expression in several tissues. Transgenic mice expressing COX-2 in mammary gland developed tumours, and these animals had reduced levels of the proapoptotic proteins Bax and Bcl-X L and elevated levels of Bcl-2 [START_REF] Liu | Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[END_REF]. In basal keratinocytes, the results are conflicting, since both epidermal hyperplasia and suppression of tumour development have been described [31;32] Transgenic mice expressing COX-2 in neurons develop age-dependent cognitive deficiencies due to an increase in neuronal apoptosis [START_REF] Andreasson | Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice[END_REF]. A recent study demonstrated hyperplastic gastric tumours in transgenic mice expressing COX-2 and microsomal prostaglandin E synthase [START_REF] Oshima | Hyperplastic gastric tumors induced by activated macrophages in COX-2/mPGES-1 transgenic mice[END_REF].

Although a number of experimental studies have demonstrated a clear positive correlation in vitro between COX-2 expression and the inhibition of apoptosis, the underlying molecular mechanisms are still not fully understood. Most of this work has been done in colon or gastric carcinoma cell lines,

where DuBois et al. [START_REF] Tsujii | Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2[END_REF] demonstrated that rat intestinal epithelial cells (RIE) stably transfected with COX-2 were resistant to butyrate-induced apoptosis. The same authors reported that these cells exhibit a delayed G 1 transit, and this may be related to the resistance to apoptosis. Our results demonstrate that COX-2 expression in liver cells is not associated to significant changes in cell-cycle distribution but is related to the inhibition of cellular apoptosis. Cell cycle arrest in G 0 /G 1 has been described in COX-2expressing endothelial, NIH 3T3, COS-7 and embryonic kidney 293 cells [START_REF] Trifan | Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism[END_REF], but this effect did not require the synthesis of prostaglandins. We did not find significant changes in G 0 /G 1 or G 2 /M cell populations, but the inhibition of apoptosis in COX-2 expressing liver cells was dependent on PGE 2 synthesis.

COX-2 inhibitors provide an indirect means of establishing a relationship between COX-2-

dependent PGs and apoptosis. However, the doses which induce apoptosis are much higher than those needed to inhibit COX-2 activity, indicating that these inhibitors may be acting via COX-2-independent effects, depending on the compound used and the cell type. Some NSAIDS, such as sulindac, potently induce apoptosis in human gastric cancer cells [START_REF] Wu | Growth inhibition and apoptosis induction of Sulindac on Human gastric cancer cells[END_REF], while others such as SC-58125 induce both apoptosis and cell cycle arrest in HCA-7 cells [START_REF] Williams | The role of cyclooxygenases in inflammation, cancer, and development[END_REF]. A prostate apoptosis response gene (Par-4) was found to be up-regulated following exposure of HCA-7 cells to NS-398 [START_REF] Zhang | Par-4, a proapoptotic gene, is regulated by NSAIDs in human colon carcinoma cells[END_REF]. Sulindac, salicylate and aspirin have been shown to inhibit NF-κB signalling by directly blocking the activity of IκB kinase, leading to reconstituted sensitivity of cancer cells to apoptosis [START_REF] Yin | The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(κ)B kinase-β[END_REF]. Additionally, indomethacin, a non-selective COX inhibitor, can act as a direct ligand of PPARγ, a suppressor of tumor cell proliferation [40].

In liver, increased COX-2 expression has recently been demonstrated in patients with HCC, especially in tissue with cirrhosis as well as in well-differentiated tumour tissue [START_REF] Koga | Hepatocellular carcinoma: is there a potential for chemoprevention using cyclooxygenase-2 inhibitors?[END_REF]. In HCC, COX-2 expression seems to be up regulated at early stages and down regulated in advanced stages. Silencing of the COX-2 gene by promoter hypermethylation has been reported to regulate the growth of some HCC cell lines [START_REF] Murata | Promoter hypermethylation silences cyclooxygenase-2 (Cox-2) and regulates growth of human hepatocellular carcinoma cells[END_REF]. However, in vitro studies revealed that NS-398, celecoxib and sulindac effectively inhibited growth of human hepatoma cell lines [11;12]. In mice implanted with hepatoma, nimesulide inhibits tumour growth by inducing apoptosis and overexpression of Bax over Bcl-2 [START_REF] Li | Nimesulide inhibits tumor growth in mice implanted hepatoma: overexpression of Bax over Bcl-2[END_REF]. Moreover, combinations of COX-2 and MEK inhibitors synergistically increase apoptosis in human HCC [START_REF] Schmidt | Novel combination of cyclooxygenase-2 and MEK inhibitors in human hepatocellular carcinoma provides a synergistic increase in apoptosis[END_REF].

Several signalling pathways have been proposed as mediators of COX-2-dependent inhibition of apoptosis. PGE 2 inhibits apoptosis in gastric mucosa cells via the mitochondrial pathway and PKA activation [29]. In cholangiocarcinoma cells, celecoxib suppressed Akt phosphorylation, released cytochrome c to the cytosol and activated caspases -9 and -3 [START_REF] Zhang | Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation[END_REF]. There are a few reports, which describe the direct effect of COX-2 expression on apoptosis in liver or HCC cells. Leng et al. [START_REF] Leng | Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis[END_REF] reported an enhanced phosphorylation of Akt in Hep 3B cells transiently and permanently transfected with a COX-2 expression vector but Bcl-2 is not induced. Celecoxib reduced Akt activation and induced caspase-9 and -3 activation with a concomitant release of cytochrome c. Our results clearly demonstrate that caspase-3 and -9 activities are inhibited in COX-2 expressing cells and the activity of caspase -8 is also decreased in CHL-C cells. The absence of cytochrome c in the cytosol of CHL-C cells after exposure to apoptotic stimulus clearly implies that COX-2 PG products act on the mitochondrial pathway to prevent apoptosis. We did not find significant changes in the expression of IAP proteins. This is in agreement with results obtained in cholangiocarcinoma cells, although cAMP-dependent induction of c-IAP2 has been reported to mediate the anti-apoptotic action of PGE 2 synthesized by COX-2 in intestinal epithelial cells [START_REF] Nishihara | Inhibition of apoptosis in normal and transformed intestinal epithelial cells by cAMP through induction of inhibitor of apoptosis protein (IAP)-2[END_REF]. The levels of Bcl-2 remained unaffected by COX-2 expression in our experiments, in agreement with previous findings in liver cells [START_REF] Kern | Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells[END_REF]. regulating the proapoptotic gene Bax and several death promoting molecules in many cell lines [START_REF] Levine | p53, the cellular gatekeeper for growth and division[END_REF].

The regulation of p53 is complex and implicated different mechanisms such as stability, nuclear accumulation and activity. COX-2 expression negatively affect p53 activity via mechanism that could involve physical interaction and COX-2 positive prostate cancer cells exhibited a decrease p53 stability, nuclear accumulation and activity after hypoxia, both via direct effects on p53 activation and via activation of Mdm2, the primary negative regulator of p53 [START_REF] Liu | Cycloxygenase-2 suppresses hypoxia-induced apoptosis via a combination of direct and indirect inhibition of p53 activity in a human prostate cancer cell line[END_REF]. In this sense, our results showed an important decrease in p53 levels in CHL-C-COX-2 expressing cells.

The results obtained in vivo from mice hydrodynamically transfected with COX-2 clearly demonstrated that PGs produced by COX-2 expression protected the liver against Fas-mediated apoptosis in agreement with the results obtained from the in vitro liver cell lines. Interestingly, the levels of Bcl-2 that are very low in liver, were up-regulated significantly after hydrodynamic transfection of COX-2.

