1 Clinica Medica 5, Università di Padova, via Giustiniani 2, 35128 Padova, Italy
2 Immunologia Clinica, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova, via Giustiniani 2, 35128 Padova, Italy
3 Clinica Medica 1, Università di Padova, via Giustiniani 2, 35128 Padova, Italy
3 Dipartimento di Scienze Mediche e Chirurgiche, Università di Padova, via Giustiniani 2, 35128 Padova, Italy
4 Xeptagen Spa, via Olivetti, 1 Pozzuoli (Na), Italy
Correspondence
P. Pontisso
patrizia{at}unipd.it
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ABSTRACT |
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MAIN TEXT |
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Surface expression of SCCA was assessed using HepG2 cells, maintained in culture in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing penicillin (100 U ml-1) and streptomycin (100 µg ml-1), supplemented with 10 % bovine calf serum (Sigma). Samples obtained 24 h after seeding and containing 106 cells were mechanically detached and incubated with 10 µg of a monospecific rabbit anti-SCCA variants antibody (Xeptagen, S.p.A, Italy) or with pre-immune rabbit serum for 20 min at 4 °C, washed with phosphate buffered saline (PBS) containing 10 % bovine calf serum (PBS10 % FBS) and further incubated with FITC-conjugated goat anti-rabbit antiserum (Santa Cruz Biotechnolgy Inc.) at a ratio of 1 : 10 for 20 min at 4 °C. To assess the overall cellular burden of SCCA, parallel immunostaining experiments were carried out in cells permeabilized with a commercially available kit (Fix & Perm, Caltag), following manufacturer's instructions.
Surface SCCA expression was also assessed in HepG2 cells pretreated with 2 % DMSO for 18 h and washed twice with PBS10 % FBS.
Cellular fluorescence was evaluated using a FACScan analyser (Becton and Dickinson) and data were processed using the CELLQuest software program (Becton and Dickinson). The difference between the mean log fluorescence intensity (MFI) of different samples was calculated using the KolmogorovSmirnov test for analysis of histograms.
Since the preS1(2147) amino acid region of HBV was previously identified as the envelope virus sequence involved in the interaction with surface SCCA, we assessed the effect of different preS1 preparations on surface expression of SCCA. The following preparations were used: a) synthetic myristylpreS1(2119) protein (adw2 subtype) (De Falco et al., 2001a); b) 4-preS1(2147) tetrameric peptide, obtained starting from a polylysine core, as described (De Falco et al., 2001b
); or c) scrambled preS1(2147) peptide used as a control (non-HBV peptide) NH2-FDPNSPNWDNGFNALAQPDPHDGPFFL-COOH. This control peptide has been designed by scrambling the amino acid sequence of preS1(2147) but maintaining its hydropathic profile, and was synthesized by solid phase chemistry on an automated peptide synthesizer. The non-HBV peptide was purified by semipreparative RP-HPLC and fully characterized by MALDI-TOF and amino acid analysis.
The preS1 synthetic protein and the peptides were dissolved at 7 µg ml-1 concentration in 3 ml PBS (pH 7·4) and added to HepG2 cells cultured in six-well plates (about 106 cells per well), as described above. As a control, cell plates were incubated with medium alone. After overnight incubation supernatant was removed, cells were washed with PBS containing 10 % FCS, mechanically detached using a scraper and surface expression of SCCA was assessed by FACS analysis, as described above.
A similar procedure was carried out with primary human hepatocytes, obtained from an explanted liver by collagenase perfusion (Rijntjes et al., 1986) and seeded on culture plates pre-coated with human liver biomatrix. The approval for experimental use of the human liver was obtained from the Medical Ethical Committee. The induction experiments with preS1 preparations were carried out one day after seeding.
