Induction of FAS ligand expression in a human hepatoblastoma cell line by HCV core protein

Virus Research 97 (2003) 103–110

Induction of FAS ligand expression in a human hepatoblastoma cell line by HCV core protein A. Ruggieri, M. Murdolo, M. Rapicetta∗ Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Received 15 May 2003; received in revised form 13 August 2003; accepted 13 August 2003

Abstract Tumour cells and virus infected cells expressing Fas ligand (FasL) can evade immune surveillance by inducing apoptosis in T cells expressing Fas. In order to characterise a possible role of hepatitis C virus (HCV) core protein in similar mechanisms during HCV infection, we investigated Fas ligand expression and activity in a human hepatoblastoma cell line (HepG2) constitutively expressing this protein. Strong FasL induction was detected by immunoblotting and flow cytometry analysis in the core expressing cell lines Hep39. In contrast, vector transfected cells or cell lines expressing HCV E1-E2 proteins did not show FasL expression. Co-cultivation experiments of Hep39 cells with a Fas-sensitive T cell line indicated that FasL induced by the core protein had apoptotic activity toward target cells. Effect of the core protein on induction of FasL promoter was further examined by co-trasfection of HepG2 cells with core-bearing plasmid and a vector in which luciferase gene expression is driven by human FasL promoter. Results of the luciferase assay indicated a positive regulation of FasL promoter by the core protein. In conclusion, HCV core protein plays a role in the induction of functional FasL in hepatoblastoma cell line and apoptosis in a target T cell line expressing Fas. Similar mechanisms may contribute, in vivo, to establishment of chronic infection and development of hepatocellular carcinoma (HCC). © 2003 Elsevier B.V. All rights reserved. Keywords: HCV; Core; FasL; HepG2

1. Introduction Hepatitis C virus (HCV), a member of the Flaviviridae family, contains a plus-strand genomic RNA of about 9600 nucleotides with a single open reading frame encoding a polyprotein that is proteolitically cleaved into 10 distinct products (Hijikata et al., 1991; Houghton, 1996). One of the main features of HCV infection is the high efficiency in establishing chronic infections which may lead to development of hepatocellular carcinoma (HCC). However, virus related mechanisms of carcinogenesis and mechanisms involved in evading host immune surveillance remain to be elucidated. Fas–Fas ligand (FasL) interaction is one of the main effector mechanisms of cytotoxic T lymphocytes (Kagi et al., 1994). FasL, expressed mainly on the surface of activated T cells, belongs to the TNF family and induces apopto∗ Corresponding author. Tel.: +39-06-49903233; fax: +39-06-49902662. E-mail address: [email protected] (M. Rapicetta).

0168-1702/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2003.08.004

sis in Fas-expressing cells (Depraetere and Golstein, 1997). In addiction, recent studies have identified FasL expression also in cells of immune-privileged sites, such as eyes and testis (reviewed in Douglas and Ferguson, 2001) which in turn can induce apoptosis of T cells. In the liver, Fas–FasL system plays an important role in regulation of apoptosis of hepatocytes (Ogasawara et al., 1993) as well as in the pathogenesis of liver diseases, including liver injury, viral hepatitis, cirrhosis and HCC (Kondo et al., 1997; Hiramatsu et al., 1994; Lee et al., 2001). Moreover, it has been postulated that tumour cells, such as melanoma and hepatocellular carcinoma, expressing FasL could evade immune surveillance by inducing apoptosis in tumour-infiltrating T cells expressing Fas (Nagata, 1996; Owen-Schaub et al., 2000). In HCCs, Fas which is expressed in normal hepatocytes, was down-regulated (Shin et al., 1998); in contrast, FasL, not present in normal hepatocytes, was expressed (Luo et al., 1997; Strand et al., 1996). Surface expression of FasL in cells of tumours such as hepatoma and melanoma has been shown to lead to the elimination of invading Fas-expressing lymphocytes (Liu et al., 2001).