In summary, the data presented in this study indicate that PGs produced by COX-2 in liver, both in vitro and in vivo, inhibit apoptosis dependent on the intrinsic mitochondrial pathway. Given the frequent expression of COX-2 in HCC, COX-2 would be expected to provide these cancers with a survival advantage. This is an important finding and may explain both the anti-apoptotichepatoprotective effect of PGE 2 after acute liver injury and the important contribution of COX-2 to the development of tumorigenesis through increased resistance to apoptosis in HCC.

Biochemical

  Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780 Copyright 2006 Biochemical Society pcDNA3 and pEGFP-N1 were obtained from Invitrogen and BD Biosciences (Clontech), respectively.

Figure 5

 5 Figure5shows the effect of COX-2 expression on the levels of several proteins governing apoptosis fate. As Fig.5A,B Cshows, p53 expression declined notably in CHL-C cells at 0 time and within 6 h of culture in medium without FBS, whereas it was maintained in CHL-V controls. Bax expression, scarcely detected in both cell lines at 10% serum, was increased in CHL-V cells from 6 h

COX- 2

 2 has been reported to promote survival of human lung adenocarcinoma cells by upregulating the level of the anti-apoptotic protein Mcl-1 and activating the PI3K/Akt-dependent pathway [43;48]. Our results demonstrate that PGE 2 produced by COX-2 does not change the levels of Mcl-1 but induces a significant increase in Akt phosphorylation, indicating a role for Akt in COX-2-dependent survival of HCC cells. p53 activation plays an important role in the induction of apoptosis by various Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780 Copyright 2006 Biochemical Society agents. Once activated, p53 negatively regulates cell cycle progression and induces apoptosis by

Fig. 1 .

 1 Fig. 1. COX-2 expression in CHL liver cells increases PGE 2 production. Chang liver cells (CHL) were stably transfected with empty pcDNA3 vector (CHL-V) or pcDNA3hCOX-2, encoding human COX-2 (CHL-C). (A) COX-2 and COX-1 protein expression in whole-cell extracts detected by Western blot. COX-2 and COX-1 expression were normalized by calculating the ratios of COX-2 and β-actin band densities, expressed in arbitrary units (a.u.). A representative experiment is shown beneath the chart. (B) Immunocytochemical analysis of COX-2 by confocal microscopy. COX-2 staining is in red and nuclear staining with Hoechst 33258 in blue. The chart shows the relative fluorescence intensities. (C) RT-PCR analysis of COX-2 mRNA expression. COX-2 mRNA amounts were calculated as arbitrary units normalized to the expression of 18S rRNA, to control for the quantity and integrity of total RNA. (D) PGE 2 production by COX-2 expressing CHL. PGE 2 concentrations were determined by ELISA in the culture media of untreated cells or cells treated at 37ºC for 30 min with 30 µM AA + 5 µM of the COX-2 inhibitor DFU and + 50 µM of the nonselective COX inhibitor indomethacin (E) Light microscopy showing morphological changes in CHL cells expressing COX-2 . All data presented are the means + SD of five independent experiments. #P< 0.05 vs. the CHL control cells. *P< 0.05 vs. the CHL-V cells. ♦P< 0.05 vs. paired AA-treated cells without DFU or indomethaccin treatment.

Fig. 2 .

 2 Fig. 2. Effect of COX-2 expression on cellular growth and cell cycle distribution.(A) Time-dependent proliferation of CHL-V and CHL-C. Cells were seeded at 1.6x10 5 on 6-cm plates in medium containing 10% FBS, and after the indicated times in culture cells were trypsinized, stained with trypan blue and counted (trypan blue negative cells). (B) Effect of FBS concentration on cell growth at 72 h. Cells were seeded as before, but in media containing differing concentrations of FBS asindicated. (C) Flow cytometry analysis of the proportion of CHL-V and CHL-C populations in S phase at 0% and 10% FBS (72 h). (D) Cell cycle analysis by FACS of CHL-V and CHL-C cells at 72 h. (E)The same experiment as in D but considering also apoptotic cells. Cells were resuspended in PBS, fixed with 70% ethanol, stained with PI and analyzed in a CyAn MLE-R cytometer. All data presented are the means + SD of four independent experiments. *P< 0.05 vs. the CHL-V control cells.

Fig. 3 .

 3 Fig. 3. COX-2 expression inhibits propidium iodine staining in liver cells. Cells resuspended in PBS were PI stained, and cell cycle analysis was determined by flow cytometry. (A) Effect of FBS concentration on the percentage of apoptotic CHL-V and CHL-C cells after 72 h of culture. (B) Time-dependent apoptosis and PGE 2 concentrations in CHL-V and CHL-C cells cultured at 0% FBS. (C) Representative DNA histograms and G1 profiles showing the PI staining from CHL-V and CHL-C cells cultured at 0 and 10% FBS for 72 h. (D) Effect of exogenous PGE 2 on the percentage of apoptotic cells. (E) Effect of DFU on the percentage of apoptotic CHL-C cells (0% FBS, 72 h of culture). (F) Western blot analysis of COX-2 expression in HepG2-V and -C. (G) Apoptosis of HepG2-V and HepG2-C cells at 0% FBS after 72 h of culture. All data presented are the mean + SD of four independent experiments. *P< 0.05 vs. paired CHL-V control cells. ♦P< 0.05 vs. cells at time = 0 (B) or untreated cells (D and E).

Fig. 4 .

 4 Fig. 4. Expression of COX-2 suppresses caspase-like activities. Activities of caspase 3 (A), 9 (B) and 8 (C), measured as DEVDase, IETDase and LEHDase activities, respectively, in CHL-V control and CHL-C COX-2-expressing cells cultured without FBS. Caspase activities were measured by fluorimetric assay with specific fluorogenic substrates (see Materials and Methods). One unit of protease activity was defined as the amount of enzyme required to release 1 pmol of AMC/min. All data presented are the means + SD of five independent experiments. *P< 0.05 vs. the corresponding CHL-V control cells.

Fig. 5 .

 5 Fig. 5. Altered expression of apoptosis indicators in COX-2-expressing liver cells. Representative Western blots showing the expression of p53, Bax, Bcl.2, xIAP, cIAP-1 and Fas in CHL-V control cells and COX-2-expressing CHL-C cultured without FBS (A,B). Densitometric analysis of the expression of p53 and Bax (C,D). The expression of target proteins was normalized to that of β-actin (whole-cell and cytosolic extracts), and the ratios are presented in arbitrary units. The data presented are the means + SD of five independent experiments. *P< 0.05 vs. the corresponding CHL-V control cells.

Fig. 6 .

 6 Fig. 6. Cytochrome c, PARP-1 and P-Akt changes in CHL-V and CHL-C cells. The presence of cytochrome c in the cytosolic and mitochondrial fractions after controlled lysis of the cells, and the levels of PARP-1 were analyzed by Western blot (A-D). Blots were normalized with βactin (whole-cell and cytosolic extracts) or Sp-1 (nuclear extracts). Total and P-Akt levels were determined in the same total cell extract in the presence or absence of 5 µM DFU (E). Results show representative Westerns (n=3) and data are the means + SD of five independent experiments. *P< 0.05 vs. the corresponding CHL-V control cells.

Fig. 7 .

