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Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA). We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family. Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration. In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.

Introduction
Pathology of the podocyte underlies several glomerular diseases, the most severe being focal segmental glomerulosclerosis, which is clinically characterized by heavy proteinuria or nephrotic syndrome and frequently progresses to renal insufficiency. The cellular mechanisms resulting in proteinuria are not completely understood, and thus the description of pathways leading to podocyte damage and repair are relevant to the identification of novel treatment strategies. The membrane transport system, known as a major transmembrane component involved in the movement of ions, small molecules, and drugs, has been extensively studied in some organs and cellular systems, such as the intestine, the liver, and the bloodbrain barrier. In the kidney, several members of the main membrane transporter families have been described in the renal tubule in the context of ion and drug transport [1]. Recently, two transporters with broad specificity for small molecules have been identified in mouse podocytes in relation to drug clearance [2,3].
Podocytes are highly differentiated cells with a complex ramified structure, the maintenance of which requires coordinated traffick-ing from the extracellular to the intracellular compartment, similar to that operating in other highly ramified cells, such as neurons [4]. Small molecule transporters constitutively expressed in the podocyte contribute to maintain the cellular structure and barrier function, possibly by interacting with endogenous substrates and metabolites; and they could also be involved in the uptake and efflux of drugs in these cells.
A better understanding of transport mechanisms in podocytes is needed to explain in part some functions of these cells and to conform to the recently issued FDA draft guidance which includes the recommendation to increase knowledge about drug transport and metabolism (www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ UCM292362.pdf).
With this background, we performed experiments using Penicillin G, a well-known beta-lactam antibiotic, which is the substrate of several transporter families that have been fully characterized in other cellular models [5]. The antibiotic was used in the context of podocyte injury by puromycin aminonucleoside (PA), a small molecule that uses membrane transporters to enter the cell [6], and induces podocyte damage which mimics lesions of minimal change disease or focal segmental glomerulosclerosis, with appearance of severe proteinuria when injected in rats [7].
We reasoned that if Penicillin G engaged transporters at the podocyte surface, the entrance and the subsequent activity of PA would be blocked, providing evidence that the two molecules use the same membrane transporter families in podocytes.

Animals and experimental design
Young male Sprague-Dawley rats (n = 30) weighing 230-250 g housed at the University of Trieste (Italy) animal facility were randomly divided into three control groups and three experimental groups (5 animals per each group, see Table 1). The rats were housed two or three per cage in an air-conditioned, lightcontrolled environment, and all animals had unrestricted access to food and water.
A committee of the Italian Health Ministry approved the experimental protocol (prot. n 166-date: April, 8,2005), in compliance with Italian regulation (D.L.vo 116/92). General anaesthesia was provided with an IP injection of 2 ml Avertin 2% and 100 ul Xilazine 2%. Local anaesthesia was provided with 100 ul of Lidocaine 2% in the site of incision and all efforts were made to minimize suffering.
In the experimental groups rats received a single intravenous injection (5 mg/100 g body weight) of puromycin aminonucleoside (PA) (Sigma) in the femoral vein, whereas rats in the control groups were treated with the same volume (1 ml) of saline. Pretreatment with Penicillin G (Penicillin G sodium salt, Sigma), at dosages of 12,500 IU or 15,000 IU was performed 10 minutes before the injection of PA or saline in the same femoral vein.
A 24h urine collection was obtained on day -1 before PA injection, then on day 5 and 10.
On day 10 the animals were sacrificed, and a blood sample was collected for creatinine and serum albumin analysis. One kidney was immediately excised to isolate glomeruli for the albumin permeability tests (Palb), and the other kidney was used for histological analyses. Creatinine and serum albumin were determined by an autoanalyzer technique (Hitachi 717, Tokyo), and proteinuria was assessed by the Comassie Brillant Blue assay.

Glomerular morphometric analysis
Kidney tissue was fixed in formalin and embedded in paraffin using standard techniques. 4 um-thick sections were deparaffinized, hydrated and stained with periodic acid-Schiff (PAS). Twenty glomeruli per animal were analyzed. Digital images were taken using an upright microscope (Olympus Italia, Segrate, Milan, Italy) equipped with a digital camera. The mean area of each glomerulus was measured by manually tracing the glomerular outline on 200X acquired images, and measured off-line by a video-based image analysis program (Sigma Scan Pro, Jandel Scientific Software).

The convective glomerular albumin permeability assay (Palb)
The method for measuring glomerular albumin permeability, adapted from the original description by Savin [8], allows the study of the glomerular response to various mediators in the absence of systemic humoral, hemodynamic, or rheologic forces [9], and has been applied extensively by our group to analyze glomerular damage [10,11,12].
Glomeruli were isolated by sieving in BSA solution; during the sieving process, they are stripped of Bowman's capsule, tubules, and associated blood vessels. In this work, two sets of experiments were performed, one comprising isolated glomeruli from control and experimental rats, and the other conducted on glomeruli isolated from untreated, healthy male Sprague Dawley rats. Glomeruli from experimental rats underwent directly Palb determination. Glomeruli from untreated animals were instead incubated with PA (0.6 ug/ml to 10.0 ug/ml) or saline for 10 minutes at 37uC. In this set of experiments Penicillin G (50 IU/ml to 700 IU/ml) was added to the incubation medium 10 min before or 10 min after PA incubation, and glomeruli were carefully washed to avoid any possible extracellular interaction between the two drugs. Penicillin G alone at the same concentration was used as a control.

Isolated glomeruli adhesion assay
Isolated glomeruli from healthy young male Sprague Dawley rats were centrifuged at 800 rpm for 2 minutes for fragment elimination. The pellet was resuspended in medium and plated on type IV collagen-coated 24 multi-well plastic plates (100 glomeruli/well). After pre-incubation for 30 minutes with Penicillin G 500 IU/ml or 700 IU/ml, the glomeruli were carefully washed and treated with PA (10, 5 and 2.5 ug/ml) or an equal volume of incubation medium as a control. After 5 days the plates were washed to eliminate unattached glomeruli and analyzed by an inverted microscope (Leica Microsystem GmbH, Germany) to evaluate morphology and number. Three independent experiments for each concentration of Penicillin G and PA were performed.

Podocyte cultures
We used a human immortalized cell line, following the procedure originally described by Doublier at al. [13], with minor modifications. Differentiated podocytes were cultured in Dulbecco's modified Eagle's high glucose medium (DMEM) containing 10% fetal bovine serum (FBS-Euroclone Ltd, Wetherby West Yorkshire, UK).

Table1. Experimental animal groups.
Group Treatment

Penicillin fluorescence in live cells
To confirm the cellular uptake of Penicillin, we performed a flow cytometry analysis. After trypsinization, podocytes (5610 5 cells/ml) were incubated with 10 ug/ml Bocillin for 20 minutes. Cells were washed with cool PBS and resuspended in Penicillinfree medium. At the indicated times 1.5610 3 cells were analyzed by flow cytometry (Bekton Dickinson FACS Elite) and untreated podocytes were used as control.

MTT assay
Human immortalized podocyte proliferation was assessed by the methylthiazoletetrazolium (MTT) assay (Sigma Company, St. Louis, MO, USA), according to the manufacturer's instructions. This non-radioactive cell proliferation assay identifies living cells based on the cellular conversion of a tetrazolium salt into a formazan product, a chromaphore which can be quantified by ELISA. Briefly, cells were plated onto 96 multi-well plates (8000 cells/well) and allowed to adhere overnight without antibiotic. Podocytes were exposed to PA alone (10, 5 and 2.5 ug/ml), Penicillin G alone (10, 50, 500 and 700 Units/ml) or PA and Penicillin G at the same concentration, in medium containing 1% fetal bovine serum (FBS). After 48 h the medium was removed and cells incubated with fresh medium containing MTT (0.5 mg/ml) for 3 hours. After removing the MTTcontaining medium, 200 ul of dimethylsulfoxide (DMSO) was added and the plates were immediately read at 570 nm. Wells containing no cells (but containing medium and MTT over the incubation period) were used to blank the plate reader. The results were the means of three separate experiments, expressed as absorbance at 570 nm.

Transporter gene expression study
Gene expression of various Penicillin G membrane transporters, selected according to the data reported for other cell types, were analyzed in human and rat podocytes. Specifically, we investigated organic anion transporters of the OATP family, one transporter of the OAT family, the peptide transporters PEPT1 and PEPT2, and  the efflux transporter P-glycoprotein. As a control, the same transporters were analyzed in neuronal (SH-SY5Y neuroblastoma) and intestinal cell lines (Caco2 colon cancer cells), as well as in whole organ mRNA extracts (intestine, liver, and kidney). Total RNA from cells was extracted using the Invitrogen Pure Link TM Micro-to-midi Total RNA Purification System. RNA concentrations were determined using Nanodrop ND 1000 (Cell Euroclone Italy). One microgram of total RNA was reverse transcribed using MMLV reverse transcriptase (Applied Biosystems). The analyses were performed by transcriptase-polymerase chain reaction (RT-PCR) and quantitative Real Time RT-PCR (QRT-PCR). Primers and annealing temperatures are reported in Table 2 and Table 3; GAPDH and beta-actin were used as housekeeping genes, as appropriate.
For the QRT-PCR the amplification steps were: pre-denaturation at 95uC for 10 min, 40 cycles of amplification with denaturation at 95uC for 15 s, annealing at proper temperature for 60s and extension at 72uC for 30 s. A final extension at 72uC for 10 min and a dissociation stage (95/60/95uC for 15 s each) were then added.
RT-PCR conditions were described by Tramonti et al. [1] and Engel et al. [14]. For the RT-PCR the ethidium bromide-stained agarose gel was visualized using a gel documentation system (PhotoDoc-it, Imaging System Digital, UVO Cambridge UK).