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Expression of Fas and FasL can be modulated by some virus gene products as a strategy to evade immune response and establish persistent infection (Li-Weber et al., 2000; Xu et al., 1999). In the case of hepatitis viruses, HBV has been shown to induce FasL in hepatoma cell lines through the expression of HBV X protein (Shin et al., 1999). Among HCV viral proteins, the hepatitis C virus core protein is likely to be important determinant in mediating pathological effects of HCV through several of its multifunctional activities (Lai and Ware, 1999; McLauchlan, 2000). These include a role of the core protein in encapsidation of viral RNA (Shimoike et al., 1999), in induction of cellular and unrelated viral promoters (Ray et al., 1995, 1997; Bergqvist and Rice, 2001) and the interactions with several cellular proteins with consequent effect on intracellular signal transduction, cell transformation and immune cell functions (Matsumoto et al., 1997; Zhu et al., 1998; Hahn et al., 2000; Aoki et al., 2000; Kittlesen et al., 2000; Jin et al., 2000). The core protein has also been shown to be able of modulating cellular apoptosis (Ray et al., 1996, 1998; Marusawa et al., 1999; Honda et al., 2000; Ruggieri et al., 1997) and is involved in cell growth promotion and immortalization (Ray et al., 1997, 2000; Ruggieri et al., in press). However, it is not known whether the core protein or other virus gene products could influence expression of the members of Fas–FasL system in hepatocytes or in liver derived cell lines. In our previous study about the role of HCV core protein in Fas-mediated apoptosis, expression of Fas receptor resulted to be not affected by the core protein expression in HepG2 cell line (Ruggieri et al., 1997). The effect of HCV core protein on the level of surface Fas ligand is so far not determined. In this study, we investigated FasL expression in a human hepatoblastoma cell line (HepG2) expressing HCV core protein, in order to elucidate the role of the core protein in establishing chronic infection and in immune evasion mechanism that lead to HCC.

2. Material and methods 2.1. Cell lines, plasmids and reagents Human hepatoblastoma cell line constitutively expressing HCV core protein (Hep39) and E1-E2 (Hep827) were established by calcium phosphate precipitation method as previously described (Harada et al., 1995). Vector pcEFswxneo without HCV insert was used as a mock control and cell lines transfected with this empty vector were indicated Hepswx. All cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 0.6 mg/ml of G418. Transient transfection experiments in reporter gene assay were performed with plasmids: pcCA39EFneo in which HCV core protein expression was driven by the CAG promoter (Aoki et al., 1998); pcCAswxEFneo plasmid

without the core insert was used as a mock control. The luciferase reporter gene driven by the human FasL gene promoter (pGL2-hFasL) was provided by Dr. M.A. Buendia (Inserm U163, Pasteur Institute, Paris, France). The pSV-␤-galactosidase vector was purchased from Promega. 2.2. Western blot analysis To detect FasL expression in established Hepswx and Hep39 cell lines, subconfluent cultures were detached with trypsin and EDTA and lysed in lysis buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 10% NP-40, 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 10 mg/ml pepstatin. Cell lysates were centrifuged for 15 min at 15,000 g. The protein concentration was measured in the supernatant by Lowry method. Equal amounts of protein (mg) were separated by SDS-PAGE using 10% or 12.5% polyacrylamide gel and electroblotted to PVDF membranes in a semidry system. After blocking with 5% non-fat milk in PBS with 0.1% Tween 20, FasL and HCV core protein were detected respectively with mAb anti-FasL (Transduction Laboratories) and mAb anti-HCV core protein (provided by Dr. T. Miyamura). Anti-␤ actin mAb was used as loading control. Antigen–antibody complexes were visualised by chemiluminescence reaction using ECL system (Amersham). 2.3. Staining of apoptotic cells by annexin V Parental HepG2, Hepswx or Hep39 cell lines were cultivated at 1 × 106 cells per well in six-wells plates. After overnight incubation, 1 × 106 Jurkat cells, which express surface Fas, were seeded onto Hep39 growing wells or parental and mock transfected cells and co-cultivated for 30 h. Non-adherent cells were then carefully collected and stained with propidium iodide and annexin V-FITC conjugate, according to the manufacturer’s instructions (Pharmingen). Fluorescence intensity of annexin positive cells, corresponding to the percentage of apoptotic cells, was measured by FACScalibur (Becton Dickinson) using CellQuest software. 2.4. Flow cytometric analysis of FasL expression After co-cultivation and collection of Jurkat cells, the remaining HepG2, Hepswx and Hep39 cell lines were detached from wells with trypsin solution and washed twice in PBS. After fixation in 2% paraformaldehyde cells were resuspended in 100 ␮l of PBS containing 1 ␮g anti-human FasL antibody and incubated at 4 ◦ C for 30 min. Following incubation with FITC-conjugated anti-mouse IgGs as secondary antibody and washings in PBS, cells were analysed by flow cytometer (FACScalibur, Becton Dickinson) by using CellQuest software. A minimum of 10,000 events were analysed for each sample.