To verify whether the observed increase of expression of SCCA was associated with different efficiency in HBV binding and/or internalization, we tested virus per cell interaction capability in cultured cells treated or not treated with trypsin after pre-incubation with preS1 preparations, the non-HBV peptide or with medium alone. HepG2 cells, grown to semiconfluence in six-well plates, were pre-incubated overnight with medium alone or with medium containing 7 µg ml-1 of preS1 synthetic protein, preS1(2147) tetrapeptide or non-HBV peptide. Medium was then removed and HBV particles were added at 4x108 genome equivalents ml-1 concentration in culture medium. After 18 h incubation at 37 °C the medium was removed, cells were washed twice with PBS10 % FBS and cells were treated with 0·5 mg ml-1 trypsin, 0·5 mM EDTA for 5 min at 37 °C to analyse virus entry. Trypsin was removed and cells were washed twice with PBS10 % FBS before cellular DNA extraction by phenol/chloroform procedure. Parallel experiments using medium alone instead of trypsin treatment were carried out to evaluate virus attachment.
To assess the efficiency of HBV attachment and entry into cells pre-incubated with preS1 preparations and treated with DMSO, similar experiments were carried out in parallel using HepG2 cells pretreated with 2 % DMSO for 18 h and washed twice with PBS10 % FBS.
The amount of HBV DNA was assessed by PCR, starting from 1 µg of cellular DNA for each sample. The primers used for nucleic acid amplification were located in the core region of HBV genome (primer 1, 5'-TTGCCTTCTGACTTCTTTCC-3'; primer 2, 5'-TCTGCGAGGCGAGGGAGTTCT-3') (Mantero et al., 1991).
The internalized viral DNA was semiquantitatively evaluated by densitometry (Gel DOC 1000, Bio-Rad, program Quantity One, Bio-Rad) after agarose gel electrophoresis in TBE buffer (0·089 M Tris, 0·089 M boric acid, 1 mM EDTA, pH 8) containing 50 µg ml-1 ethidium bromide. Serial dilutions of a known amount of HBV DNA, amplified in parallel, were used as a reference standard and a roughly linear detection between 103 to 107 genomes ml-1 was obtained (data not shown).
The expression of SCCA was assessed by FACS analysis in cell samples obtained 24 h after seeding and the results obtained in both HepG2 cells and primary human hepatocytes were similar. The addition of DMSO treatment did not influence basal expression of surface SCCA, as shown in Fig. 1. Pre-incubation of cells with preS1 containing reagents increased surface expression of the serpin, while the scrambled peptide did not affect the basal cell fluorescence signal (Fig. 2
), confirming that the observed biological effect was strictly sequence dependent. Intracellular staining for SCCA, carried out in parallel experiments with permeabilized cells, did not show a significant increase in the total fluorescence intensity after incubation with preS1 reagents, suggesting a redistribution of the serpin at the surface level, rather than an increase in overall expression of SCCA.
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As shown in Fig. 3, an increased binding and percentage of internalization was documented in cells pre-incubated with preS1 tetrameric peptide and the amount of HBV genomic equivalents was duplicated after pre-incubation with synthetic preS1, while non-HBV scrambled peptide did not affect HBV per cell interaction. The greatest effect of synthetic preS1 protein compared to preS1 peptide is probably due to its higher affinity for surface SCCA, as a consequence of partial folding of this myristylated synthetic protein, previously documented by circular dichroism measurement (De Falco et al., 2001a
).
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SCCA has recently been found to be overexpressed in hepatocellular carcinoma (HCC) (Pontisso et al., 2003), as occurs in other tumours of epithelial origin (Kato, 1996
). Whether the SCCA surface overexpression induced by the preS1(2147) motif is one of the mechanisms by which HBV aids HCC development is still an open question and studies are currently ongoing in our laboratories.
In conclusion, the HBV preS1(2147) sequence is able to induce overexpression of SCCA at the cell surface facilitating virus entry, whilst the increased efficiency of HBV entry following DMSO addition is not mediated by SCCA.
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ACKNOWLEDGEMENTS |
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Received 30 January 2003;
accepted 21 November 2003.
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