Transporter immunofluorescence analysis
An indirect immunofluorescence method was utilized on primary cells fixed in acetone or paraformaldehyde, depending on the subsequent application, and plated on Thermanox plastic coverslips (Nunc, Rochester, NY, USA). F-actin was detected directly by phalloidin-FITC or phalloidin-rhodamine (Sigma). Mouse monoclonal anti-[C219] P-glycoprotein (Abcam), rabbit anti-synaptopodin and rabbit anti-vinculin (Sigma) were the primary antibodies used for the study. As secondary fluorescentlabelled antibodies, we used FITC-conjugated goat anti-mouse IgG (Pierce), FITC-conjugated goat anti-rabbit IgG (DAKO A/S, Nenmark) and Alexa Fluor 488 donkey anti-rabbit (Invitrogen). Slides were examined by a fluorescence microscope (Leica Microsystem, Germany). Controls with no primary antibody showed no fluorescence labelling. P-glycoprotein expression was analyzed semi-quantitatively measuring fluorescence intensity by image analysis software (Image ProPlus 6.3 Software, Media-Cybernetics, SilverSpring, MD, USA). Results were expressed as relative intensity comparing treated cells to controls. For each experimental point, 10 to 20 microscopic fields were examined.
Flow Cytometry Analysis of P-glycoprotein activity P-glycoprotein-related transport activities were investigated by using the fluorescence probe Rho123 (Sigma Company, St. Louis, USA) in an efflux assay, as described by Chen et al. [15]. After PA

Membrane transporters' blockade
As transport inhibitors we used cyclosporine A and rifampicin (Sigma Company, St Louis, UAS). While rifampicin blocks the OATPs/Oatps transporters, cyclosporine A exerts its inhibiting activity on both OATPs/Oatps and P-glycoprotein. The two inhibitors were alternatively utilized in combination with fluorescent penicillin. More in detail, to assess effects on OATPs/Oatps activity, cells were first treated for 30 minutes with Cyclosporine 1 uM, or rifampicin 1 uM-10 uM. Then, fluorescence penicillin (50 ug/ml) was added for 30 minutes; after washes, live cells were imaged.
To study P-glycoprotein blockade, cells were first incubated with fluorescent penicillin 50 ug/ml for 30 min, and images were taken after addition of cyclosporine 1 uM for 30 min.
Fluorescence intensity was measured on 100 cells per condition by image analysis software (Image ProPlus 6.3 Software) and results were expressed as relative change of intensity compared to controls arbitrarily given a value of 1. Experiments were repeated trice per each condition.

Statistical analysis
Data are presented as mean 6 SD or mean 6 SEM. Comparison of means was performed by ANOVA test.

Results
Penicillin G pre-treatment in isolated glomeruli before PA incubation PA incubation at concentrations ranging from 10 ug/ml to 0.6 ug/ml increased P alb of isolated healthy glomeruli (Fig 1a). The effect was abolished in a dose-dependent manner by 15 min pre-incubation with Penicillin G and complete inhibition appeared at a concentration of 700 IU/ml, but a significant effect was already present at a concentration of 500 IU/ml (Fig. 1b).
Incubation of glomeruli with Penicillin G alone did not affect P alb (Fig. 1c). When isolated glomeruli were exposed to PA (10 ug/ ml) before Penicillin (700 Unit/ml), P alb remained elevated (0.7560.05 vs. 0.8960.20) (Fig. 1d), supporting the hypothesis that the Penicillin effect is exerted by blocking PA entrance into the cell.

Effect of PA and Penicillin G on glomerular adhesion and cellular outgrowth on collagen IV substrate
In control conditions 8565% of isolated glomeruli adhered to the collagen IV substrate, and a cellular outgrowth was observed around the glomerular structure ( Fig. 2A-a). The incubation with PA alone diminished the number of adhered glomeruli at all PA concentrations compared to control; the outgrowth from glomeruli was limited, and the cellular morphology was altered ( Fig. 2A-b,  2B).
A complete inhibition of the PA effect was present when Penicillin G 700 IU/ml was pre-added, i.e. the number of attached glomeruli and the outgrowth from glomeruli were similar to the control experiment ( Fig. 2A-c, 2B). A partial inhibition of PA effect was observed for Penicillin G 500 IU/ml (Fig 2B). Glomeruli treated only with Penicillin at the same concentrations demonstrated a behavior similar to the control (data not shown).

Penicillin G prevents PA-induced podocyte damage
A preliminary time-course study was first conducted with human podocytes incubated with PA for different periods (12, 24, 32 and 48 hours); decreased viability was observed after 48 hours of incubation; therefore, this time point was selected for further investigation.
Penicillin G alone did not affect cell viability as compared to medium alone, whereas PA alone diminished viability by about 50% (Fig 3a). Pre-incubation with Penicillin G prevented partially or completely the decrease of viable cells induced by PA; the effect was statistically significant at the concentration of 700 IU/ml of Penicillin G (Fig 3a).
As illustrated in Figure 3B, in control condition actin stress fibers were prominent in the podocyte cell body. The focal adhesion molecule vinculin showed a peripheral and regularly distributed pattern of expression and co-localized with actin. These features were dose-dependently lost after PA incubation (2,5 and 10 ug/ml) for 48h, whereas preincubation with Penicillin G partially (PA 10 ug/ml) or completely (PA 2.5 ug/ml) prevented PA-induced cytoskeletal remodeling.

Internalization of fluorescent Penicillin in human podocytes
The above described results suggest that Penicillin G may block PA entry by engaging membrane transporters. If so, it would be possible to find Penicillin G inside the cell. To test this hypothesis, cells were incubated with the Penicillin fluorescent analogue Bocillin. Fluorescent intracellular puncta evenly distributed throughout the cytoplasm (Fig. 4a,b,c) and in the cell processes (Fig 4d, e) were clearly observed after 30 minutes incubation. Intracellular fluorescence was detected in 100% of cells also by flow cytometry (Fig 4f).
Penicillin G pre-treatment in the PA rat model As expected [16], a single injection of PA in three-month old Sprague-Dawley rats caused proteinuria by day 10   In control condition, vinculin is periodically expressed at the cell periphery, at the tip of actin stress fibers, as shown by co-localization with actin (left panels). These features are modified after incubation with either 2.5 and 10 ug/ml PA; vinculin is irregularly dispersed in the cell body, actin stress fibers are progressively reduced by 2.5 and 10 ug/ml PA, and co-localization of the two molecules occurs in the cell body and is absent at the cell periphery (middle panels). Preincubation with Penicillin G (500 Units/ml) partially (PA 10 ug/ml) or completely (PA 2.5 ug/ml) preserves the cytoskeletal structure from PA damage (right panels); the antibiotic prevents loss of actin fibers even with PA dosages of 10 ug/ml. With 2.5 ug/ml PA, vinculinpositive focal adhesions are also maintained at their location. Scale bars = 20 um. doi:10.1371/journal.pone.0066159.g003 associated with increased serum albumin (Table 4). Penicillin pretreatment also affected the kidney-to-body weight ratio and glomerular size. Control animals, receiving Penicillin G followed by saline, did not differ from saline-injected controls ( Table 4). The analysis of P alb on isolated glomeruli from animals sacrificed at the end of the study (day 10) showed that Penicillin G pre-treatment preserved glomerular albumin permeability in the normal range and demonstrated a strict correlation with the urinary protein values observed in the in vivo experiments (Fig 5).

Podocytes express the mRNA of species-specific membrane transporters
The inhibitory effect of Penicillin G on PA-induced glomerular injury both in vitro and in vivo, and the entrance of Penicillin into the cell, suggested the possible engagement by Penicillin of the same membrane transporters used by PA. Therefore, RT-PCR and quantitative RT-PCR experiments were performed to analyze components of the membrane transport families that have been associated with Penicillin G membrane transport in other cell systems.
As summarized in Fig 6, human podocytes expressed OATP-A, OATP-B, OATP-D and OATP-E from the OATP family; in contrast, OATP-C (liver specific transporters), OAT3 and PEPT1  were not detected, and PEPT2 showed a low expression level (Fig  6a, b). In primary rat podocytes, we studied the OATP transporter family (Oatp1, Oatp2, Oatp3, OatpE, Oatp4c1), one member of the Oat family (Oat3), and the peptide transporters PepT1 and PepT2 (Fig 6c,d). In agreement with the results obtained in human cells, we found expression of all genes that codify for Oatp transporters but Oatp3, and absence of the other analyzed membrane transporters.
Considering the high expression of the OATP transporters, we searched for their possible variation induced by PA. Though a significant variation was only found for Oatp2, that was increased in rat podocytes after PA treatment, also other transporters exhibited expression changes, particularly OATP-A in human cells and Oatp1 in rat podocytes (Fig 7a,b,c).

Podocytes express a specific efflux transporter Pglycoprotein
The gene expression of OATP/Oatp influx transporters on the podocyte surface implies the co-expression of efflux membrane transporters, such as P-glycoprotein, as described by Kim [17].
Our results show that the P-glycoprotein transporter gene (MDR1) is indeed expressed by human podocytes (Fig 8a), and increases in PA-treated cells, particularly after 48 hours (P,0.001, Fig 8c).
Correspondingly, the rat MDR1 homologues, Mdr1a and Mdr1b, are both expressed in rat podocytes (Fig 8b), where 48 hour PA incubation was able to increase Mdr1a expression ( Fig  8d). P-glycoprotein analysis by immunofluorescence confirmed the increased expression after 48 hour PA incubation (Fig 9 a,b,c), when PA also caused remodeling of the podocyte cytoskeleton, as shown by the alteration of synaptopodin (Fig 9e), and the almost complete disappearance of actin stress fibers (Fig 9g).
To establish if P-glycoprotein expressed by podocytes was functionally active, and if its activity could be modified by PA, we used its specific fluorescent substrate Rho 123 (Fig 10) and the specific inhibitor cyclosporine A (CSA) [15]. In control experiments, after Rho 123 incubation and replacement with Rho 123free medium, mean fluorescence intensity decreased by approximately 35%, indicating Rho 123 efflux from the cells (Fig 10a). PA incubation caused an increase of Rho 123 efflux, as shown by further reduction of fluorescence as compared to the control cells (Fig. 10b), whereas CSA consistently abolished the efflux of the substrate.

Effects of transport blockade
Confirming the use of OATPs/Oatps by penicillin G to enter the cell, blockade of these transporters by either cyclosporine or rifampicin, inhibited the entry of fluorescent penicillin ( Figure  11a,b) into podocytes.
When cyclosporine was added after penicillin internalization was completed, P-glycoprotein blockade was confirmed by persistent cell fluorescence, whereas untreated cells were mostly devoid of penicillin (Figure 11c).