 7 Fig. 7. Hydrodynamic transfection of mice with GFP-COX-2 inhibits Fas-induced liver apoptosis. Animals were transfected with 100 µg of plasmid encoding GFP or GFP-COX-2. Control animals were treated with saline. (A) GFP fluorescence in liver sections taken 24 h after transfection to verify the expression of GFP or GFP-COX-2 fusion protein. (B) (ALT) serum activity determined as an index of transfection-induced liver injury after 24 h. (C) Intrahepatic PGE 2 concentrations were determined 24 h after transfection. (D) Caspase-3 activity (DEVDase) in liver extracts taken 5 h after intraperitoneal injection with the Fas-activating antibody Jo2 (0.3 µg/g body weight). (E) Apoptosis in liver sections taken 5 h after injection with Jo2. The fluorograms show representative results of TUNEL analysis (red fluorescence) and nuclear staining with Hoechst 33258 (blue). The bar chart shows the percentage of TUNEL positive cells (F). Protein levels of P-Akt and Bcl-2/Bax ratio in liver extracts from saline, GFP and GFP-COX-2 mice (G). All data presented are the means + SD of eight animals per condition. *P< 0.05 vs. GFP-transfected animals.

  Figure 5

  Figure 6

  Figure 7

Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780

Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780 Copyright 2006 Biochemical Society cell

  /cytosolic extracts) or Sp1 (nuclear extracts). Different exposure times were performed on each blot to ensure linearity of the band intensities. Densitometric analysis was expressed in arbitrary units.

	5 minutes in Laemmli sample
	buffer, and equal amounts of protein (20-30 µg) were separated by 10-12% SDS-PAGE. The relative
	amounts of each protein were determined in total, cytosolic or nuclear cellular extracts as appropriate
	with the following polyclonal or monoclonal antibodies: COX-2 and COX-1 (Cayman), inhibitors of
	apoptosis (IAPs) (R & D Systems), Bcl-2 family proteins (Santa Cruz), cytochrome c and FAS (BD
	Pharmingen), p53 (Santa Cruz), PARP-1 (Alexis), and Akt/P-Akt (S473) (Cell Signaling) After
	incubation with the corresponding anti-rabbit or anti-mouse horseradish peroxidase conjugated
	secondary antibody, blots were developed by the ECL protocol (Amersham). Target protein band
	densities were normalized by calculating the ratio to the corresponding densities of β-actin (whole-

m 200 m

Acknowledgements: A.F.M. and R.M. were supported by CNIC-BANCAJA fellowships. This work was supported by grants SAF2003-00342, SAF2004-00957 and SAF2005-03022 from CICYT, Spain and by a grant from Instituto de Salud Carlos III (Red de Centros C03/01). Biochemical Journal Immediate Publication. Published on 27 Jun 2006 as manuscript BJ20060780 Copyright 2006 Biochemical Society


Nina Kureishy

Daisy Faruque

Dr Colin D Porter
email: colin.porter@icr.ac.uk

Primary attachment of murine leukaemia virus vector mediated by particle-associated heparan sulphate proteoglycan

Keywords: murine leukaemia virus, heparan sulphate proteoglycan, virus attachment, retroviral vector, glycosaminoglycan CA, viral capsid protein, DMEM, Dulbecco's modified Eagle's medium, FCS, foetal calf serum, GAG, glycosaminoglycan, GPI, glycosylphosphatidylinositol, HS, heparan sulphate, HSPG, heparan sulphate proteoglycan, MLV, murine leukaemia virus, PI-PLC, phosphatidylinositol

Target cell entry of murine leukaemia virus vectors proceeds via primary attachment, independent of the viral envelope protein, and subsequent envelope/receptor interaction. Whilst much attention has been paid to modifying the latter for target cell specificity the initial binding interaction has been overlooked, despite its opposing involvement both in providing the virus available for receptor binding and in depleting free virus. As a first step towards modifying primary attachment, both to provide specificity and to enhance vector availability, we sought to determine the nature of this interaction. Following an initial screen of glycosaminoglycans for their ability to inhibit virus binding and transduction we have shown that production of virus from cells in which glycosaminoglycan-sulphation is inhibited, or treatment of virus with heparinase III, reduces both particle attachment and infection. Detection in purified virus preparations of a neo-epitope generated by heparinase III confirmed the presence of virus-associated heparan sulphate proteoglycan, acquired from the producer cell. We propose that host-acquired cell surface heparan sulphate proteoglycan (potentially including syndecan-2) provides a means of virus attachment to target cells that precedes specific receptor interaction and membrane fusion.

Inhibition of heparan sulphate biosynthesis may provide a sufficiently reduced background of primary binding such that novel mechanisms of attachment, ideally with appropriate target cell-specificity, can be introduced.

INTRODUCTION

The major limitations to in vivo gene delivery with retroviral vectors, aside from recent safety concerns associated with proviral integration, are efficiency and specificity. Transduction with murine leukaemia virus (MLV) vectors is routinely enhanced in vitro by the inclusion of polycations, such as polybrene, to overcome the electrostatic effects of apposing negatively-charged virus and target cell membranes; cationic agents such as liposomes not only enhance virus binding in this way but can modify the efficiency of membrane fusion consequent on virus envelope/receptor interaction. However, such approaches will be of limited value in vivo: ideally, especially for applications requiring systemic administration, the retroviral particles would be endowed with intrinsic means of rapid, specific association with the intended target cells without significant interaction with non-target cells.

Strategies to achieve such specificity have focused on modification of the surface component (SU) of the viral envelope protein, by addition of targeting moieties (with or without replacement of the original receptor-binding domain), although most have been hampered by the difficulties of retaining fusogenic capacity in the resulting chimaeric protein [1]. Such studies have laboured under the premise that the crucial interaction for the normal route of infection is that of the virus envelope with its cellular receptor, leading to membrane fusion and release of the viral core to the cytoplasm. However, although MLV infectivity is indeed dependent upon a cognate envelope/receptor interaction for entry, viral particles initially adsorb to cells independently of their envelope protein [START_REF] Pizzato | Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction[END_REF]. Since adsorption is equivalent with/without envelope protein [START_REF] Pizzato | Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction[END_REF][START_REF] Porter | Rescue of retroviral envelope fusion deficiencies by cationic liposomes[END_REF], direct binding to the receptor (Pit-2 in the case of the commonly-used amphotropic pseudotype) must be minimal; rather, it is the surface-adsorbed virus that is available for receptor interaction. In vitro, agents such as polybrene enhance transduction by non-specifically increasing the level of receptor-independent virus adsorption and thus the pool available for receptor-binding [START_REF] Davis | Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes[END_REF]. However, understanding the nature of the initial association in the absence of such agents is crucial to developing novel binding specificities suitable for in vivo application; previous studies, concentrating on modifying envelope protein properties, have failed to address primary attachment as an issue that can negate such efforts at retargeting [START_REF] Pizzato | Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells[END_REF]. In consequence, such envelopes can be considered to coat viruses for Copyright 2006 Biochemical Society which fusion, rather than binding, is conditional upon target recognition and the intended strategy will only become evident when combined with ablation of envelope/receptor-independent binding. Better still would be replacement of the latter with a novel means of primary attachment specific to the intended target cell.

A second reason for seeking to modify the nature of the initial virus association with cellular membranes is that widespread adsorption of current vectors is a formidable problem for their efficient delivery via systemic administration: rapid depletion of the available circulating virus pool means that much of the injected dose fails to reach the target site. Thus, in addition to providing targeting, controlling the specificity of primary attachment will increase virus circulating times and therefore the accumulation of vector at the target site.

In this study we have investigated the role of glycosaminoglycans (GAGs) in virus attachment to the cell surface and subsequent transduction by amphotropic MLV retroviral vectors (MLV-A). We show that sulphated GAG, specifically heparan sulphate (HS) acquired from the producer cells, is a component of the virus particles that is involved in infection of TE671 cells.

EXPERIMENTAL

Cell culture and virus production

Human rhabdomyosarcoma TE671 and producer cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum (FCS).