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2.5. Transient transfection and dual-luciferase reporter assay HepG2 cells were transfected by calcium phosphate precipitation as previously described (Pharmacia Biothec) (Harada et al., 1995). The 5 × 105 culture of HepG2 cells seeded onto 60 mm culture dish 24 h before transfection were transfected with 5 ␮g of each of following plasmids: pcCA39EFneo, pcCAwtEFneo, pGL2-hFasL luciferase reporter plasmid. Transfected cell lines were indicated as follow: HepCA39: HepG2 cells co-transfected with the core expressing plasmid and pGL2-hFasL; HepCAwt: cells co-transfected with pcCAwtEFneo and pGL2-hFasL plasmids; HepG2LG: HepG2 cells transfected with pGL2-hFasL and pSV-␤-galactosidase vectors. The pSV-␤-galactosidase vector (1.5 ␮g) was co-transfected as a control of transfection efficiency. After 48 h of incubation, cell lysis and luciferase quantifications were performed using commercial reagents (Promega) and measuring in a Lumat LB 9501 Berthold Luminometer. In transfection experiments, the background level for luciferase or ␤-galactosidase was evaluated using extracts from cells transfected with DNA plasmid lacking luciferase or ␤-galactosidase reporter genes, respectively. Results are expressed as relative light units (RLU), which were calculated as the ratio of luciferase to ␤-galactosidase activities within each sample. Transfections were performed in duplicate and the mean and standard deviation were calculated for each experiment, which was performed three times.

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of FasL in core expressing Hep39 cells was demonstrated by the expression of a 37 Kda band specifically recognised by anti-huFasL mAb. As expected, parental HepG2 cells did not express FasL. A very faint band, of 37 Kda was detected in Hepswx cell line. Thus, the possible induction of FasL associated to transfection with the empty vector was very weak. 3.2. Surface expression of FasL by flow cytometry analysis Based on the assumption that Fas–FasL interaction occurs at cell surface and only FasL expressed on cell membrane is biologically active in inducing apoptosis upon binding to surface Fas receptor we further examined Hep39 cells for surface FasL expression. The quantitation by immunofluorescence and flow cytometric analysis with the same anti-huFasL mAb used for immunoblotting was performed. The results reported in Fig. 2 show a distinct FasL positive cell population among Hep39 cell lines. This was not detected in mock transfected (Hepswx) or in parental HepG2 cells indicating that Hep39 cells were positively stained for surface FasL. The presence of 33% of FasL-expressing cells among the core transfected cell population was estimated by flow cytometry. This percentage was significantly higher compared to HepG2 and vector transfected cells (1.3 and 1.8% respectively). The lack of FasL detection in a cell line expressing other HCV structural proteins (Hep827 constitutively expressing E1-E2) further supported that FasL expression on HepG2 cell line was associated to the core protein expression.

3. Results 3.1. FasL induction in HepG2 cells To verify the effect of HCV core protein expression on FasL induction we analyzed human hepatoblastoma cell line, previously characterised as FasL-negative, after transfection for stable expression of the core protein. The results of immunoblotting analysis by using specific anti-FasL mAb are reported in Fig. 1. A significant induction

Fig. 1. FasL protein expression in Hep39 cell line by immunoblot analysis. Total cell lysates (40 ␮g) from HepG2, vector transfected (Hepswx) and core expressing (Hep39) cell lines were subjected to 10% SDS-PAGE and probed with the specific anti-human FasL monoclonal antibody as described in Section 2. Detection of ␤-actin on the same blot was used as loading control.