Discussion
The presence of aminoacid transporters in podocytes has been known for some time [4], whereas drug transport has been largely investigated only in tubular cells [5]. As a basis for the present experiments, we speculated that substances which are harmful to the podocyte, such as Puromycin Aminonucleoside (PA), require a drug transporter system to enter and damage the cell.
The membrane transporters OATLP1 and mOCTL1 have been described in podocytes, and have been shown to contribute to trans-epithelial molecular transfer in these cells [2,3]. More recently, Xia e al. [6] suggested that PA uses a membrane transporter to enter the podocyte, and our expression data demonstrate that, besides the organic cation transporter PMAT identified by these authors, podocytes do express and utilize other transporters for which Penicillin G is a known substrate. Further support to our results comes from a series of microarray expression data obtained from mouse primary podocytes or mouse podocyte cell lines, publicly available as GEO data sets (for instance: GSM253173, GSM253174, and GSM833242, GSE17145, and GSE39441). They show that podocytes indeed express a complete     The potent Penicillin G prevention of PA-induced increased glomerular permeability and of the accompanying harmful consequences on podocyte cytoskeletal structure can only be explained by the sharing of the same transport systems by the two substances. In line with this hypothesis, fluorescent Penicillin was observed inside the cell, while administration of Penicillin after PA was ineffective, still consistent with a cis-inhibition of the uptake mechanisms.
Drug transporter molecules have been classified into influx transporters, related to the uptake of substances into the cell, and efflux transporters, which are involved in the transport of materials outside the cell, and are typically located either at the basolateral or the apical membrane in polarized cells [17].
The OATP transporters have been considered key determinants in the cellular uptake of many endogenous and exogenous chemicals and are expressed in various tissues [17,18]. They were long considered only transporters of organic anions, but have also been shown capable of transporting cation, neutral, zwitterionic as well as anionic compounds [17]. Recently, the OATP family has been identified as the main transporter of Penicillin G in humans [19].
The proton-coupled oligopeptide transporters PEPT1 and PEPT2 have a significant role in conservation and regulation of peptide-bound amino acids and also in the absorption, availability and efficacy of a variety of peptidomimetic drugs, such b-lactam antibiotics [1].
Our results show that the mRNA of components of the OATP/ Oatp families, and at a much lesser extent those of the PEPT family, are expressed by both human and rat podocytes. Expression of OATP members is affected by PA treatment, both in vitro and in vivo. In contrast to podocytes, renal tubular cells handle Penicillin G mainly by the OAT3 transporter, and accordingly, Oat3 knockout mice exhibit altered clearance and distribution of the antibiotic [20]. An exact homology between OATP transporters in humans and Oatp transporters in rodents does not exist, and several components of this transport family are still unknown. Rat Oatp1 and Oatp2 are abundantly expressed in liver and kidney and much less in the brain, whereas Oatp3 is absent in the liver but highly expressed in the kidney [21]. According to our data, podocytes seem to express both Oatp1 and Oatp2, but not Oatp3. Human OATPE and rat OatpE have an overall aminoacid sequence homology of 72.6% and have been found in the retina [22]. In contrast, the expression of Oatp4c1 was described specifically in rat glomeruli [23], in agreement with our data.
Concerning the efflux transporters, Kim et al. [17] showed that a number of OATP transporters share drug substrates with certain efflux transporters, such as P-glycoprotein (MDR1), and this coexpression is described in several organs involved in drug disposition such as the liver, the intestine, and kidney tubular cells, as well as in barrier systems, such as the blood brain barrier. P-glycoprotein acts as a membrane-bound, ATP-consuming drug efflux pump that actively extrudes a variety of structurally unrelated compounds across the plasma membrane as a defense mechanism [1]. Thus, the coordinated activity between an OATP influx transporter and an efflux transporter may determine the net cellular drug entry or efflux of shared substrates [17]. Functionally, two different types of P-glycoprotein have been described, but only class I has been related to drug transport [24]. Humans carry a single class I group gene (MDR1, or ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1), while rodents carry two class I genes (mdr1a and mdr1b) (1); all encoded proteins are capable of drug transport, and Penicillin G is their main substrate [25].
We discovered a significant expression of the genes encoding Pglycoprotein in human and rat podocytes, and, similarly to OATP/Oatp molecules, their expression changed after PA treatment. In vitro, we could also confirm increased extrusion activity after PA treatment, which could be an important mechanism of protection from pathological insults, albeit not podocyte-specific, because mesangial cells have been shown to possess this protein as well [25].
Our studies with specific inhibitors of confirm that both influx and efflux transporters are actively engaged by Penicillin G on the podocyte surface. Therefore, based on results obtained in this study, we can summarize our working hypothesis as represented in Figure 12; podocytes constitutively express specific membrane transporters which regulate small solute passage across the membrane and maintain cellular homeostasis. For example, low dosages of Penicillin G are dealt by these transporters and podocytes do not exhibit morphological and molecular changes. Due to its chemical characteristics, PA uses the same membrane transporters as Penicillin G to enter podocytes, but PA entry causes podocyte damage and variation of expression of the transporters themselves. Instead, when membrane transporters are already engaged by Penicillin G, PA entry is blocked and cell damage is prevented.
In conclusion, our study has demonstrated the existence and function of some influx and efflux membrane drug transporters on podocytes and their modification in response to PA. Further research is certainly needed to completely clarify their function, which could be relevant for cell pathophysiology and the choice of treatment for glomerular diseases. Considering that membrane transporters are involved in drug metabolism, we believe that specific polymorphisms of these molecules may be relevant to drug response in nephrotic syndrome.


extfieldsofstudy:
Medicine
Computer Science

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GPCRDB: an information system for G protein-coupled receptors

For the past 20 years, the GPCRDB (G protein-coupled receptors database; http://www.gpcr.org/7tm/) has been a ‘one-stop shop’ for G protein-coupled receptor (GPCR)-related data. The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data, such as multiple sequence alignments and homology models. The GPCRDB also provides visualization and analysis tools, plus a number of query systems. In the latest GPCRDB release, all multiple sequence alignments, and >65 000 homology models, have been significantly improved, thanks to a recent flurry of GPCR X-ray structure data. Tools were introduced to browse X-ray structures, compare binding sites, profile similar receptors and generate amino acid conservation statistics. Snake plots and helix box diagrams can now be custom coloured (e.g. by chemical properties or mutation data) and saved as figures. A series of sequence alignment visualization tools has been added, and sequence alignments can now be created for subsets of sequences and sequence positions, and alignment statistics can be produced for any of these subsets.

INTRODUCTION
G protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors. They regulate a wide range of cellular processes, including those associated with taste, smell and vision, and they control myriad intracellular systems, ranging from neurotransmission to hormone signalling. GPCRs are major targets for the pharmaceutical industry, as reflected by the fact that more than a quarter of all FDA-approved drugs act on a GPCR (1). At present, only 30 of the 350 genes that code for non-olfactory receptors in the human species (2) are truly validated therapeutic targets (3), indicating this family's immense potential for future drug development. An increasing number of drugs have been found to display polypharmacology, i.e. activity through multiple receptor targets (4). However, endogenous ligands for 135 of the so-called orphan receptors have so far eluded researchers.
Early releases of the GPCRDB (5-8) focused on the compilation and homogeneous presentation of many types of heterogeneous data, with the aim of providing the four main facilities needed in an information system: browsing, querying, retrieval and inference. The first three of these facilities have been available ever since the start of the project, but received a major boost when the GPCRDB was coupled to an intelligent PDF reader (9) that puts all relevant aspects of the GPCRDB non-intrusively in a side-bar in the PDF reader window. Inference really only started when a number of interactive tools were added (10), enabling bioinformaticians to interact with multiple sequence alignments, together with derived data such as entropy and variability scores, in an integrated environment. For example, these tools were successfully applied in the 2010 GPCR-Dock competition (11). In the past, computing facilities were aimed at expert GPCR bioinformaticians. In contrast, the new interactive tools are readily accessible to non-expert users and allow faster execution of visualization and analysis tasks.

Crystal structure browser
In recent years, X-ray crystallography of GPCRs has revealed the sites and mechanisms for binding ligands, lipids, and G proteins, as well as the conformations of *To whom correspondence should be addressed. Tel: +31 24 3619390; Fax: +31 24 3619395; Email: vriend@cmbi.ru.nl, Gerrit.Vriend@radboudumc.nl activated states. The GPCRDB structure browser includes manually annotated key data for all ligand-receptor complexes and at least one (the highest resolution) representative file for receptors solved only as apo structures. The structure browser can select crystal structures based on a series of filters for receptors, ligands, activation states, G protein presence, PDB and Pubmed identifiers, resolution, structure completeness and so forth. Sequence similarities can be retrieved by specifying a receptor reference, facilitating template selection for homology modelling.
Structure-based sequence alignments and homology models GPCR transmembrane (TM) helices are known to contain many irregularities. However, it was only recently realized, with the availability of new crystallographic data, that GPCRs contain many more -bulges than those in helix II and helix V. The -bulges in GPCRs are relatively nonconserved. As an example of this non-conservation, Figure 1 shows the -bulges in helix V of rhodopsin and the adenosine 2A receptor, and Figure 2 shows the alignment of the middle part of helix II in 60 trace amine receptors. The receptors are on average 70% sequence identical, but the bulge is only present in around half of all family members.
The abundance of -bulges required a novel residue numbering approach that involved rewriting the DSSP secondary structure analysis software (15) and the GPCR-specific alignment software. This, in turn, meant that >65 000 homology models had to be constructed again. All GPCR sequences are now modelled twiceonce using as template the most sequence-similar inactive form, and once using the most similar active form. Modelling was done with YASARA (16), aligning the model with the template contained in the GPCRDB and using default values for all other parameters and options. All GPCR alignment profiles were manually updated to reflect our latest knowledge about thebulges, and all 1272 alignments were regenerated. It seems likely that this exercise will need to be repeated in the coming years as new GPCR structure data reveal new -bulge patterns.