Serum-free culture supernatants containing MLV-A for transducing the βgalactosidase gene were obtained after overnight incubation of TELCeB6/AF producer cells [START_REF] Cosset | High-titer packaging cells producing recombinant retroviruses resistant to human serum[END_REF] in OptiMEM (Invitrogen, Paisley, UK). Supernatants were filtered (pore size 0.45 µm) and frozen at -80 °C before use. MLV-A titre on TE671 cells was 1-3 x 10 7 infectious units/ml in the presence of 8 µg/ml polybrene.

Viral transduction assay

TE671 cells were plated at 2.5 x 10 4 cells per well in 24-well plates for 18 h before infection by exposure to retrovirus for 40 min at 37 °C. Before use, retrovirus preparations were diluted 1:10 in OptiMEM (to prevent saturation), of which 50 µl in a total volume of 250 µl was used per well. For some experiments, target cells were pre-exposed to soluble GAGs (see below). Limiting the exposure period to 40 min, after which virus was removed and replaced with DMEM/FCS, ensured that the readout was a reflection of the initial kinetics of transduction [START_REF] Porter | Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo[END_REF]. At 48 h after viral infection cells were histochemically stained for β-galactosidase expression by washing with PBS, fixing with 0.5 % glutaraldehyde in PBS for 15 min at room temperature, washing again and incubating in PBS containing 2 mM MgCl 2 , 0.01 % sodium deoxycholate, 0.02 % Igepal CA-630, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.1 % X-Gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside) for 4 h at 37 °C. The total number of cells and the number of blue cells were counted in multiple microscopic fields of view and all experiments were conducted in triplicate. The degree of transduction was determined as the average number of transduction events per cell (typically 0.05-0.1 for untreated controls), as previously described [START_REF] Porter | Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo[END_REF]. The statistical significance of differences between groups was evaluated for 95 % and 99 % confidence by 1-way analysis of variance (ANOVA), using pairwise comparison versus untreated control and Dunnett's error protection (Analyse-it Software, Leeds, UK).

Virus binding assays

Cells were detached by treatment with versene and resuspended in OptiMEM at 2 x 10 6 cells/ml, from which 50 µl aliquots were placed in polypropylene tubes. For some experiments, target cells were pre-exposed to soluble GAGs (see below). Virus (200 µl) was added and the mixture incubated for 40 min at 37 °C. Controls were cells or virus alone. The cells were washed by the addition of 10 ml PBS and pelleted by centrifugation at 1,200 rpm (300 g) for 5 min; they were resuspended in 5 ml PBS, pelleted again, resuspended in 1 ml PBS and transfered to an Eppendorf tube. The cell pellet was dissolved in 30 µl SDS-PAGE sample buffer containing 5 % βmercaptoethanol. All experiments were conducted in triplicate thus far; replicates were combined before separating proteins by SDS-PAGE. Detection of MLV capsid protein (CA) was achieved by Western blotting, as described below. Aliquots of the input virus were included as a guide to the efficiency of binding; moreover, the proportionality of CA signal to virus concentration enabled assessment of quantitative differences in virus binding, as reported previously [START_REF] Porter | Rescue of retroviral envelope fusion deficiencies by cationic liposomes[END_REF]. Quantitation of reduced virus binding due to experimental intervention was confirmed by repeat Western blot analysis of the experimental sample alongside 2-fold serial dilutions of the control sample. Additionally, the results reported below were experimentally reproducible.

Treatment of cells with soluble GAGs

All GAGs were purchased from Sigma (Poole, UK): chondroitin sulfate A (chondroitin-4-sulphate, C4S) from bovine trachea (Sigma catalogue number C-9819), chondroitin sulfate B (dermatan sulfate, DS) from porcine intestinal mucosa (C-3788), chondroitin sulfate C (chondroitin-6-sulphate, C6S) from shark cartilage (C-4384), HS from bovine kidney (H-7640), heparin from bovine intestinal mucosa (H-0777), or hyaluronic acid (HA) from bovine vitreous humor (H-7630); stock solutions were prepared at 5 mg/ml in PBS. Target cells in a 24-well plate for infection (see above) were incubated for 1 h at 37 °C in 200 µl OptiMEM containing 50 µg/ml GAG. Virus was then added (50 µl per well, resulting in a final GAG concentration of 40 µg/ml), as described above. In a parallel experiment the GAG solution was replaced with OptiMEM, after washing the cells, prior to addition of virus. Suspension cells for virus binding assay were similarly pre-exposed to 200 µg/ml GAG, prior to addition of virus (200 µl per 50 µl cell suspension, resulting in a final GAG concentration of 40 µg/ml), as described above.

Inhibition of sulphation

Target cells were incubated for a total of 4 days in DMEM/FCS supplemented with 35 mM sodium chlorate to desulphate proteins and proteglycans [START_REF] Mislick | Evidence for the role of proteoglycans in cation-mediated gene transfer[END_REF]; they were plated at 2.5 x 10 4 cells per well in 24-well plates 18 h prior to addition of virus. Similarly, virus producer cells were incubated in DMEM/FCS containing sodium chlorate for 4 days before harvesting virus for infection of untreated target cells. Sodium chlorate is a potent inhibitor of all cellular sulphation reactions but has no effect on protein synthesis or other post-translational modifications [START_REF] Baeuerle | Chlorate--a potent inhibitor of protein sulfation in intact cells[END_REF]; nevertheless, it was important to control for possible effects on virus production, for which reason Western blot analysis of the viral envelope glycoprotein (SU) and capsid (CA), as described below, was used to monitor virus harvested from treated producer cells.

Digestion of GAGs

TE671 cells (5 x 10 5 cells) were detached using versene and washed twice in PBS, 0.1% BSA. To digest HS, cells were resuspended in PBS-ABC, 0.1 % BSA and treated with heparinase III (up to 10 U/ml; Sigma H-8891) for 1 h at 37 °C. Cell pellets were washed twice in PBS then resuspended in 600 µl OptiMEM before infection with 150 µl retrovirus (1:10 diluted) for 40 min at 37 o C. Cells were then washed, resuspended in 3 ml DMEM/FCS and 3 x 1 ml aliquots were plated into 24well plates. Virus-associated HS was digested with heparinase III (up to 20 U/ml) for 4 h at 37 °C by combining equal volumes of virus in OptiMEM and enzyme in PBS-ABC, 0.1 % BSA. Digested virus was subsequently diluted 1:5 (equivalent to 1:10 dilution of the original stock) for use in the transduction assay, or used without dilution for assay of virus binding. Chondroitinase ABC (up to 10U/ml; Sigma C-3667) was similarly used to digest C4S, C6S, DS and (less efficiently) HA.

Phosphatidylinositol-phospholipase C (PI-PLC, up to 4 U/ml; Sigma P-8804) was used at 30 o C to digest glycosylphosphatidylinositol (GPI)-anchored proteoglycans.

The preparation was then diluted to give a final 1:10 dilution of virus before addition (50 µl) to the cells for infection for 40 min at 37 °C. The cells were histochemically stained for β-galactosidase expression 48 h after viral infection, as described above. It was important to exclude potential protease exposure during GAG digestion, for which reason Western blot analysis of SU and CA, as described below, was used to monitor treated virus: being on the surface of the virus particle (in contrast to CA, protected within the viral core), SU is an indicator of exposure to protease.

Analysis of cell surface GAG by flow cytometry

Cells were detached using versene, treated to digest specific cell surface GAGs as indicated, washed and resuspended in PBS. Aliquots (2 x 10 5 cells) were pelleted, resuspended in 50 µl primary antibody and incubated on ice for 30 min. Primary antibodies used were murine monoclonal antibodies 10E4 (Calbiochem, San Diego, CA) and 3G10 (Seikagaku Corporation, purchased from AMS Biotechnology Europe, Abingdon, UK) diluted 1:100 for detection of HS, CS-56 (Sigma) diluted 1:100 for detection of CS and CBL471 (Cymbus Bioscience, Southampton, UK) diluted 1:20 for detection of human CD55. Following washing, cells were similarly incubated in 50 µl 1:10 diluted FITC-conjugated goat anti-mouse F(ab') 2 secondary antibody (Dako, Ely, UK). Staining was analysed by FACS, using propidium iodide to exclude non-viable cells.

