1 Leadd BV, Leiden, The Netherlands
2 BFSC, Leiden Institute of Chemistry, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands
3 Research Laboratories of Schering AG, Berlin, Germany
Correspondence
Mathieu H. M. Noteborn
m.noteborn{at}chem.leidenuniv.nl
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ABSTRACT |
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MAIN TEXT |
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TTV can infect not only hepatocytes but also extrahepatic tissues (Zhong et al., 2002; Suzuki et al., 2001
), although most of its replication appears to occur in the liver. Most of the literature shows that the infection of TTV seems to have few if any pathogenic effects (reviewed, for example, by Bendinelli et al., 2002
), but in certain cases it seems that TTV can still damage liver tissues (Hu et al., 2002
). In addition, various reports have described that, although TTV does not seem to contribute to the development of human hepatocellular carcinoma (HCC) from chronic liver disease, it may be a risk factor for the development of HCC in patients with type C liver disease in the F4 stage (Yoshida et al., 2000
; Pineau et al., 2000
; Tagger et al., 1999
). The irregular regeneration of hepatocytes in TTV-positive patients was significantly higher than that in TTV-negative patients (Moriyama et al., 2001
), and Camci et al. (2002)
described a high prevalence of TTV in cancer patients.
TTV harbours a circular single-stranded DNA genome, and although there are significant differences, there are also striking similarities to chicken anaemia virus (CAV) in its genomic organization (Fig. 1a; Miyata et al., 1999
; Mushahwar et al., 1999
; Erker et al., 1999
; Noteborn et al., 1991
, 1999
). Since CAV encodes a protein that is uniquely apoptotic in cancer cell lines, we were interested in whether TTV contained a similar entity. A detailed comparative analysis of the putative TTV ORF 3 encoding 105 aa (Miyata et al., 1999
; GenBank accession no. AB008394) with Apoptin showed that, although their overall sequence homology seemed limited, intriguing similarities existed both in sequence (Fig. 1b
) and predicted structure (not shown).
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The synthesis of the Myc-tagged TAIP was first analysed by transfection of COS-1 cells with plasmid pCMV-TAIP (Van der Vorm et al., 1994; Danen-Van Oorschot et al., 2000
). Two days after transfection, the cells were lysed and Western blot analysis was carried out using antibody 9E10. The COS-1 cells transfected with pCMV-TAIP were shown to synthesize a specific Myc-tagged product with the expected size of approximately 15 kDa. Control lysates of mock-transfected COS-1 cells did not contain a protein product reacting with 9E10 (Fig. 1c
).
Next, we examined whether the TAIP harboured apoptotic activity in human HCC cells and compared this with the effects of either Apoptin or LacZ as positive and negative controls, respectively. The human HCC-derived cell lines HepG2 (p53+), HUH-7 (p53 mutant) (both from ATCC) and Hep3B (p53; Puisieux et al., 1993) were transiently transfected with pCMV-TAIP encoding the Myc-tagged TAIP, pCMV-Apoptin encoding Apoptin and pcDNA3.1-MycHis-LacZ encoding the LacZ protein (Zhuang et al., 1995
; Danen-van Oorschot et al., 2000
). Two and 5 days after transfection, the cells were screened for the production of the specific transgene by indirect immunofluorescence (Noteborn et al., 1990
). Apoptosis was analysed by nuclear DNA staining with DAPI (Telford et al., 1992
). Two days after transfection, the putative TAIP was mainly detected in the cytoplasm, whereas Apoptin was mainly found in the nucleus of the transfected HCC lines. The negative control LacZ protein was present in the cytoplasm (Fig. 2
a). Five days after transfection, all three analysed HCC cell lines transfected with the TAIP transgene as well as those expressing Apoptin underwent high levels of apoptosis (Fig. 2b
), whereas the cells expressing LacZ only underwent a low level of cell death. These data clearly show that TAIP can induce apoptosis in human HCC cells. Furthermore, the fact that TAIP was able to kill Hep3B cells indicated that TAIP, like Apoptin, induces apoptosis in a p53-independent way.
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The difference in efficacy for TAIP-induced death in HCC versus non-HCC lines is probably not caused by a technical or cell culture difference, since no similar difference was observed in the other transfections. In conclusion, TAIP seems to induce apoptosis preferentially in HCC cells, unlike Apoptin, which induces high levels of apoptosis independent of the tumour origin.