Translation of generic and receptor-specific residue numbers
We wanted to take proper care of the -bulges that are widely present in six of the seven GPCR TM helices without excessively changing the commonly used generic residue numbering schemes. The Oliveira numbering (17) and the B&W numbers (18) were maintained as far as possible, whereas bulge residues were given the same number as the residue directly N-terminal in the sequence but with a digit added that reflects the number of the bulge. The Utopia-GPCRDB PDF reader automatically takes these new numbers into account, whereas our new sequence indexing tool provides computational access to all generic and receptor-specific residue numbers for selected receptors.

Snake-like and helix box diagrams
Snake-like diagrams now include the full loops and terminal sequences ( Figure 3A). New helix box diagrams present the TM helices as seen from 'above' (Figure 3A). These diagrams are similar to previously used helical wheel plots but orient the amino acids in better agreement with the 3D structures. Hovering the computer mouse pointer over the TM amino acids displays their residue numbers. Amino acids can be coloured to illustrate their physicochemical properties, or the presence of mutation data or the mutation effects. The diagrams can be downloaded as picture files or in scalable vector graphics format to allow further editing.

Residue conservation and property statistics
In functional or evolutionary studies of specific amino acids or their properties, it is typically relevant to find  out which additional receptors share the conserved amino acids or properties (e.g. specific charge or hydrophobicity). Therefore, sequence alignments are augmented with a series of statistics. For each position, the following data are listed: the consensus sequence, the percentage of each of the 20 amino acids present, and percentages for relevant properties such as aromaticity, acidity or hydrogen bonding capability.

Alignment sub-site/domain selections
The alignment and similarity tools integrated into GPCRDB offer users the ability to select arbitrary combinations of helices, residue positions or predefined sets of residues, for example, the amino acids in the TM binding cavity (19). By focusing on a given functional site rather than the full sequence, the receptor similarities will better reflect the structural features involved in, for example, receptor dimerization, ligand binding or G protein binding.
Similarity search with a reference receptor (one-to-all similarities) Similarity searches are conducted by specifying a target of interest, a set of receptors and the residue positions of interest. Results are presented as a sequence alignment, in which the target is followed by a list of hits in order of sequence identity, similarity or alignment score. The data can be downloaded as either an alignment file or a spreadsheet.

Trees (all-to-all similarities)
Neighbour-joining trees (20) can be generated based on any sub-site/domain and set of receptors. Trees can be calculated with up to 100 bootstraps, displayed in circular and ladder representations, and downloaded in Newick format for use with stand-alone tree software.

Sequence motif search (conserved and non-conserved separation)
The sequence motif search tool generates more precise and discriminative results, by allowing residues to be matched for relevant amino acid properties (21), e.g. their hydrophobicity, hydrogen bond donor capability or size. Relevant applications for this tool include rationalization of observed polypharmacology, receptor panel selection for off target screening and ligand inference from old to new targets.

CONCLUSIONS
The 20th yearly release of the GPCRDB includes a large number of novel discoveries. The solved structures (see http://gpcr.scripps.edu/) reveal the presence of manybulges that are not conserved among or even within GPCR subfamilies. We have updated all the alignments and homology models, together with the residue numbering schemes, to ensure agreement between the contents of the GPCRDB and new insights obtained by studying all the available structure data. Additionally, the new GPCRDB release includes a powerful yet user-friendly computational toolbox that provides users with crystal structure browser, receptor visualization and alignment analysis tools, plus options to study receptor similarity both quantitatively and graphically.


Anticancer Activity of Acetone Extract of Quercus infectoria Olivier Fagaceae in 1,2 Dimethyl Hydrazine Induced Colon Cancer

Adenoma of colon and rectum (colorectal cancer) is one of the commonly occurring cancers reported to cause 655,000 deaths worldwide. It is fourth most common form of cancer in United States and third leading cause of cancer-related deaths in the Western world (WHO 2006, NCI 2009). In India also the incidence of colon cancer is very large. During a 32-year period (1941–1972), 555 cases of colorectal cancer were recorded at the Tata Memorial Hospital, Mumbai[1]. In contrast a total of 560 cases of colorectal cancer were treated at the same institution in 2006 alone. Colon cancer as compared to rectal cancer occurs more frequently as a result of mutations in the signaling pathway that artificially increases signaling activity. The mutations can be inherited or acquired and occur in the intestinal crypt stem cell [2]. Aberrant crypt foci (ACF) are putative preneoplastic lesions of colonic neoplasia in rodents and humans. During the process of colon carcinogenesis, ACF appears in the early stages and subsequently develop into polyps, adenomas and eventually carcinomas [3]. Hence ACF development is commonly used as a parameter for assessment of colorectal cancer even in animals.

Introduction
Adenoma of colon and rectum (colorectal cancer) is one of the commonly occurring cancers reported to cause 655,000 deaths worldwide. It is fourth most common form of cancer in United States and third leading cause of cancer-related deaths in the Western world (WHO 2006, NCI 2009). In India also the incidence of colon cancer is very large. During a 32-year period , 555 cases of colorectal cancer were recorded at the Tata Memorial Hospital, Mumbai [1]. In contrast a total of 560 cases of colorectal cancer were treated at the same institution in 2006 alone. Colon cancer as compared to rectal cancer occurs more frequently as a result of mutations in the signaling pathway that artificially increases signaling activity. The mutations can be inherited or acquired and occur in the intestinal crypt stem cell [2].
Aberrant crypt foci (ACF) are putative preneoplastic lesions of colonic neoplasia in rodents and humans. During the process of colon carcinogenesis, ACF appears in the early stages and subsequently develop into polyps, adenomas and eventually carcinomas [3]. Hence ACF development is commonly used as a parameter for assessment of colorectal cancer even in animals.
Current medical treatment in colon cancer consists of chemotherapy, hormonal treatment and targeted therapy. They are sophisticated, expensive and not widely available. Therefore, a search for novel anticancer agents from natural products may provide an alternative and cost-effective treatment modality [4]. The herbal drug Quercus infectoria is reputed plant in Ayurvedic system of medicine and commonly known as "Majuphul" in Ayurvedic literature. Tannins(gallotannic acid) is one of the main constituent found in the galls of Quercus infectoria in 50%-70%. Gallotannic acid a tannic acid is known for its anti-mutagenic, anticancer and antioxidant properties [5]. In present investigation we have studied the anticancer potential of Q. infectoria using 1,2-dimethylhydrazine induced colon cancer model in rats.

Materials and Methods
Materials 1,2-dimethylhydrazine (DMH) was purchased from Sigma Chemical Company. ELISA kit for TNF-alpha, TGF β and VEGF from Nucleus inc. Ahmedabad. All other chemicals including solvents were of high purity analytical grade marketed by Dutt enterprise chemicals, Gujarat, India.
Dried galls of Q.infectoria were obtained from a commercial supplier in Ahmedabad. It was authenticated by Dr. Hitesh A. Solanki, Reader Department of Botany, Gujarat University, Ahmedabad. Acetone extract of Q.infectoria was prepared. The yield of extract was 46.6%. It was standardized by HPTLC taking gallotannic acid as marker.

Induction of colon cancer
Male albino Wistar rats weighing 150-200 gms obtained from Zydus research center Moraiya. Animals were cared for in compliance with the principles and guidelines of Council for the purpose of control and supervision of experimental animals (CPCSEA), ministry of environment and animal welfare, Govt. of India and approved by institutional animal ethical committee, in accordance with the Indian National Law on Animal Care and Use (Registration number: 1338/C/10/CPCSEA). The animals were housed 4 per polypropylene cage with a wire-mesh top and a hygenic bed of husk in a specific pathogen-free animal room under controlled conditions of a 12 h light/12 h dark cycle with temperature of 24 ± 2°C and relative humidity of 50 ± 5% until the end of the experimental period. The rats were held in quarantine for 1 week and had access to food and tap water ad libitum. Commercial pellet diet containing 4.2% fat was powdered and mixed with 15.8% peanut oil, making a total of 20% fat. This modified diet was fed to all rats throughout the 16 week experimental period.

Administration of carcinogen
Colon cancer was induced by subcutaneous injections of 1,2 dimethylhydrazine (DMH) at 20 mg/kg once a week for the first 4 consecutive weeks. DMH was dissolved in 1 mM EDTA; the pH was adjusted to 6.5 with 1 mM NaOH.

Treatment with acetone extract of Q. infectoria (AEQI) to animals
After 1 week of acclimatization, animals were randomly divided into six groups each containing ten rats. Group I: Control Group II: Rats treated with DMH 20 mg/kg sc weekly for four weeks. Group III: Rats treated with DMH and AEQI 450 mg/kg p.o for 16 weeks. Throught the period body weight of animals and mortality were recorded. At the end of sixteen weeks blood samples were collected and then rats were sacrificed.

Assessment of anti cancer activity
Animals were sacrificed by ether anesthesia at the end of fourth month from start of the experiment. The colons were removed, cleaned with saline solution (0.9%) and slit open longitudinally from cecum to anus. Each colon was cut into proximal, middle and distal portion of equal length, and fixed flat between two pieces of filter paper in 10% neutral-buffered formalin for at least 24 h. Later, they were stained with 5% methylene blue for 5 to 10min, and they were then placed on a microscopic slide; the mucosal side was observed through light microscope at 40x magnifications. ACF were counted by the method of Bird (1987) [6]. Crypts or distinct foci of crypts were counted as an ACF if they displayed at least two of the following characteristics [7]: (i) occupy a greater area than surrounding crypts; (ii) have a thickened epithelial lining; (iii) have elongated or altered shape of luminal opening; and (iv) have an increased pericryptal zone separating the crypt or foci from surrounding crypts.

Detection of tissue vascular endothelial growth factor
Yet another part of colon tissue was homogenized in 50 mM/l Tris buffer (pH 8.0) and centrifuged at 10000 RPM for 10 min. 50 µL supernatant were used for VEGF measurement. VEGF concentration were assayed by ELISA. Total protein was measured by Lowry's method [12].

Detection of serum TNF α and TGF β
At the end of 16 weeks before the animals were sacrificed blood samples were collected from retro-orbital plexus of the eye from 12 h fasted rats into vacutainer clotted tubes, where sera were separated by centrifugation within 30 min at 4000 rpm at 4°C for 10 min using centrifuge, then divided into aliquots and kept at -80°C for further assay of TNF-a and TGF β immediately. Serum TNF-α and TGF β were estimated by enzyme-linked immunosorbent assay (ELISA) [13].