Virus purification and concentration

Harvested virus (40 ml) was purified by low-speed centrifugation in 4 x 50 ml polypropylene tubes at 3,500 rpm (2,500 g) at 4 °C for 16 h. The majority of the supernatant was removed and the remaining 500 µl in each tube was combined.

OptiMEM (3 ml) was then added to the preparation; this constituted purified virus.

For evaluating virus-associated heparan sulphate proteoglycan (HSPG) by Western blotting, 400 µl purified virus was treated with heparinase III, pelleted by ultracentrifugation using a Beckman SW41 Ti rotor (30,000 rpm (150,000 g) at 4 o C for 1 h) and dissolved in 50 µl SDS-PAGE sample buffer containing 5 % βmercaptoethanol.

Preparation of cell lysates and cellular membrane proteins

Cells (2 x 10 6 ) were detached and treated with 10 U/ml heparinase III as above, following which they were pelleted, resuspended in 100 µl 1 % Triton X-100, 10 mM HEPES, 3.5 mM MgCl 2 , 1 mM PMSF and incubated on ice for 30 min. Nuclei were Copyright 2006 Biochemical Society pelleted by centrifugation for 15 min in a microfuge and the supernatant (lysate) was combined with an equal volume of 2 x SDS-PAGE sample buffer containing 5 % βmercaptoethanol. To prepare cellular microsomal membranes, 2 x 10 8 TE671 cells were detached with versene, washed, resuspended in 2 ml PBS-ABC, 0.1 % BSA with 10 U/ml heparinase III and incubated for 2 h at 37 o C; the cells were subsequently pelleted and lysed in 4 ml hypotonic buffer (10 mM Tris pH 7.4, 2 mM MgCl 2 , 1 mM CaCl 2 ) on ice for 30 min. Cellular debris was pelleted and subjected to repeat hypotonic lysis, following which the supernatants from both rounds were combined.

Membrane fragments were pelleted by ultracentrifugation using a Beckman SW55Ti rotor (35,000 rpm (150,000 g) at 4 o C for 1 h), resuspended in 1 ml 10 mM Tris pH 7.4 and combined with an equal volume of 2 x SDS-PAGE sample buffer containing 5 % β-mercaptoethanol.

Western blot analysis

Electrophoresis of samples was performed under reducing conditions using 12% SDSpolyacrylamide gels prior to electrophoretic transfer to nitrocellulose (Hybond ECL; Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked for 16 h in 5 % non-fat milk powder in Tris-buffered saline (TBS) then exposed to primary antibody for 1 h in TBS, 5 % milk, 0.1 % Tween-20. MLV CA and SU proteins were detected using goat anti-Rauscher leukaemia virus p30 at 1:2,000 dilution and anti-Rauscher leukaemia virus gp70 at 1:1,000 dilution, respectively (Quality Biotech Inc., Camden, NJ). Proteins from which HS had been removed by heparinase III-digestion were detected using murine monoclonal antibody 3G10 (Seikagaku Corporation) at 1:1,000 dilution. Syndecans were detected using rabbit polyclonal antibodies for human syndecan-2 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution and for human syndecan-4 (Zymed, San Francisco, CA) at 1:100 dilution. Membranes were washed and incubated for 1 h in the appropriate peroxidase-conjugated rabbit anti-goat, rabbit anti-mouse or swine anti-rabbit secondary antibody (Dako) at 1:2,000 dilution, followed by enhanced chemiluminescence detection (Amersham Biosciences) using Kodak Biomax Light Film.

RESULTS

Amphotropic-pseudotyped retroviral vector was obtained from a producer cell line developed for clinical suitability (ie. production of high-titre, helper-free, serumresistant recombinant retrovirus) [START_REF] Cosset | High-titer packaging cells producing recombinant retroviruses resistant to human serum[END_REF] and assayed for the involvement of GAGs in attachment to adherent human TE671 target cells. The primary assay used was of transduction (by expression of β-galactosidase) -for which virus attachment is a prerequisite -owing to the greater ability compared to direct assay of binding to quantitate the effect of the various treatments; findings were corroborated by assay of virus binding. The initial transduction rate was determined by limiting exposure of target cells to 40 min since the degree of transduction (ie. the number of transduction events/number of cells exposed) has linear kinetics through this period for a range of virus concentrations and degrees of binding [START_REF] Porter | Rescue of retroviral envelope fusion deficiencies by cationic liposomes[END_REF][START_REF] Porter | Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo[END_REF]. Moreover, transduction rate correlates with binding [START_REF] Porter | Rescue of retroviral envelope fusion deficiencies by cationic liposomes[END_REF]. Detection of cell-associated viral capsid protein (CA) was subsequently used to verify that effects following GAG inhibition/modification were indeed due to altered binding.

Inhibition of transduction and virus binding with soluble GAGs

As a starting point to investigate the potential role of GAG chains in mediating virus attachment, target cells were incubated with soluble GAGs before exposure to virus.

At 40 µg/ml, heparin was the most potent inhibitor of transduction of TE671 cells by MLV-A (>10-fold), followed by HS and DS (Figure 1A); similar results were obtained when virus was incubated with GAG before adding to cells (data not shown).

Inhibition due to HS and DS was reduced at lower concentrations of GAG but heparin remained just as potent at 5 µg/ml (data not shown). Since MLV-A infectivity can be inhibited by anions [10], a potential complication of interpreting such studies is that the soluble GAGs are anionic, although their differential inhibition suggests biochemical specificity. Furthermore, when cells were exposed to GAGs and washed prior to addition of virus, significant inhibition was seen for heparin only (Fig 1A ), indicating a specific, stable cell surface/GAG interaction.

Virus binding to cells in the presence of soluble GAGs was also directly evaluated.

The nature of the assay, based on determining cell-associated viral CA that is integral to the virus particle, necessitated the use of suspension target cells and a greater concentation of virus, under which conditions binding was strongly inhibited by heparin only (Figure 1B). These results suggest the involvement of a GAG of the heparin/HS structure in mediating virus binding to cells. Heparin and HS have the same basic disaccharide repeat but differ in the degree of sulphation: although HS is less modified overall than heparin it can contain heparin-like sub-domains since the sulphation density is heterogeneous [11]. For this reason, and given the possible differences between human HS and the use of soluble bovine HS in these experiments, it is not possible to distinguish between the involvement of heparin or HS. However, since heparin is the highly-sulphated form found in mast cells it can be concluded that the GAG involved is HS, and that the interacting domain is such that heparin can compete more effectively than bovine kidney HS.

Inhibition of GAG sulphation

Sodium chlorate (NaClO 3 ) was used to inhibit GAG sulphation, by inhibition of ATPsulphurylase, an enzyme required for the synthesis of the sulphate donor, 3'phosphoadenosyl-5'-phosphosulphate [START_REF] Mislick | Evidence for the role of proteoglycans in cation-mediated gene transfer[END_REF][START_REF] Baeuerle | Chlorate--a potent inhibitor of protein sulfation in intact cells[END_REF]. Incubation of target cells in 35 mM NaClO 3 for 4 days had no effect on transduction. In contrast, addition to producer cells prior to and throughout virus preparation consistently reduced the initial transduction rate (Figure 2A) without discernable effect on virus particle number (ie.