The original hypothesis that TTV causes cryptogenic hepatitis has not been borne out (Bendinelli et al., 2002). On the other hand, it has been shown that, in particular cases, TTV seems to be involved in liver damage (Hu et al., 2002
). Here, we have reported that the putative TTV-derived TAIP induced cell death in three liver-derived human cell lines but significantly less in three non-liver-derived cell lines. First indications from experiments using normal rat hepatocytes microinjected in the nucleus with plasmids encoding TAIP, Apoptin or the non-apoptotic LacZ protein indeed showed that neither Apoptin nor TAIP induced apoptosis in these cells (data not shown). Although this is interesting, further experiments with, for example, normal human liver cells will be required before concluding that TAIP causes cell death in a tumour-specific manner, similar to Apoptin (Noteborn, 2002
).
Besides genomic homologies between CAV and TTV (Miyata et al., 1999; Bendinelli et al., 2002
), we have provided evidence here that at least one TTV strain also encodes a potential apoptotic activity, which has some characteristics reminiscent of Apoptin. Only two TTV isolates have been reported with 100 % homology to TAIP, the source of our TAIP sequence, TA278, and VT 416, both type 1 genotypes. A further eight genotypes (all type 1) showed 9099 % homology at the protein level, but until the relevant experiments have been performed we cannot know whether even single amino acid variants contain the same activity as TAIP. This restricted distribution might indicate that TAIP (activity) is not essential for TTV or that it may be responsible for some strain-specific cytopathogenic effect. In addition, there may be tissue-specific effects of TTV infection, since in the limited cases where pathology has been observed this was always restricted to the liver of TTV-infected individuals (e.g. Hu et al., 2002
), despite some reports of replication in additional sites in the body (Xiao et al., 2002
; Bando et al., 2001
). This may be relevant to the finding by Kamahora et al. (2000)
, who found expression of only three transcripts when full-length TTV was transfected into COS cells, none of which resembled TAIP. However, if TTV-related pathology is restricted to liver, it seems possible that cell-type/tissue-specific additional transcripts may exist. In light of the possibility of tissue- and/or strain-specific pathology, it seems important to identify more complete TTV coding sequences and to sample several tissues in order to characterize its pathogenic potential adequately. In addition, our results do not exclude the possibility that further (putative) ORFs of newly identified TTV isolates may also induce apoptosis (Peng et al., 2002
). It is even possible that other TTV proteins harbour (further) apoptotic potential, analogous to the CAV protein VP2 (Noteborn, 2002
). Finally, it might be of interest to determine whether the TTV-like mini virus, which is an intermediate between CAV and TTV (Tahahashi et al., 2000
; Hino, 2002
), harbours apoptotic activity.
TAIP and Apoptin induced p53-independent apoptosis; however, unlike Apoptin, TAIP only induced low levels of apoptosis in non-HCC lines. This apparent contradiction may be explained by a characteristic phenomenon of Apoptin a tumour-specific phosphorylation site at Thr-108 (Rohn et al., 2002). Asabe et al. (2001)
described a TTV ORF3 gene, not homologous to TAIP, which generates two variants of a protein with a different serine-phosphorylation state, similar to the hepatitis C virus non-structural protein. It might well be that small TTV-derived proteins derived from this ORF3 gene and possibly also TAIP are regulated by a specific kinase, as is the case for Apoptin. TAIP might be more active in cells that contain such a kinase. To test this, one has to examine the phosphorylation state of TAIP, for example, in HCC versus normal liver cells.
Apoptin has been shown to be a promising anti-cancer gene therapeutic agent in various animal models (Pietersen et al., 1999; Van der Eb et al., 2002
). Our initial TAIP studies suggest that TAIP shows potential as a basis for treatment of HCC tumours. Furthermore, TAIP promises to be another agent with which to dissect (disturbed) tumour-specific cell death pathways, as it appears to have some specificity in which tumours it kills preferentially. Finally, the heterogeneic nature of TTV, coupled with the potentially different pathogenic sequences, such as TAIP, indicate that it may be of clinical relevance which TTV strain has actually infected the patient.
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ACKNOWLEDGEMENTS |
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Received 10 November 2003;
accepted 6 February 2004.
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