Statistical Analysis
Results have been expressed as mean ± standard error of the mean (SEM). Data were analyzed as a completely randomized design using One Way Analysis of Variance (ANOVA) followed by Dunnett's Post Hoc Test. The statistical significance was set at P < 0.05.

Result Body weight and mortality
Body weight of animals treated with DMH were found to be significantly reduced after 4 weeks and 16 weeks study period. There was also high mortality found in these animals. Treatment with AEQI significantly prevented not only the loss of body weight (Table 1) but also mortality in these animals ( Table 1).

Effect of AEQI on development of ACF induced by DMH
There was development of colon ACF and AC in rats treated with DMH ( Figure 1). AEQI treatment for 16 weeks significantly reduced the number of AC, ACF and crypt multiplicity as compared to DMH group (Table 2).

Oxidative stress parameters
Treatment with DMH produced significant increase in MDA, MPO, NO levels associated with significant decrease in colonic SOD as compared to control animals. Treatment with AEQI significantly prevented DMH induced increase in MDA, MPO and NO levels further it also produce increase in SOD levels ( Figure  2).

Serum TNF α, TGF β and tissue VEGF
Serum TNF α, TGF β and tissue VEGF levels were increased with DMH administration to rats. This up regulation was prevented by treatment with AEQI. DMH caused elevation in TNF α, TGF β and tissue VEGF levels. Oxidative stress could be the additive factor which was induced by administration of DMH (Figure 3).

Discussion
In the present study rats treated with DMH were found to show presence of ACF and severity of dysplasia which indicates development of carcinomatous changes. Our findings are in consonance with those reported earlier.
ACF are indeed the earliest manifestations of colon cancer, and then ACF could provide insight into the earliest events of colon tumorogenesis. The genesis of colon tumor is a multistep process, and ACF have been proposed to precede the earliest adenoma [6,14,15].
DMH is used to produce Colon cancer, which is metabolized into the carcinogenic metabolite without previous metabolism by other tissues or colon bacteria in rat [16,17]. The ultimate carcinogenic metabolite of DMH is responsible for methylation of the DNA bases of various organs, including epithelial cells in the proliferative compartment of the crypts, which results in a great loss of colonic cells by apoptosis, an increase in proliferation, and an apparent increase in mutations of colonic epithelial cells [18]. All these alterations are likely to produce increase in inflammatory growth factors such as TNF α, TGF β and VEGF as well as oxidative stress. There was also an increase in mortality and decrease in body weight of the animals treated with DMH, which may be due to induction of cancer by DMH.
Weight loss is an important prognostic factor in cancer; the higher the extent of weight loss, the shorter the survival time. The prognostic effect of weight loss is greatest in patients with a more favorable prognosis [19]. Treatment with AEQI reduces weight loss and DMH induced mortality in animals showed good prognosis in colon cancers.   ACF and AC were found to significantly reduced in colon of rats treated with both DMH as well as AEQI, thus suggesting that AEQI possesses activity against DMH induced colon cancer. This is further strengthened by observation that there was significant reduction in mortality of rats treated with AEQI and significant prevention of the loss of body weight in rats treated with AEQI in addition to DMH.
The phytochemical work carried by Umachigi et al., (2008) revealed that tannin is one of the active compounds which may be responsible for the antiproliferative activity [20]. Srivastava 2000 reported that gallotannic acid is antimutagenic in nature [5]. Several antioxidants in plants have been suggested to contribute to the anti carcinogenic effects and other such as flavanols have been able to inhibit cancer cell proliferation in vitro [21]. Inhibit ACF and AC generation by AEQI in colon may be due to presence of tannic acid.
Important to carcinogenesis, the unregulated or prolonged production of cellular oxidants has been linked to mutation (induced by oxidant-induced DNA damage), as well as modification of gene expression. In particular, signal transduction pathways, including AP-1 and NFκB, are known to be activated by reactive oxygen species, and they lead to the transcription of genes involved in cell growth regulatory pathways [22].
In addition to these many other chemical mediators are involved in carcinogenesis such as TNF-α, which is an acute phase reactive protein which is responsible for many inflammatory diseases including caner of colorectal metastases confined to the liver [23] Further in colon cancers, loss of TGF-β sensitivity is frequently accounted for by loss or mutation of known components of the TGF-β signaling pathway, notably TβRII and Smad4. Moreover VEGF expression was closely related with biological behavior of colon cancer and significantly associated with high intratumoral microvessel density (MVD) [24].
As mentioned above increased luminal activities of NO might be the aiding factor in tumor development. Taylor et al (1996) reported that iNOS gene expression is mediated by NFкB [25].
Malondialdehyde is final product of oxidative stress and is good indicator for extent of oxidative stress [26]. Myeloperoxidase catalyses the conversion of proportionally more stable hydrogen peroxide to unstable hydrochlorus acid. This in turn promotes oxidative stress and additionally it induces neutrophil infiltration on mucosal area causing further damage to the tissue [27]. Preventive anti-oxidant, such as superoxide dismutase (SOD) enzyme is the first line of defense against reactive oxygen species [28]. Superoxide dismutase (SOD) is widely distributed in cells with high oxidative metabolism and has been proposed to protect such cells against the deleterious effect of superoxide anion [29].
In our studies we found anti oxidant and anti inflammatory po-  Each value is mean ± SEM for 6 replicates in each group. * P <0 .05 significant different from control and # P <0 .05 significant different from DMH treated control.
Each value is mean ± SEM for 6 replicates in each group. * P <0 .05 significant different from control and # P <0 .05 significant different from DMH treated control. tential of Q. infectoria. Kaur et al., (2003) showed anti oxidant and anti inflammatory potential of Q. infectoria which supports our finding [30]. Thus our data suggest that antioxidant effect of AEQI might be due to presence of tannic acid.
TNF is a member of a family of cytokines that are important for immune function and tissue homeostasis. Over expression of these ligands could contribute to tumor development by providing a defense that protects tumor cells by killing host immune cells. Similarly TGF β induces angiogenesis and metastasis at late stage of tumor growth [31]. Guetz et al., 2006 said that overexpression of VEGF in colon cancer tissue indicated poor prognosis [24]. All these are important proinflammatory cytokines and their expression is under the transcriptional regulation NFκB. In our studies we found that there was significant increase in TNF α, TGF β and VEGF. These fairly support the contention that TNF α, TGF β and VEGF are involved in colon cancer.

Conclusion
Data from our study suggest that Q. infectoria exerts a chemopreventive effect in DMH-induced experimental carcinogenesis in rats indicating its potential as an anticancer drug for the treatment of colon cancer. The anticarcinogenic property of Q. infectoria in DMH induced colon carcinogenesis may be due to antioxidant property and reduction in the level of TNF α, TGF β and VEGF.


A genetic variation of the p38β promoter region is correlated with an increased risk of sporadic colorectal cancer

p38 plays a critical role in the proliferation, survival, migration and metastasis of colorectal cancer (CRC) cells. The present study assessed the correlation between a single nucleotide polymorphism (SNP) in the p38β promoter region (rs2235356, -1628A>G) and the predisposition of individuals to sporadic CRC in a case-control study. A genotyping method was developed to detect this SNP, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. A logistic regression analysis was used to determine the odds ratio (OR) and 95% confidence interval (CI). It was revealed that the -1628G variant allele was correlated with an increased risk of CRC (OR, 1.99; 95% CI, 1.60–2.47; P<0.0001). An in silico analysis revealed several transcription factors that either acquired or lost the ability to bind to -1628AA in the p38β promoter region due to the SNP. Therefore, this allelic variant may be a genetic modifier for CRC susceptibility.

Introduction
Colorectal cancer (CRC) is the third most frequently diagnosed type of cancer worldwide, accounting for >1 million cases and 639,000 mortalities every year (1). The incidence of CRC is currently markedly increasing in developing countries, including China (2). In a recent study, CRC was ranked in the top five causes of mortality in urban and rural areas (9.78 and 5.96 fatalities per 100,000, respectively) of China (3). CRC is a heterogeneous disease affected by genetic and environmental factors (4). It is widely accepted that the mutation of the adenomatous polyposis coli (Apc) gene is important in the early stages of the transformation of the colonic epithelium. The Apc gene encodes a tumor suppressor protein that is linked to 80% of cases of sporadic CRC, and which is involved in the inherited condition, familial adenomatous polyposis syndrome (5). Besides the loss of the APC protein and exposure to environmental factors, including cigarette smoke and alcohol (6), a poor diet and high body mass index (BMI) (7) also contribute to the risk of CRC. The mitogen-activated protein kinase (MAPK) pathway is known to transduce these signals, leading to various biological outcomes, including apoptosis, inflammation and tumorigenesis (8). The MAPK family of proteins comprises extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38. The p38 subfamily consists of four isoforms (p38α, -β, -γ, and -δ) that are defined by the threonine-glycine-tyrosine (Thr-Gly-Tyr; TGY) dual phosphorylation motif, and exhibit significant homology at the amino acid level. p38α and -β are ubiquitously expressed and are thought to have overlapping functions, while p38γ and -δ are differentially expressed, depending on the tissue type. p38 is activated through phosphorylation at the Thr 180 -Gly-Tyr 182 motif by MAP kinase kinase 3 (MKK3), MKK4 and MKK6 (9). The activation of p38 results in various cellular changes, including the regulation of transcription, protein synthesis, cell surface receptor expression, cell cycle progression and apoptosis. The oncogenic function of p38 is context-dependent. In CRC, p38α is required for the proliferation and survival of CRC cells, and its inhibition leads to cell cycle arrest and autophagic cell death (10). p38 has been demonstrated to be involved in CRC cell migration and metastasis in animal models (11,12). Furthermore, it has been revealed that the inhibition of p38 results in a loss of CRC cell resistance to chemotherapy drugs (13). Therefore, p38 activity is important in CRC tumorigenesis and resistance to chemotherapy.
Regardless of its importance, few studies have examined the contribution of p38 genetic polymorphisms to the risk of CRC. The present study investigated the functional impor-tance of an SNP located in the promoter region of p38β, and correlated its presence with the risk of sporadic CRC. To the best of our knowledge, the study demonstrates for the first time that the p38β promoter region SNP (rs2235356, -1628A>G) is correlated with an increased risk of sporadic CRC, in a study cohort.