CA content) or SU envelope protein incorporation (Figure 2B). The addition of NaClO 3 to control virus had no direct effect on transduction. Although 35 mM NaClO 3 is typical for inhibition of the sulphurylase [START_REF] Mislick | Evidence for the role of proteoglycans in cation-mediated gene transfer[END_REF], the effect of using higher concentrations was also evaluated: there was a dose-dependent reduction in transduction rate, without impact on virus production, to 10-fold at 50/75 mM NaClO 3 (Figure 2A andB).

The effect of treatment on virus binding paralleled that on transduction: viruses prepared from NaClO 3 -treated producer cells with equivalent particle numbers showed a dose-dependent reduction in binding (Figure 2C). The magnitude of the effect on binding was estimated to be ~5-fold by comparison to the signal intensities of dilutions of cell-associated control virus; this is similar to the effect on Copyright 2006 Biochemical Society transduction, given the experimental differences between the assays. These data are consistent with the presence in virus preparations of a producer cell-derived sulphated component that promotes cell attachment, and hence transduction. In principle, such a component could be intrinsic to the virus particle or an unassociated factor in the medium conditioned by the producer cells. However, since exogenous sulphated GAGs are inhibitory, the reduced sulphation of such molecules endogenous to the conditioned medium would be unlikely to reduce binding and transduction, suggesting that the necessary component is virus-associated.

Removal of GAG by enzyme treatment

Target cells and virus preparations were independently treated with heparinase III or chondroitinase ABC to remove HS or CS/DS, respectively. Because enzyme digestion tended to detach adherent cells, the target cells were used in suspension for this experiment and attached after virus exposure. The efficacy and specificity of cell surface HS and CS removal was validated by FACS (Figure 3A). HS expression was assessed using two monoclonal antibodies: 10E4 staining was lost upon treatment with heparinase III, whilst strong 3G10 staining of the whole cell population was apparent only after digestion (Figure 3A). The latter antibody detects a neo-epitope (desaturated uronate) generated by removal of HS with heparinase III, whilst 10E4 is specific for a particular HS domain that may not be represented in all HSPGs [START_REF] David | Developmental changes in heparan sulfate expression: in situ detection with mAbs[END_REF].

Target cell digestion was ineffective with respect to transduction (data not shown). In contrast, incubation of virus preparations with heparinase III before addition to adherent target cells consistently resulted in reduced transduction (Figure 3B).

Although the presence of enzyme at reduced concentration could in principle affect target cell surface GAG during infection, the lack of effect of digesting cells prior to virus exposure allows the result to be attributed to an effect upon the virus.

Chondroitinase ABC treatment of virus was ineffective, ruling out the involvement of C4S, C6S or DS (Figure 3B). These results, indicating the involvement of HS in the virus preparation, support the above conclusion that a sulphated component is necessary and are consistent with the potent inhibition by heparin.

To reach this conclusion it was important to exclude potential protease exposure during enzymatic treatment for GAG removal, since this would confound To distinguish between the potential involvement of HS attached to transmembrane versus GPI-tethered proteoglycan, virus was treated with PI-PLC to digest the latter; although the conditions used effectively digested cell surface CD55 (a GPI-linked protein; Figure 3A), there was no effect on transduction of treating virus (Figure 3B).

The effect of heparinase III-digestion of virus was dose-dependent but maximal for ≥1 U/ml, resulting in 3-fold reduction in transduction (Figure 3C). Impaired transduction was matched by a comparable reduction in virus binding for treatment with heparinase III, but not chondroitinase ABC or PI-PLC (Figure 3D). Consistent with the above, these experiments indicate the involvement of a non-GPI-linked HSPG in mediating MLV-A attachment to TE671 cells.

Virus purification

Infectivity using crude preparations of virus, as in the experiments described above, has been reported in some instances to be affected by soluble components, including shed SU and chondroitin sulphate proteoglycan [13]. Although TE671 conditioned medium does not have non-specific inhibitory activity and TE671-derived producers are relatively free of soluble SU [START_REF] Slingsby | Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency[END_REF] it was important to establish that the experiments implicating HSPG in attachment were unaffected by soluble components of the producer cell conditioned medium containing the virus. MLV-A was purified by lowspeed centrifugation to concentrate viral particles; the method is sufficiently gentle to prevent the particle aggregation and loss of SU associated with ultracentrifugation and avoids concentrating soluble SU or non-viral proteins [15]. This resulted in recovery of 50% of the viral particles, as judged both by infectious titre and relative CA content. For comparison with unpurified preparations the purified virus was diluted to give an equivalent particle number on this basis.

Virus purified following production from cells treated with 35 mM NaClO 3 showed reduced transduction, equivalent to that seen without purification (Figure 4A).

Similarly, heparinase III digestion of purified virus reduced transduction to the same degree as virus without purification (Figure 4B). As described for the experiments above in Figure 2 and Figure 3, reduced transduction was paralleled by virus binding (data not shown). These data confirm that the producer-acquired HSPG involved in viral attachment is particle-associated.

Detection of virus-associated and cell surface HSPG

As shown above, HS detection on TE671 cells using antibody 10E4 was considerably weaker than that using 3G10 after heparinase III-digestion (Figure 3A); identical results were obtained for the TE671-derived virus producer cells. In contrast, but consistent with the variable presence of the 10E4 epitope, the cell line MDA-MB157 showed strong staining with 10E4 in addition to strong post-digestion staining with 3G10 (data not shown), from which it can be concluded that TE671 cells have abundant surface HS that contains relatively little of the 10E4 epitope. Since creation of the uronate neo-epitope is the more definitive indication of the presence of HSPGs, heparinase III-digestion and Western blot analysis using antibody 3G10 was used to assess virus-associated HSPG.

Western blot analysis of producer cell lysates with 3G10 stained a band of 35 kDa and a doublet of higher molecular-weight, dependent upon heparinase III digestion; virus (ie. cell supernatants) contained heparinase III-dependent 3G10-positive 35 kDa and doublet 50-60 kDa bands, the first of which was equivalent to the species in the cell lysate (Figure 5A). Additional digestion with chondroitinase ABC had no effect (data not shown). The same bands were obtained, respectively, for lysates of treated TE671 cells and their digested conditioned medium, the latter indicating an abundance of soluble HSPGs in the supernatant. Consistent with this, the majority of the 3G10reactive material was retained in the centrifugation supernatant of the virus preparations purified for the experiment described above (Figure 5B). Because the sensitivity to digestion of these purified viruses suggested that some HSPG must have co-purified, heparinase III-digested virus was further concentrated by ultracentrifugation prior to Western blotting; predominantly, the 35 kDa band was detected (Figure 5B). In subsequent experiments the higher molecular-weight material was variably present, in addition to the greatly-enriched 35 kDa species (Figure 5C); identical treatment of TE671 conditioned medium did not detect this band, which was, however, present for preparations of virus lacking the viral envelope protein.

Reasoning that syndecan-2 and syndecan-4 [16,[START_REF] Oh | Syndecans-2 and -4; close cousins, but not identical twins[END_REF] were likely candidates for hostacquired HSPG mediating virus attachment consistent with the foregoing data, microsomal (predominantly plasma) membrane preparations from heparinase IIIdigested TE671 cells were analysed by Western blotting with antibodies for the core protein of each of these in parallel with 3G10; moreover, blots used with the syndecan antibodies were stripped and re-used with 3G10. The syndecan antibodies each detected several bands, the smallest of which corresponded to the core protein size; multiple higher molecular weight species are likely due to heterogeneity of GAG modification and heparinase III-susceptibility, and to SDS-resistant dimerisation.