3.3. FasL-expressing Hep39 cell lines-induced apoptosis of Fas-bearing cells The identification of the correlation between the core protein expression and FasL up-regulation in cultured HepG2 cells prompted us to examine whether the induced FasL was able to bring about apoptosis of a target T cell line. To investigate this functional activity of FasL expressed on Hep39 cells, these were co-cultivated with Fas-bearing Jurkat T cell line, which is known to be sensitive to Fas-mediated apoptosis. Apoptotic death induced by Hep39 cells was analysed by quantitation of annexin V positive cell population. As shown in Fig. 3, when Jurkat cells were co-cultured with core expressing cells they underwent apoptosis, as indicated by the appearance of a percentage of annexin-positive cell population (18.13%), which was significantly increased above the background percentage of apoptosis in control Jurkat cells (2.89%). In contrast, HepG2 as well as Hepswx did not induce apoptosis of Jurkat cells. This result suggested that FasL expressed in core transfected cell line was biologically active and induced apoptosis of a Fas-sensitive cell line.

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Fig. 2. Cytofluorimetric analysis of FasL expression in established cell lines. Immunofluorescence and flow cytometry analysis of FasL indicating a significative induction of FasL positive cell population among Hep39 cells. Percentage of FasL positive cells calculated by using CellQuest software is indicated in parenthesis. The diagrams show the analysis of flurescencce intensity against the side scattering properties of cell populations.

3.4. Induction of FasL promoter by HCV core protein Since HCV core protein has been shown to have transactivation activity the direct regulation of FasL expression by the induction of FasL promoter was investigated. To this purpose, HepG2 cell line was co-transfected for transient expression with the core-bearing plasmid (pcCA39EFneo) and pGL2-hFasL vector, in which luciferase gene expression is driven by human FasL promoter. The results reported in Fig. 4 indicated a significant induction of FasL promoter activity in HepG2 cells expressing HCV core protein compared to the mock transfected and parental cell lines, as the RLU in Hep39 cells was two-fold than in control cell lines (see Fig. 4A). This suggests a positive regulation of FasL promoter by the core protein. Aliquots of same cell lysates in which induction of luciferase activity was measured were analysed by immunoblotting to monitor HCV core protein expression in order to ensure expression of the effector (see Fig. 4B). We found a significant expression of 21 Kda band corresponding to full length core protein that was specifically recognised by anti-core mAb.

4. Discussion Results from present study suggest that HCV core protein expressed in HepG2 cell system induces the expression of FasL and the activity of FasL promoter in the same cell

line. A biological activity toward a target T cell line was also demonstrated indicating that the core protein provided HepG2 cells with apoptotic activity. The virus related expression of FasL in infected cells is considered one of the possible mechanisms that could lead to viral persistence. In fact, cells expressing surface FasL can protect themselves against CTL-mediated injury by actively destroying them via the same Fas ligand–Fas receptor pathway that CTL use to kill their target cells. Similar activity has been reported for viral gene products from several viruses which have evolved this strategy to avoid their own death while eliminating nearby cells. Among RNA viruses, SIV and HIV nef proteins have been reported to induce FasL both in vitro in virus infected cells and in vivo in CD4+ T cells which in turn induced apoptosis of CTL and consequently evade the immune system (Xu et al., 1997). The immunosuppressive function of HIV tat protein has also been shown to be accompanied by induction of FasL on the infected macrophages; moreover FasL mRNA is up-regulated in PBMC from HIV seropositive individuals (Mitra et al., 1996). Among DNA viruses immediate-early gene product 2 (IE2) of cytomegalovirus (HCMV) has been shown to up-regulate FasL in infected retinal epithelial cells (Chiou et al., 2001). This may be a potential mechanism for the pathogenesis of HCMV retinitis. With regard to viral hepatitis, Fas–FasL system is involved in the pathogenesis of acute and chronic liver disease (reviewed in Pinkoski et al., 2000) and hepatocytes

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Fig. 3. Induction of apoptosis in Jurkat cells by Hep39 cell line. FITC-conjugated annexin V staining of Jurkat T cell line after cocultivation on Hep39, Hepswx, HepG2 cell lines. Fluorescence intensity of annexin positive cells correspond to the percentage of apoptotic cells measured by flow cytometry and analysed by CellQuest software. The numbers in upper-right side of the panels represent percentage of apoptotic cells.