Materials and methods
Study subjects and sample collection. The study was approved by the Institutional Review Board of Sun Yat-Sen University (Guangzhou, China). The study cohort consisted of 855 patients with histologically confirmed sporadic CRC and 871 cancer-free control subjects, who were genetically unrelated Han Chinese individuals from Guangzhou and the surrounding regions of Southern China (14). The response rate was 95%. The exclusion criteria removed individuals with familial adenomatous polyposis and those fulfilling the Amsterdam criteria for hereditary nonpolyposis CRC from the study. The 871 cancer-free control subjects were randomly selected from a pool of >10,000 individuals who had participated in the health check-up programs in the community health stations in Guangzhou during the same period of time as when the cases were recruited. The response rate was 85%. The controls were frequency-matched to the cases by gender and age (±5 years). Following the receipt of written informed consent, each participant was scheduled for an interview that used a structured questionnaire to collect information on various factors, including smoking status, alcohol use and the family history of cancer. The definitions of these factors corresponded with those used in a previous study by our group (14). The present study used the BMI cutoff points suggested by the Cooperative Meta-Analysis Group of the Working Group on Obesity in China (15). Subjects whose BMI was <18.0 kg/m 2 were categorized as being underweight; whereas 18.0-25.0 kg/m 2 constituted normal body weight and >25.0 kg/m 2 was classed as overweight. Each subject was requested to donate 5 ml blood.
Genotyping. The p38β (rs2235356, -1628A>G) polymorphism was detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In brief, the genomic DNA from the peripheral blood of the participants was extracted by the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and subjected to PCR using the forward primer, 5'-CCGAGGTTTTGTGCAGAGTT-3', and the reverse primer, 5'-CAGGTCAGCTCAGTGATGAGA-3'. The PCR conditions used were as follows: 94˚C for 5 min, 35 cycles of 94˚C for 30 sec, 60˚C for 45 sec and 72˚C for 60 sec, and a final extension step of 72˚C for 10 min. The PCR amplification was further digested with FokI (New England Biolabs, Inc., Ipswich, MA, USA) overnight at 37˚C, followed by agarose gel electrophoresis to reveal the genotype of either -1628AA (two bands of 157 and 228 bp), -1628GG (one band of 385 bp) or the heterozygous -1628AG (three bands of 157, 228 and 385 bp) (Fig. 1). A GeneRuler Low Ranger DNA Ladder (100 bp; Thermo Scientific, Waltham, MA, USA) was used. Two researchers who were blinded to the CRC status of the participants independently evaluated the gel electrophoresis results. The PCR-RFLP assay was repeated in 10% of    the samples, which yielded identical results (data not shown). For each target genotype the PCR products were purified and confirmed by direct sequencing (Figs. 2-4). An in silico analysis was performed using Genomatix (Genomatix Software GmbH, Munich, Germany) to identify transcription factors that either acquired or lost the ability to bind to -1628AA in the p38β promoter region due to the SNP.
Statistical analysis. A two-sided χ 2 test was used to evaluate the differences in the distributions of age, gender, smoking status, alcohol use, BMI and family history of cancer between the CRC cases and the cancer-free controls. The Hardy-Weinberg equilibrium (HWE), determined by a χ 2 goodness of fit test, was used to compare the expected and the observed genotype frequencies in the cancer-free controls. Unconditional logistic regression was used to calculate the odds ratios (ORs) and 95% confident intervals (CIs) to estimate the correlation between the presence of the SNP and the CRC risk, with and without adjustments for age, gender, smoking status, alcohol drinking status, BMI and family history of cancer. A logistic regression model was also used for the trend test. In the stratification analysis, the main effects of the p38β polymorphisms were assessed in each subgroup, along with the effect of potential interactions between the p38β polymorphisms and selected variables on cancer risk. All two-sided statistical tests were performed using SAS software version 9.1 (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

Characteristics of patients with CRC and the control subjects.
The demographic characteristics of the 855 CRC cases and 871 cancer-free controls are presented in Table I. There was no significant difference in the distributions of age (P= 0.5773) and gender (P= 0.9319) between the two groups. However, the patients with CRC were more likely to consume alcohol (46.8% in CRC cases, compared with 23.4% in the controls; P<0.0001) and, in female participants, be menopausal (80.5% in CRC cases, compared with 57.9% in controls; P<0.0001). Furthermore, there were a greater number of CRC cases with an abnormal BMI or a family history of cancer compared with the controls (P= 0.0245 and P= 0.0379, respectively). These variables were therefore adjusted in the multivariate logistic regression analysis, as well as in the stratification and gene-environment interaction analysis, to reveal the effect of the studied SNP on the risk of CRC.
Distribution of the p38β promoter region SNP and the risk of sporadic CRC. The genotypic and allelic distributions of the p38β -1628A>G polymorphism among the cases and controls are summarized in Table II. The observed genotype frequencies of this SNP were in agreement with the Hardy-Weinberg equilibrium in the control subjects (P= 0.796). As demonstrated in Table II, the logistic regression analysis indicated that the subjects carrying the -1628G variant allele (-1628AG and -1628GG) had a 1.99-fold increased risk of sporadic CRC compared with those who were homozygous for the -1628A allele (95% CI, 1.6-2.47; P<0.0001). The increase in CRC risk was particularly evident in the subjects who were heterozygous for the -1628G allele, with a 2.22-fold increase in risk (95% CI, 1.78-2.79; P<0.0001). There was a significant trend for the allele dose effect on the risk of CRC (P trend <0.0001). The correlation between the -1628G variant allele and the risk of CRC was also statistically significant at a 5% type I error level, having adjusted for multiple tests using a Bonferroni correction (P<0.0001).

Stratification analyses of the p38β promoter region SNP and the risk of CRC.
A stratification analysis of the correlations between the p38β variant genotypes and the risk of sporadic CRC was performed by subdividing the subjects by age, gender, smoking and alcohol consumption statuses, family history of cancer and BMI (Table III). With the exception of the subjects who had a family history of cancer, the subjects carrying the -1628G variant allele (-1628AG and -1628GG) were correlated with an increased risk of CRC in all remaining subgroups (Table III).
In silico analyses of the affect of the p38β promoter region SNP on p38β gene expression. The presence of the SNP in the p38β promoter region may affect the gene expression of p38β. An in silico analysis was performed using Genomatix (Genomatix Software GmbH, Munich, Germany), to identify the transcription factors that had either acquired or lost the ability to bind to this locus due to the SNP. It was determined that the -1628G variant allele was correlated with a loss of binding ability for chorion-specific transcription factor (GCMa), but also with an acquired binding ability for basic Krüppel-like factor (KLF3), erythoid Krüppel-like factor (EKLF), the rat C2H2 zinc finger protein (involved in olfactory neuronal differentiation), Wilms' tumor suppressor, the zinc finger with Krüppel-associated box (KRAB) and SCAN (named after SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA) domains 3 (data not shown).

Discussion
The present study investigated the correlation between a putative functional SNP of the p38β promoter region (rs2235356) and the risk of CRC in a Chinese population, with a sample size of 855 patients with sporadic CRC and 871 cancer-free control subjects. We developed a PCR-based method for detecting this SNP, and demonstrated that the -1628G allele was correlated with an increased risk of sporadic CRC in our study cohort (95% CI, 1.6-2.47; P<0.0001). p38 promotes the survival of CRC cells, primarily through its functions in the DNA repair pathway and in autophagy. p38 is one of the effector kinases of the DNA damage sensor system, following the activation of ataxia telangiectasia mutated (ATM) kinase, ataxia telangiectasia and Rad3-related (ATR) kinase and DNA-dependent protein kinase (DNA-PK) (16). Therefore, p38 may enhance the DNA repair response following chemotherapy in colon cancer cells, resulting in drug resistance. Furthermore, p38 has been demonstrated to inhibit the autophagy and cell death of CRC cells (17), indicating that p38 activity is necessary for the survival of these cells. The present study identified an SNP in the p38β promoter region that may be biologically significant in the tumorigenesis of sporadic CRC, which is consistent with the importance of p38 in mediating CRC.
One of the primary downstream effectors of p38 is activator protein-1 (AP-1), a heterodimeric transcription factor composed of proteins belonging to the c-fos, c-Jun and    activating transcription factor (ATF) families. In CRC, AP-1 activity is activated by ERK and JNK (18), or by the hyperactivation of the Wnt pathway. p38 phosphorylates ATF-2, which forms a heterodimer with the Jun family transcription factors to form active AP-1 (19). Aberrant c-Jun activity has been observed in CRC, which is consistent with the function of p38 in the regulation of AP-1 (20). Abnormal p38 activity may increase the risk of CRC by modulating AP-1. In addition to JNK and ATF-2, AP-1 activity is also regulated by the level of c-fos. The expression of c-fos is dependent on sterol regulatory element, which interacts with ternary complex factors (TCFs). Sap-1a is a TCF that is phosphorylated by p38, thus AP-1 activity may also be regulated by p38 through Sap-1a. Therefore, aberrant levels of the p38 pathway constituents may promote CRC through the activation of the AP-1 transcription factor.
In conclusion, the present study has provided evidence that the -1628A>G genetic variation in the p38β promoter region may contribute to the susceptibility to CRC in Chinese populations. It would be of interest to determine if this SNP is correlated with the risk of sporadic CRC in other ethnic groups. Furthermore, future efforts are required to determine the functional importance of this SNP in controlling the level of p38β.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Polymorphisms of the core, NS3, and NS5A proteins of hepatitis C virus genotype 1b associate With development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the common sequelae of hepatitis C virus (HCV) infection. It remains controversial, however, whether HCV itself plays a direct role in the development of HCC. Although HCV core, NS3, and NS5A proteins were reported to display tumorigenic activities in cell culture and experimental animal systems, their clinical impact on HCC development in humans is still unclear. In this study we investigated sequence polymorphisms in the core protein, NS3, and NS5A of HCV genotype 1b (HCV‐1b) in 49 patients who later developed HCC during a follow‐up of an average of 6.5 years and in 100 patients who did not develop HCC after a 15‐year follow‐up. Sequence analysis revealed that Gln at position 70 of the core protein (core‐Gln70), Tyr at position 1082 plus Gln at 1112 of NS3 (NS3‐Tyr1082/Gln1112), and six or more mutations in the interferon/ribavirin resistance‐determining region of NS5A (NS5A‐IRRDR≥6) were significantly associated with development of HCC. Multivariate analysis identified core‐Gln70, NS3‐Tyr1082/Gln1112, and α‐fetoprotein (AFP) levels (>20 ng/L) as independent factors associated with HCC. Kaplan‐Meier analysis revealed a higher cumulative incidence of HCC for patients infected with HCV isolates with core‐Gln70, NS3‐Tyr1082/Gln1112 or both than for those with non‐(Gln70 plus NS3‐Tyr1082/Gln1112). In most cases, neither the residues at position 70 of the core protein nor positions 1082 and 1112 of the NS3 protein changed during the observation period. Conclusion: HCV isolates with core‐Gln70 and/or NS3‐Tyr1082/Gln1112 are more closely associated with HCC development compared to those with non‐(Gln70 plus NS3‐Tyr1082/Gln1112). (HEPATOLOGY 2013;58:555‐563)