There was no congruence between the bands detected by the syndecan-4 antibody and those detected by 3G10; in contrast, there was overlap of the bands detected with the 3G10 and syndecan-2 antibodies, although the latter detected additional unique bands (Figure 5D).

DISCUSSION

Recombinant MLV initially binds to target cells independently of its specific envelope protein [START_REF] Pizzato | Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction[END_REF][START_REF] Porter | Rescue of retroviral envelope fusion deficiencies by cationic liposomes[END_REF]. Our experiments indicate that the major mechanism of such primary attachment involves particle-associated heparan sulphate and that proteoglycans containing such glycosaminoglycan chains are detectable in highly purified virus preparations. This conclusion is supported by a variety of experiments that are consistent in the effects of GAG manipulation in both direct virus binding and transduction assays. Notably, inhibition of GAG sulphation and heparinase III digestion were as effective on purified virus as they were on crude producer cell virus harvests. An important aspect of these experiments is that virus exposure of the target cells was sufficiently short such that the read-out provided a reflection of the initial kinetics of the assay; this is of greater relevance to in vivo application than is the consequence of the extended exposure which is often employed in vitro.

Inhibition of sulphation resulted in up to 10-fold reduction of binding/transduction without effect on virus particle number or envelope incorporation, whereas maximal heparinase III digestion was somewhat less effective. The likely explanation of this difference is that, whereas sulphation of the whole GAG chain will be inhibited, digestion removes only a portion of each chain. HS consists of alternating hexosamine and uronic acid monosaccharide units but there is considerable heterogeneity, including the nature of each unit (D-glucosamine or N-acetyl-Dglucosamine; D-glucuronic acid or L-iduronic acid) and their modifications (notably sulphation at several potential positions) [11]. Heparinase III is specific for hexosamine-glucuronic acid linkages so digestion may leave significant (although shorter) HS chains intact. Importantly, sulphation is critical for molecular interaction with HS [11, [START_REF] David | Developmental changes in heparan sulfate expression: in situ detection with mAbs[END_REF][START_REF] Esko | Order out of chaos: assembly of ligand binding sites in heparan sulfate[END_REF]. HS heterogeneity also underlies the antibody data by FACS and Western blot: whilst all 10E4 epitopes are heparinase III-sensitive, not all generate 3G10 epitopes upon digestion of the hexosamine-glucuronic acid linkage, depending on the nature of the adjacent hexosamine; similarly, not all heparinase III-sensitive linkages that do generate 3G10 epitopes originate from 10E4-reactive sites (additionally dependent on the sulphation status). Thus, 10E4 and 3G10 staining are not superimposable although there is some overlap [START_REF] David | Developmental changes in heparan sulfate expression: in situ detection with mAbs[END_REF]. An important implication is that 3G10 does not detect all digested HSPGs, dependent on the exact nature of the non-reducing end (ie. the heparinase III substrate site closest to the protein core). This was evidenced by the lack of reactivity in our experiments of 3G10 with multiple species detected by antibodies to the syndecan-2/4 core proteins. The presence of the 3G10-positive 35 kDa band in the purified, heparinase III-treated virus preparations can thus be taken as indicative of the virus containing HSPG but this species is not necessarily responsible for virus attachment. Virus incorporation of HSPGs was equivalent in the absence of the envelope protein indicating that acquisition is passive at the time of budding from the producer cell plasma membrane. Despite there being a relative abundance of soluble HSPGs detected in virus preparations compared to those incorporated into the virus particles the equivalence of virus behaviour following purification indicates that the soluble material does not affect infectivity; this suggests that the HS motif that mediates virus attachment is specifically present in the acquired HSPG.

Virus attachment by means of low affinity capture leading to high-affinity interaction and entry has many precedents [19]. Cell surface HS has been implicated in many cases as assisting primary attachment, for viruses from diverse taxonomic groups; eg. specific HS monosaccharide sequences are important mediators of herpesvirus binding by viral glycoproteins B and C or HIV binding by the envelope glycoprotein gp120, in each case preceding interaction with protein entry receptors [START_REF] Liu | Cell surface heparan sulfate and its roles in assisting viral infections[END_REF]. HS involvement as the virus-associated component of the interaction, as the experiments reported herein indicate to be the case for MLV, is a novel finding. Previously, however, Lei et al have reported that HS associated with MLV mediates binding to fibronectin [21]; fragments of fibronectin containing both the HS-binding domain and one (or both) integrin-binding domain(s) have been used as molecular bridges to enhance transduction of non-adherent cells [START_REF] Bajaj | High efficiencies of gene transfer with immobilized recombinant retrovirus: kinetics and optimization[END_REF], particularly for ex vivo gene delivery to bone marrow stem cells [START_REF] Moritz | Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors[END_REF][START_REF] Hanenberg | Efficient gene transfer to human epidermal keratinocytes on fibronectin: in vitro evidence for transduction of epidermal stem cells[END_REF] but additionally for epidermal stem cells [25]. Lei et al showed that HS was not important as a target cell component for fibronectinassisted transduction and that immobilisation of MLV from both murine and human packaging cells, regardless of envelope protein expression, was sensitive to treatment of virus with heparinase III but not chondroitinase ABC; in the absence of fibronectin, transduction of NIH3T3 target cells was dependent on inclusion of polybrene and so the potential involvement of viral HS interaction with target cells was not explored [21]. Our data regarding a direct interaction of viral HS with TE671 cells are consistent with this report, indicating parallels between HSPG-mediated fibronectinimmobilisation and cellular capture of MLV.

In contrast to the above, a report by Walker et al described MLV binding to heparin and suggested this as the mechanism of primary attachment to cells [26]. However, their use of extended (72 h) exposure of cells to virus complicates interpretation of their transduction experiments and the suggestion is contradicted by their own data that showed equivalent binding (independent of envelope) to proteoglycan-deficient and wild-type cells, both of which were inhibitable by heparin; in contrast, these data are consistent with the conclusion of our studies that virus-associated HS mediates binding to cells. Importantly, our data derive from short (40 min) exposure of target cells, during which the transduction kinetics are linear, enabling conclusions to be drawn concerning the major means of primary attachment of relevance to practical application of MLV vectors, without complication by slow mechanisms that are of no such significance. Another important feature is that our experiments avoided the use of agents such as polybrene, inclusion of which complicates the conclusion of some studies [START_REF] Guibinga | Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells[END_REF] given that polybrene promotes a dominant electrostatic interaction [START_REF] Davis | Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes[END_REF]21] that is itself dependent on cell surface GAG [START_REF] Guibinga | Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells[END_REF] and that polybrene/GAG/MLV complexation enhances transfer of virus to the cell surface [28] (Kureishy and Porter, unpublished data); again, such agents are not relevant to in vivo application of MLV vectors, despite their utility in vitro.

Many viruses acquire host proteins within or on the virion surface [29]; HIV has been widely studied in this regard [30], for which incorporation of biologically-active HLA-DR [START_REF] Cantin | The presence of host-derived HLA-DR1 on human immunodeficiency virus type 1 increases viral infectivity[END_REF] and ICAM-1 [START_REF] Fortin | Hostderived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity[END_REF] into the HIV envelope can assist infectivity. MLV has been shown to acquire specific heterologous proteins upon budding from the host cell plasma membrane [33]; in an experimental system, incorporation of such proteins was less efficient than that of the homologous envelope protein and reflected surface abundance [START_REF] Suomalainen | Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus[END_REF]. There are therefore precedents for proposing viral incorporation of host proteins -in this case HSPG(s) -that are responsible for cellular attachment.

Given that binding is independent of envelope protein expression there can be no envelope-dependence for incorporation of such factors. The HSPGs remain unidentified, although our data exclude GPI-linked glypicans; on the basis of some It is probably simplistic to imagine that primary attachment of MLV involves a single molecular interaction, but that described above appears to be the major one.