can be induced to express FasL during an inflammatory response (Galle et al., 1995). However, it is not clear whether FasL-mediated mechanisms may contribute to virus persistence. In the case of hepatitis viruses, X protein of HBV has been reported to induce FasL expression in hepatoma cell lines and a possible mechanism for the escape from immune surveillance was suggested (Shin et al., 1998). It is worth to notice that induction of FasL in hepatoma cells is engendered by X protein of HBV and by the core protein of HCV, to which either an oncogenic potential is attributed. Present finding about induction of FasL by the core protein might explain the reported expression of FasL in HCCs from HCV infected patients, that was suggested to be involved in immune evasion by tumour cells (Nagao et al., 1999). Several studies have indicated that the core protein of HCV may have immunomodulatory function. The suppression of T-lymphocyte responsiveness was found in transgenic mice (Geissler et al., 1998; Soguero et al., 2002). The binding to the cytoplasmic tail of TNFR1, lymphotoxin-␤-receptor and Fas in co-cultured cell lines (Zhu et al., 1998; Matsumoto et al., 1997) was suggested to

affect the immune cell functions. In addition, inhibition of IL-12 and nitric oxide (NO) production by the core protein was found in a murine macrophages cell line. All these findings suggest that a role for the core protein in suppression of Th1 immunity may be present following HCV infection (Lee et al., 2001). Furthermore, the core protein was shown to be able to increase the sensitivity of a T cell line to Fas-mediated apoptosis (Chang et al., 2000). That is consistent with the increased Fas-mediated apoptosis observed in PBMC from chronically infected patients (Taya et al., 2000). In this study, we examined a mechanism different from those reported above by which the core protein may influence the immune evasion with consequent contribution to virus persistence and HCC development. Analysis of FasL expression and activity associated to the core protein expression was favourably performed in HepG2 cell line, which is a FasL negative cell line of liver origin and thus suitable to examine the effect of proteins encoded by hepatropic viruses such as HCV. Induction of FasL in vector transfected cells (Hepswx),was very weak and it was not

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Fig. 4. Activation of FasL promoter by HCV core protein. (A) Activation of FasL promoter was analysed by transient co-transfection assay with the core expressing plasmid (HepCA39neo) and pGL2-hFasL in which luciferase gene expression is driven by human FasL promoter. HepCAwt: HepG2 cells co-transfected with pcCAwtEFneo and pcGL2-hFasL plasmid; HepG2LG: HepG2 cells transfected with pGL2-hFasL plasmid. Results are expressed as relative light units (RLU) calculated as the ratio of luciferase to ␤-galactosidase activities within each sample. Data shown are the mean ± standard deviation of three independent experiments, each with triplicate luciferase results. (B) HCV core protein was detected by immunoblotting analysis in aliquots of the same lysates in which luciferase activity was measured. The expression level of ␤-actin was used as an internal control.

confirmed by FACS analysis of surface FasL that showed that the core protein was strictly responsible for FasL induction in HepG2 cells. Furthermore, consistent with the lack of surface FasL, Hepswx cells did not induce apoptosis in target cells upon co-culture. According to previous reports showing susceptibility of HepG2 cells toward Fas-mediated apoptosis (Ruggieri et al., 1997), we can observe that in the same cell system the HCV core protein could either sensitise HepG2 cells to apoptotic death or enable these cell lines with killing activity toward target cells; that is consistent with the multiple functions associated to the core protein as well as to different roles of this viral protein in various stages of infection and disease associated to HCV infection. Moreover, it is known that HCV core protein acts as transactivator and up-regulates various host genes (reviewed in Ray and Ray, 2001). Consistently, we found that induction

of FasL resulted from activation of FasL promoter by the core protein in HepG2 transfectants. Up to now cellular factors regulating FasL expression are only partially described (Lacanà and D’Adamio, 1999; Bauer et al., 1998); Nur77 gene, whose induction associates to FasL induction (Weith et al., 1996), has been reported to be activated in hepatoma cell line by HBV X protein or by HTLV-1 tax (Lee et al., 2001; Chen et al., 1998). Whether the core protein acts synergistically or modulates expression of Nur77 or other cellular factors awaits further investigation. In conclusion, it was shown from this work that the intracellular expression of HCV core protein plays a role in the induction of FasL in hepatoblastoma cells. These data suggest a possible role of HCV core protein in immune escaping by induction of apoptosis in T cells expressing Fas. Furthermore, present results support the hypothesis that the