H epatitis C virus (HCV) is a major etiologic agent of chronic hepatitis worldwide, with the estimated number of infected individuals being more than 180 million. Approximately 15% to 20% of chronically infected individuals undergo liver cirrhosis in a decade or so after infection, with hepatocellular carcinoma (HCC) arising from cirrhosis at an estimated rate of 1% to 4% per year. [1][2][3] Several host factors such as male gender, older age, elevated a-fetoprotein (AFP) level, advanced liver fibrosis as well as nonresponsiveness to interferon (IFN) therapy have been reported as important predictors of HCC development. 4,5 Recently, a host genetic factor, i.e., the DEPDC5 locus polymorphism, was reported to be associated with progression to HCC in HCV-infected individuals. 6 On the other hand, it remains controversial as to whether HCV itself plays a direct role in the development of HCC. Experimental data suggest that HCV contributes to HCC by modulating pathways that promote malignant transformation of hepatocytes. HCV core, NS3, and NS5A proteins were shown to be involved in a number of potentially oncogenic pathways in cell culture and experimental animal systems. 7 HCV core protein rendered cultured cells more resistant to apoptosis 8,9 and promoted ras oncogene-mediated transformation. 10,11 Moreover, transgenic mice expressing the HCV core protein in the liver developed HCC. 12 However, the clinical impact of HCV proteins on HCC development in humans and whether all HCV isolates are equally associated with HCC is yet to be determined. In a clinical setting, HCV core protein mutations at positions 70 (Gln 70 ) and/or 91 (Met 91 ) were closely associated with HCC development. [13][14][15][16] Gln 70 and/or Met 91 were also linked to resistance to PEG-IFN/ribavirin (RBV) treatment. [17][18][19][20] In addition, we and other investigators reported that an Nterminal part of the NS3 protein has the capacity to transform NIH3T3 and rat fibroblast cells 21,22 and to render NIH3T3 cells more resistant to DNA damage-induced apoptosis, which is thought to be a prerequisite for malignant transformation of the cell. 23 Also, the NS5A protein is a pleiotropic protein with key roles in both viral RNA replication and modulation of the host cell functions. 24 In particular, the links between NS5A and the IFN responses have been widely discussed. It was proposed initially that sequence variations within a region in NS5A spanning from amino acids (aa) 2209 to 2248, called the IFN sensitivity-determining region (ISDR), were correlated with IFN responsiveness. 25 Subsequently, in the era of PEG-IFN/RBV combination therapy, we identified a new region near the C-terminus of NS5A spanning from aa 2334 to 2379, which we referred to as the IFN/RBV resistance-determining region (IRRDR). 26,27 The degree of sequence variations within the IRRDR was significantly associated with the clinical outcome of PEG-IFN/RBV therapy. In the context of HCC, several retrospective studies suggested that IFN-based therapy might reduce the risk of HCC development. 4,[28][29][30] In an attempt to clarify whether viral factors, in particular those within the core, NS3, and NS5A proteins, are involved in HCC development, we carried out a comparative analysis of the aa sequences obtained from HCV patients who developed HCC and those who did not. In addition, we studied the sequence evolution of these genes in the interval between chronic hepatitis C and HCC development over a period of 15 years.

Patients and Methods
Ethics Statement. The study protocol, which conforms to the provisions of the 1975 Declaration of Helsinki, was approved beforehand by the Ethic Committees in Akashi City Hospital and Kobe University Graduate School of Medicine, and written informed consent was obtained from each patient enrolled in this study.
Patients. A total of 49 HCV-infected patients who developed HCC (HCC group) were retrospectively examined. They were followed up (from 1988 to 2003) with an average period until HCC development being 6.5 6 2.9 years. Paired serum samples at the time of chronic hepatitis C (pre-HCC sample) and HCC development (post-HCC sample) were collected. As a control group, 100 HCV-infected patients who were followed up over a period of 15 years (from 1988 to 2003) without HCC development were retrospectively examined. Serum samples of the control group were available at the time of first visit to the clinic. All patients enrolled in this study were chronically infected with HCV genotype 1b (HCV-1b). HCV subtype was determined as reported previously. 31 Serum HCV RNA titers were quantitated by reversetranscription polymerase chain reaction (RT-PCR0 with an internal RNA standard derived from the 5 0 noncoding region of HCV (Amplicor HCV Monitor test, v. 2.0, Roche Diagnostics, Tokyo, Japan). All patients underwent liver biopsy and were diagnosed as chronic hepatitis. All HCC and 68% (68/100) of non-HCC patients received IFN-monotherapy, either natural IFN alpha (Sumiferon, Dainipponsumitomo Pharmaceutical, Osaka, Japan) at a dose of 6 million units (MU) or recombinant IFN alpha 2b (Intron A; Schering-Plough, Osaka, Japan) at a dose of 10 MU, 3 times a week for 6 months. All HCC patients were nonresponders (NR), who had detectable viremia during the entire course of IFN treatment. On the other hand, 18 (26%) of the 68 non-HCC patients treated with IFN achieved HCV RNA negativity at the end of treatment followed by rebound viremia within 6 months after the treatment and, therefore, they were referred to as relapsers. The other 50 IFN-treated, non-HCC patients were NR. The remaining 32 non-HCC patients did not receive IFN. All patients were seen every 2 months and tested for liver function markers during the follow-up period.
Sequence Analysis of HCV Core, NS3, and NS5A Proteins. HCV RNA was extracted from 140 lL of serum using a commercially available kit (QIAmp viral RNA kit; Qiagen, Tokyo, Japan). The core, NS3, and NS5A regions of the HCV genome were amplified as described elsewhere. 26,[32][33][34] The sequences of the amplified fragments were determined by direct sequencing. The aa sequences were deduced and aligned using GENETYX Win software version 7.0 (GENETYX, Tokyo, Japan). The numbering of aa was according to the polyprotein of the prototype of HCV-1b; HCV-J. 35 Statistical Analysis. Statistical differences in the baseline parameters of HCC and control groups were determined by Student's t test for numerical variables and Fisher's exact probability or chi-square tests for categorical variables. Likewise, statistical differences in viral mutations between HCC and control groups were determined by Fisher's exact probability test. Kaplan-Meier analysis was performed to estimate the cumulative incidence of HCC. The data obtained were evaluated by the log-rank test. Univariate and multivariate logistic analyses were performed to identify variables that independently associated with HCC development. Variables with P < 0.1 in univariate analysis were included in a backward stepwise multivariate logistic regression analysis. The odds ratios and 95% confidence intervals (95% CI) were calculated. All statistical analyses were performed using SPSS v. 16 software (Chicago, IL). Unless otherwise stated, P < 0.05 was considered statistically significant.
Nucleotide Sequence Accession Numbers. The sequence data reported in this article have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB719460 through AB719842.