Consequently, MLV-A entry -dependent on the SU/Pit-2 interaction for membrane fusion -can be understood to proceed via a series of steps involving (i) cell-free motion of virus particles, leading to (ii) primary attachment via associated HSPG to HS-binding cell surface components as a means of virus concentration on the 2dimensional cell membrane to enhance (iii) cognate receptor interaction. The first step represents a significant limitation in vitro/ex vivo, for which Brownian particle motion in the medium provides an adequate model [START_REF] Chuck | -MLV-A Non-env Control Biochemical Journal Immediate Publication[END_REF]36]; it has been addressed in many ways -eg. use of cations [START_REF] Davis | Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes[END_REF][START_REF] Porter | Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo[END_REF], centrifugation during virus exposure [37], CaPO 4 co-precipitation [38], flow-through transduction [39] and the use of fibronectin fragments (see above) -all of which seek to promote juxtaposition of the virus and target cell. The opposite problem pertains for in vivo application requiring systemic administration, for which rapid depletion due to virus proximity to, and adsorption on, non-target cell surfaces (mostly unproductive for infection) limits availability and therefore efficient delivery to distant sites.

The identification of virally-associated HS as a natural mediator of primary attachment is important for controlling this key step in the infectious process. Since there may be multiple HSPGs contributing the HS responsible for virus attachment, their identification and eventual elimination represents a difficult challenge and inhibition might best be approached by blocking HS biosynthesis during virus production, either by inhibition of sulphation (as in the experiments above) or mutation/knock-down of critical synthetic enzymes, such as EXT1 [40]. Controlling primary attachment is key both to maintaining virus availability and to efficient capture once the target site is reached: replacement of the promiscuous mechanism with one specific for the target cell will be of greater importance for achieving specific delivery than will modification of the final step in the infectious process. Indeed, introduction of sufficient specificity at the level of primary attachment will obviate complex envelope engineering, and the natural receptor interaction can be retained for efficient fusion. In conclusion, we have shown that MLV-associated

Biochemical

  Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825

Biochemical

  Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825 Copyright 2006 Biochemical Society interpretation. Because treatment exposes viral surface proteins but not core proteins, digested virus was examined by Western blot for CA and SU abundance versus untreated controls; reduced SU abundance, or its degradation, would be indicative of exposure of the viral particle surface to protease. Both CA and SU abundance were invariant upon digestion, with or without prior virus purification (see below), confirming the absence of exposure to protease during treatment for GAG removal.

Biochemical

  Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825 Copyright 2006 Biochemical Society congruence with 3G10 staining, syndecan-2 is a candidate [16]. The cellular "receptors" similarly remain unknown.

Figure 1

 1 Figure 1 Inhibition of MLV-A attachment by soluble GAGs(A) An indirect assay of the effect of GAGs on attachment was provided by determining the effect on the initial transduction rate (by assessing transduction resulting from 40 min exposure to virus). Virus stocks were diluted 1:10 to prevent saturation, resulting in transduction efficiencies up to 10%. Cells were incubated with GAGs, which were either left in the medium during virus exposure (light grey bars -'GAGs') or washed off the cells prior to addition of virus (dark grey bars -'GAGs washed'). In the former case all GAGs resulted in significant inhibition (p<0.01, 1way ANOVA versus control), most markedly for heparin. For washed cells, only prior exposure to heparin resulted in significant inhibition (p<0.01); there was no significant effect due to exposure to other GAGs (p>0.05). Data shown are mean ± SE (n=6). (B) Determination of cell-associated CA by Western blot was used as a direct assay of the effect of GAG exposure on virus binding. Negative controls for the binding assay were virus alone (V) and cells without virus exposure (C). Levels of virus binding for all GAGs except heparin resulted in a signal comparable to that due to loading 1% of the virus to which the cells were exposed (1% control lane). HS, heparan sulphate; Hep, heparin; HA, hyaluronic acid; C4S/C6S, chondroitin-4/6sulphate; DS, dermatan sulphate.

Figure 2

 2 Figure 2 Inhibition of sulphation during MLV-A production reduces subsequent attachment (A) Transduction rate assay of the effect of incubating producer cells in the indicated concentrations of sodium chlorate (NaClO 3 ). All concentrations resulted in significant inhibition (p<0.01, 1-way ANOVA versus control); use of 50 mM NaClO 3 was significantly different compared to use of 35 mM NaClO 3 (p<0.01), but there was no additional benefit for use of higher concentrations (p>0.05). Data shown are mean ± SE values (n=3). (B) Virus preparations during inhibition of sulphation that were used in part A were assayed by Western blot for CA to control for the possible effect of NaClO 3 on virus particle number, and for SU to control for possible reduction in envelope incorporation (and thus reduction in transduction rate independent of attachment). Virus particle number (and consequently SU content) was reduced only at the highest concentration of NaClO 3 , itself a reflection of reduced producer cell

Figure 3 6 U

 36 Figure 3 Effect of enzymatic digestion of GAGs(A) TE671 cells were analysed by FACS for cell surface HS, without/with digestion for 1 h at 37 o C with 10 U/ml heparinase III (panels 1 and 2: '-HepIII' and '+HepIII', respectively), using monoclonal antibodies 10E4 (thick line) or 3G10 (thin line); data are overlayed on those resulting from use of the secondary antibody alone (solid grey profile). 10E4 staining for HS was eradicated by heparinase III-digestion, whilst 3G10 staining for the neo-epitope generated upon digestion was dependent on such treatment. Similarly, detection of CS (panel 3: '+/-C'ABC') in control cells (thick line) was eradicated by treatment with 10 U/ml chondroitinase ABC for 1 h at 37 o C (thin line), and detection of CD55 (panel 4: '+/-PI-PLC') in control cells (thick line) was eradicated by treatment with 4 U/ml PI-PLC for 1 h at 30 o C (thin line). Enzyme treatment was specific for the appropriate target; additionally, 4-fold serial dilutions of the amount of enzyme used indicated that CS removal was equally effective at 0.6 U/ml C'ABC, whilst residual HS and CD55 were detected following digestion with 2.5 U/ml HepIII and 1 U/ml PI-PLC, respectively (data not shown). (B) Transduction rate assay of the effect of incubating virus for 4 h at 37 o C without enzyme (control) or with 4 U/ml heparinase III (HepIII) or chondroitinase ABC (C'ABC), or for 4 h at 30 o C with 4 U/ml PI-PLC. Treatment with HepIII resulted in significant reduction (p<0.01, 1-way ANOVA versus control), whilst C'ABC and PI-PLC were ineffective (p>0.05). Data shown are mean ± SE values (n=3). (C) The effect on the transduction rate of treating virus with HepIII was dose-dependent on enzyme concentration. All concentrations resulted in significant inhibition (p<0.01, 1-way ANOVA versus control) but there was no additional benefit for use of concentrations above 1 U/ml (p>0.05). Data shown are mean ± SE values (n=3). (D) Determination of cell-associated viral CA by Western blot was used as a direct assay of the effect of enzyme treatment of virus as in part B on subsequent binding. Negative controls for the binding assay were virus alone (V) and cells without virus exposure (C).

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825

Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825 Copyright 2006 Biochemical Society Swaney

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ACKNOWLEDGEMENTS

This work was funded by the Medical Research Council. Biochemical Journal Immediate Publication. Published on 8 Aug 2006 as manuscript BJ20060825 Copyright 2006 Biochemical Society

heparan sulphate mediates cellular capture and propose that modification of this interaction will be essential for practical application by maintaining vector availability and promoting specific attachment.