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core protein of HCV may contribute to the establishment of HCC. Acknowledgements We particularly aknowledge Dr. T. Miyamura for helpful discussion and for providing anti-HCV core monoclonal Ab. We thank Dr. M.A. Buendia and Dr. C.V. Paya for providing the plasmid pGL2-hFasL; Dr. T. Harada for helping in preparation of stable cell lines. We are grateful to Giulia Cecere and Sabrina Tocchio for secretarial assistance and to Alessandro Spurio and Roberto Gilardi for computer graphic assistance. This work was supported by the grant “National Hepatitis Project” from the Istituto Superiore di Sanità, Italy and “Viral Vaccins” Ministero della Sanità, Italy. References Aoki, Y., Aizaki, H., Shimoike, T., Tani, H., Ishii, K., Saito, I., Matsuura, Y., Miyamura, T., 1998. A human liver cell line exhibits efficient translation of HCV RNAs produced by a recombinant adenovirus expressing T7 RNA polymerase. Virology 250 (1), 140–150. Aoki, H., Hayashi, J., Moriyama, M., Arakawa, Y., Hino, O., 2000. Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1. J. Virol. 74, 1736–1741. Bauer, M.K., Vogt, M., Los, M., Siegel, J., Wesselborg, S., Schulze-Osthoff, K., 1998. Role of reactive oxygen intermediates in activation-induced CD95 (APO-1/Fas) ligand expression. J. Biol. Chem. 273 (14), 8048–8055. Bergqvist, A., Rice, C.M., 2001. Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein. J. Virol. 75, 772–781. Chang, S.H., Young, G.C., Beom-Sik, K., Isabel, M., Lester, I.M., Young, S.H., 2000. The HCV core protein acts as a positive regulator of Fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology 276, 127–137. Chen, X., Zachar, V., Chang, C., Ebbesen, P., Liu, X., 1998. Differential expression of Nur77 family members in human T-lymphotropic virus type1-infected cells: transactivation of the TR3/nur77gene by Tax protein. J. Virol. 72, 6902–6906. Chiou, S.H., Liu, J.H., Hsu, W.M., Chen, S.S.-L., Chang, S.-Y., Juan, L.-J., Lin, J.-C., Yang, Y.-T., Wong, W.-W., Liu, C.-Y., Lin, Y.-S., Liu, W.-T., Wu, C.-W., 2001. Up-regulation of fas ligand expression by human cytomegalovirus immediate-early gene product. 2. A novel mechanism in cytomegalovirus-induced apoptosis in human retina. J. Immunol. 167 (7), 4098–4103. Depraetere, V., Golstein, P., 1997. Fas and other cell death signaling pathways. Semin. Immunol. 9, 93. Douglas, R.G., Ferguson, T.A., 2001. The role of Fas ligand in immune privilege. Nature 2, 917–924. Galle, P.R., Hofmann, W.J., Walczak, H., Schaller, H., Otto, G., Stremmel, W., Krammer, P.H., et al., 1995. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J. Exp. Med. 182, 1223–1230. Geissler, M.K., Tokushige, T., Wakita, V.R., Zurawski, J.R., Wands, J.R., 1998. Differential cellular and humoral immune responses to HCV core and HBV envelope proteins after genetic immunizations using chimeric constructs. Vaccine 16, 857–867. Hahn, C.S., Cho, Y.G., Kang, B.-S., Lester, I.M., Hahn, Y.S., 2000. The HCV core protein acts as a positive regulator of Fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology 276, 127–137. Harada, T., Kim, D.W., Sagawa, K., Suzuki, T., Takahashi, K., Saito, I., Matsuura, Y., Miyamura, T., 1995. Characterization of an established

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