Demographic Characteristics of HCC and Control
Groups. The clinical characteristics of HCC and control groups are shown in Table 1. The HCC group had significantly higher titers of ALT, AST, and AFP, and higher fibrosis staging score than that of the control group. There was no significant difference in viremia titers between the two groups.
Correlation Between Core Protein Sequence Polymorphism and HCC Development. HCV core protein sequences were obtained from all (49/49) and 94% (94/100) of pre-HCC and control patients' sera, respectively. Comparative sequence analysis revealed that 22 (45%) of 49 HCV isolates in the pre-HCC sera (pre-HCC isolates) and 59 (63%) of 94 HCV isolates from the control group (control isolates) had wild-core (Arg 70 /Leu 91 ) ( Table 2). The difference between HCC and control groups was hovering at a statistically significant level (P ¼ 0.05). When the sequence pattern at position 70 alone was examined, a stronger association with HCC was observed. We found that 21 (43%) of 49 pre-HCC isolates had Gln 70 while only 13 (14%) of 94 control isolates did (P ¼ 0.0002). On the other hand, there was no significant correlation between sequence pattern at position 91 and HCC. Thus, a single mutation at position 70 (Gln 70 ) was the only polymorphic factor within core protein that was significantly associated with HCC development. It should be noted that there was no significant correlation between Gln 70 and the degree of fibrosis progression (data not shown).
Correlation Between NS3 Protein Sequence Polymorphism and HCC Development. Sequences of NS3 serine protease domain (aa 1027 to 1146) were obtained from 94% (46/49) and 93% (93/100) of pre-HCC and control isolates, respectively. We found that 29 (63%) of 46 pre-HCC isolates had Tyr and Gln at positions 1082 and 1112, respectively (Tyr 1082 / Gln 1112 ), while 39 (42%) of 93 control isolates did ( Table 2). The difference in the proportion between pre-HCC and control isolates was statistically significant (P ¼ 0.029). On the other hand, there was no significant correlation between Tyr 1082 /Gln 1112 and the degree of fibrosis progression (data not shown).
Correlation Between NS5A Protein Sequence Polymorphism and HCC Development. NS5A protein sequences were obtained from 92% (45/49) and 74% (74/100) of pre-HCC and control isolates, respectively. Twenty-four (53%) of 45 pre-HCC isolates had IRRDR of 6 or more mutations (IRRDR6) while only 15 (20%) of 74 control isolates did ( Table  2; P ¼ 0.0003). We also found that pre-HCC isolates tended to have a higher degree of sequence heterogeneity in ISDR than control isolates, although not statistically significant due probably to the small number of cases examined; 11 (24%) of 45 pre-HCC isolates and 8 (11%) of 74 of control isolates had ISDR with three or more mutations (P ¼ 0.07). Moreover, Asn at position 2218 (Asn 2218 ) within the ISDR was found in 24% (11/45) of pre-HCC isolates and only in 4% (3/ 74) of the control isolates (P ¼ 0.002), suggesting that Asn 2218 is significantly associated with development of HCC.
Cumulative HCC Incidence on the Basis of Core-Gln 70 , NS3-Tyr 1082 /Gln 1112 , NS5A-IRRDR6, and NS5A-Asn 2218 . Follow-up study revealed that the cumulative HCC incidence in patients infected with HCV-1b isolates with core protein of Gln 70 and those of non-Gln 70 , respectively, was 29% and 5% at the end of 5 years, 56% and 23% at the end of 10 years, and 63% and 26% at the end of 15 years (Fig. 1A), with the differences between the two groups being statistically significant (P < 0.0001; Log-rank test). Likewise, the cumulative HCC incidence in patients infected with HCV-1b isolates with NS3 of Tyr 1082 / Gln 1112 and those of non-(Tyr 1082 /Gln 1112 ), respectively, was 15% and 7% at the end of 5 years, 37% and 24% at the end of 10 years, and 45% and 24% at the end of 15 years (P ¼ 0.02) (Fig. 1B). Also, the cumulative HCC incidence in patients infected with HCV-1b isolates of IRRDR6 and those of IRRDR5, respectively, was 18% and 10% at the end of 5 years, 59% and 22% at the end of 10 years, and 63% and 27% at the end of 15 years (P ¼ 0.0002) (Fig. 1C). Similarly, the cumulative HCC incidence in patients infected with HCV-1b isolates of Asn 2218 and those of non-Asn 2218 , respectively, was 31% and 9% at the end of 5 years, 77% and 28% at the end of 10 years, and 77% and 33% at the end of 15 years (P ¼ 0.0003) (Fig. 1D).
Identification of Independent Factors Correlated With HCC Development by Univariate and Multivariate Logistic Regression Analyses. In order to identify significant independent factors associated with HCC development, all available data of baseline patients' parameters and core, NS3, and NS5A polymorphic factors were first analyzed by univariate logistic analysis. This analysis yielded eight factors that were significantly associated with HCC development: core-Gln 70 , NS3-(Tyr 1082 /Gln 1112 ), NS5A-IRRDR6, NS5A-Asn 2218 , increased levels of ALT (>165 IU/L), AST (>65 IU/L), and AFP (>20 ng/L), and fibrosis staging score (3). Subsequently, those eight factors were entered in multivariate logistic regression analysis. This analysis identified two viral factors, core-Gln 70 and NS3-(Tyr 1082 /Gln 1112 ), and a host factor, AFP levels (>20 ng/L), as independent factors associated with HCC development ( Table 3).
Evolution of the Sequences of the Core, NS3, and NS5A Proteins During the Follow-up Period From Chronic Hepatitis to HCC Development. Finally, we investigated sequence evolution of the core protein, NS3 and NS5A (IRRDR and ISDR) during the followup period from chronic hepatitis to HCC development by comparing the sequences between pre-HCC and     IRRDR and ISDR showed a high degree of sequence evolution. IRRDR sequences were different between pre-HCC and post-HCC isolates in 66% (25/38) of cases analyzed (Fig. 3). IRRDR sequences tended to be more polymorphic at the time of HCC occurrence. Frequency of HCV isolates with IRRDR6 was significantly higher in post-HCC isolates than in pre-HCC isolates; IRRDR6 was found in 47% (18/38) of post-HCC isolates compared to 24% (9/38) of pre-HCC isolates (P ¼ 0.03). On the other hand, ISDR3 was found in 21% (8/38) of post-HCC isolates compared to 11% (4/38) of pre-HCC isolates, with the difference between the two groups being not statistically significant (P ¼ 0.3).

Discussion
HCC is one of the common long-term complications of HCV infection. However, whether HCV itself plays a direct role in the development of HCC and whether all HCV isolates are equally associated with HCC development remain to be determined. HCV core, NS3, and NS5A proteins have been reported to affect a wide variety of potentially oncogenic pathways in cell culture and experimental animal systems. 7 In the present study, we demonstrated that HCV isolates with core-Gln 70 , NS3-Tyr 1082 /Gln 1112 or NS5A-IRRDR6 were closely associated with HCC development. In addition, a follow-up study revealed that sequence patterns at position 70 of the core protein and positions 1082 and 1112 of NS3 did not significantly alter during the progression from chronic hepatitis to HCC while NS5A-IRRDR showed a significantly higher degree of sequence heterogeneity in post-HCC than in pre-HCC isolates.
Correlation between polymorphisms at positions 70 and 91 of HCV-1b core protein and IFN-based treatment outcome was extensively studied, especially in a Japanese population. [17][18][19][20] Interestingly, the same mutations were also associated with progression to HCC in the Japanese population with HCV-1b infection. 13 Results obtained in the present study confirmed and emphasized the significant association between the mutation at position 70 (core-Gln 70 ), but not at position 91, and HCC development (Tables 2, 3; Fig.  1A). Despite the clinical evidence that strongly supports the correlation between core-Gln 70 and HCC development, the molecular mechanism underlying this correlation is still obscure. Delhem et al. 36 found that tumor-derived HCV core proteins, but not nontumor-derived ones, interact with and activate doublestranded RNA-dependent protein kinase (protein kinase R or PKR), which might modulate viral persistence and carcinogenesis. Gln 70 was found in two of the three tumor-derived sequences, whereas Arg 70 was found in two of the three nontumor-derived ones.
As for the NS3 protein of HCV, the possible link between an N-terminal portion of NS3 encoding viral serine protease (aa 1027 to 1146) and hepatocarcinogenesis was reported. 21,22 However, information about the relationship between NS3 sequence diversity and HCC development is still limited. We previously reported a significant correlation between predicted secondary structure of an N-terminal portion of NS3 and HCC development. 34 In the present study, we demonstrated that HCV patients infected with HCV isolates with NS3-(Tyr 1082 /Gln 1112 ) were at a higher risk to develop HCC than those infected with HCV isolates with non-Tyr 1082 /Gln 1112 (Tables 2, 3; Fig.  2B). Computer-assisted secondary structure analysis of NS3 revealed that Tyr 1082 was associated with the presence of a turn structure at around position 1083 while Phe 1082 was associated with the absence of the turn structure. 34 Notably, the catalytic triad of NS3 serine protease consists of His 1083 , Asp 1107 , and Ser 1165 . 37 Since positions 1082 and 1112 are in close vicinity of the catalytic triad, sequences diversity at these positions might influence the serine protease activity and also pathogenicity of HCV. Large-scale, multicenter clinical studies as well as more detailed experimental studies at the molecular and cellular levels are needed to clarify the importance of sequence diversity at positions 1082 and 1112 of NS3 in HCVmediated hepatocarcinogenesis.
HCV heterogeneity in NS5A-ISDR and NS5A-IRRDR are correlated with IFN-responsiveness. 17,18,25,26 As IFN-based therapy reduces the risk of HCC development, 4,28-30 we were interested to investigate whether there is a correlation between sequence heterogeneity in NS5A and development of HCC. Our present results revealed that a high degree of sequence heterogeneity in IRRDR (IRRDR6) was closely associated with HCC development (Table 2). We previously reported that IRRDR6 was significantly associated with good responses to PEG-IFN/ RBV combination therapy. 26,27 These results collectively suggest that oncogenic properties and PEG-IFN/ RBV responsiveness are independent viral characteristics and that PEG-IFN/RBV therapy helps eliminate oncogenic HCV isolates, thus reducing the risk of HCC development.
Position 2218 of NS5A, located within ISDR, appears to tolerate a wide range of aa substitutions as observed in different HCV-1b isolates. 25,38,39 Interestingly, Asn at position 2218 (Asn 2218 ) was detected significantly more frequently in pre-HCC isolates than in the control isolates. Further studies are needed to determine the possible importance of this residue in hepatocarcinogenesis.
Another focus of attention is how the sequences of the core protein, NS3, and NS5A-IRRDR evolve during the interval between chronic hepatitis and HCC development. One of the significant advantages of the present study was that we could conduct a longitudinal investigation by analyzing the target sequences of preand post-HCC isolates. We found that core-Gln 70 and NS3-(Tyr 1082 /Gln 1112 ) were well conserved in each paired sample. This indicates that core-Gln 70 and NS3-(Tyr 1082 /Gln 1112 ) were already present before the development of HCC. Non-Gln 70 of the core protein and non-Tyr 1082 and non-Gln 1112 of NS3 were also well conserved in each paired sample. These results imply the possibility that these sequence patterns were not a result of HCC but, rather, they were a possible causative factor for the development of HCC. We hypothesize, therefore, that HCV isolates with core-Gln 70 and/or NS3-(Tyr 1082 /Gln 1112 ) are highly oncogenic, whereas those with non-(Gln 70 plus NS3-Tyr 1082 /Gln 1112 ) are less oncogenic. It is not clear yet as to whether these oncogenic mutations were present from the very beginning of HCV infection or if they emerged at a certain timepoint (before the initiation of follow-up) during the longterm persistence through an adaptive viral evolution in the host. More comprehensive follow-up study is needed to address this issue. In any case, the core-Gln 70 and NS3-(Tyr 1082 /Gln 1112 ) would be considered an index for prediction of HCC development. On the other hand, IRRDR in NS5A is more tolerant for sequence evolution. IRRDR in post-HCC isolates showed a significantly higher degree of sequence heterogeneity compared with that in pre-HCC isolates. This observation suggests that IRRDR is under strong selective pressure during the course of HCV infection and that the high degree of IRRDR heterogeneity (IRRDR6) in HCV isolates from patients with HCC may not be a causative factor for development of HCC.
In conclusion, the present results suggest the possibility that patients infected with HCV isolates with core-Gln 70 and/or NS3-(Tyr 1082 /Gln 1112 ) are at a higher risk to develop HCC compared to those with non-(Gln 70 plus NS3-Tyr 1082 /Gln 1112 ).