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Cell Biology International (2006) 30, 915–919 (Printed in Great Britain)
Different transformation pathways of murine fibroblast NIH 3T3 cells by hepatitis C virus core and NS3 proteins
Irina S. Smirnovaab*, Nikolai D. Aksenova, Maksim S. Vonskya and Maria G. Isaguliantsb
aInstitute of Cytology RAS, Tikhoretsky Avenue 4, 194064 St. Petersburg, Russian Federation
bSwedish Institute for Infectious Disease Control, Stockholm, Sweden


Abstract

The oncogenic potential of both Hepatitis C virus (HCV) core and HCV NS3 proteins has been demonstrated, but these proteins induce transformation of immortal murine fibroblasts NIH 3T3 via different pathways. As long-term expression (50–100 passages) of HCV core triggers neoplastic transformation of NIH 3T3 through crisis of growth, HCV NS3 induces transformation shortly after transfection. We explain this distinction by different effects of core and NS3 on p53-mediated transactivation: inhibition by NS3 and activation by core protein.


Keywords: Cell transformation, Core, NS3, HCV, p53.

*Corresponding author. Institute of Cytology RAS, Tikhoretsky Avenue 4, 194064 St. Petersburg, Russian Federation. Fax: +7 812 297 03 41.


1 Introduction

Hepatocellular carcinoma (HCC) is one of the most common human cancers worldwide. Persistent infection with hepatitis C virus (HCV) can be considered as a critical risk in the development of HCC. The molecular mechanisms of HCC are still obscure. The central question regarding HCC is which viral gene products are necessary for its establishment. The lack of efficient cell culture systems that support high-level HCV replication limits research into the expression of individual viral proteins. It has been reported that the core proteins, NS3 and NS4B, have oncogenic potential (Ray et al., 2000; Moriya et al., 1998; Sakamuro et al., 1995; Zemel et al., 2001; Park et al., 2000). HCV core and NS3 proteins, that play key roles in the life cycle of virus and interaction with host cellular proteins (Tellinghuisen and Rice, 2002; Block et al., 2003), are the subjects of our investigation.

Both proteins have demonstrated their tumorigenicity by transforming some established cells in vitro as well as by inducing tumors in vivo in nude mice (Ray et al., 2000; Moriya et al., 1998; Sakamuro et al., 1995; Zemel et al., 2001). Nevertheless their oncogenic potential is still controversial and the precise mechanisms of their transformation are unknown. It has been shown that these proteins could modulate cell growth and apoptosis processes (Takamatsu et al., 2001; Kwun et al., 2001; Ray and Ray, 2001). p53 is a transcriptional factor required for the transactivation of a number of genes involved in growth control as well as in apoptosis (Levine, 1997). The modulation of p53 transcriptional regulatory activity by core and NS3 has also been shown (Lu et al., 1999; Otsuka et al., 2000; Kwun et al., 2001; Kao et al., 2004).

We focused our attention on interactions of NS3 and core proteins with tumor suppressor p53. Our research has shown that different pathways of interaction of HCV proteins with p53 lead to different ways of transformation – via crisis (in the case of core protein) and without crisis (in the case of NS3 protein).

2 Materials and methods

2.1 Stable cell lines, determination of growth rate

Construction of pCMVcore191neor and pCMV NS3 neor-plasmids expressing, respectively, HCV core and HCV NS3 proteins (genotype 1b) were described previously (Isaguliants et al., 2004). NIH 3T3 was maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS; Life Technologies) and replated 1:5 every 3 days to avoid spontaneous transformation (Chow and Rubin, 1999).

Cells were transfected with each plasmid using Lipofectamine (Life Technologies) and selected on geneticin-containing medium (G418, 500μg/ml). Stable core and NS3-transfectants were tested for expression of each protein by indirect immunofluorescence with primary anti-HCV core (Isaguliants et al., 2004) and anti-HCV NS3 (Isaguliants et al., 2003) antibodies, respectively. Secondary antibodies, anti-rabbit Ig, conjugated to Cy3 (Nanoprobes Inc., NY, USA), were used for detection of core and NS3 proteins by fluorescence microscopy.

For the determination of cell growth rate 2×104 cells were plated in 35mm dishes (Nunc). Cells were fed DMEM with 10% FCS every day. The doubling time was calculated from the growth curve. The saturation density was determined as the number of cells at confluence per cm2.

2.2 Anchorage-independent growth in soft agar

Soft agar dishes (50mm) were prepared with an underlayer of 0.5% agar (Difco, Detroit, MI, USA) in DMEM containing 10% FCS. Cells were seeded in the dishes in DMEM containing 10% FCS and 0.33% agar at a density of 4×103cells/dish. The dishes were examined microscopically for colony formation and photographed. The data were expressed as the percentage of colonies containing more than 200 cells after incubation for 14 days.

2.3 Flow cytometry

Cells were harvested, washed in PBS, permeabilized with 0.1% saponin for 0.5h, washed in PBS, stained with a solution containing propidium iodide (50mg/ml) and RNase (2U/ml), and analysed on ODAM ATC-3000 flow sorter-analyser (Bruker Spectrospin, Wissembourg, France) by one parameter FACScanning. The analysis rate was 400–600cells/s. A total of 15,000–20,000 cells were analysed in each sample. For serum starvation, cells in exponential growth phase (60–80% confluence) were rinsed with PBS, and cultured in DMEM with 0.5% FCS for up to 14 days. Ploidy was assessed using diploid mouse splenocytes as an internal standard. Clones were assessed in three independent runs. Data were processed using ModFit 2.0 software (Verity Software House Inc., Topsham, ME, USA). The cells were irradiated in an RUM 17 at 6Gy.

2.4 Luciferase assay

Reporter plasmid pG13-luc carrying 13 repeats of the consensus wild-type p53 binding sequence was kindly provided by el-Deiry et al. (1993). Plasmid pSVβ-gal expressing β-galactosidase was purchased from Promega (Madison, WI, USA). Core and NS3 stable transfectants were co-transfected with reporter plasmids pG13-luc and pSVβ-gal, NIH 3T3 were co-transfected with reporter plasmids pG13-luc and pSVβ-gal alone or with pCMVcore191 or pCMV NS3. After 48h, cells were lysed with Luciferase Cell Culture Lysis Buffer (Promega). Soluble proteins were recovered and quantified using Bradford assay (BioRad, Hercules, CA, USA). Luciferase activity in soluble fractions was measured using Luciferase Assay System (Promega), and β-galactosidase activity was measured using ortho-nitrophenyl-galactopyranoside (4mg/ml; Sigma). The p53-dependent transactivation was represented as a mean ratio of luciferase to β-galactosidase activity in three independent experiments.

3 Results

Two cell lines expressing HCV core and two cell lines expressing HCV NS3 were established. We obtained similar results for each of two core cell lines and for each of two NS3 cell lines, so we described for simplicity a representative one for each. The expression of core and NS3 genes was confirmed by immunofluorescence staining using core-specific and NS3-specific polyclonal antibodies. Immunofluorescence analysis revealed strong perinuclear granular staining and weaker nuclear staining in both cases (Fig. 1).


Fig. 1

Indirect immunofluorescence analysis of core and NS3 proteins expressed in NIH 3T3 cells. NIH 3T3 (a) and core-transfectants (b) were stained with core-specific rabbit antibodies; NIH 3T3 (c) and NS3-transfectants (d) were stained with NS3-specific antibodies. Then the cells were stained with cy3-conjugated anti-rabbit IgG and observed under fluorescent microscope. The cells are presented at 100th passage.


It was shown that contrary to HCV core-induced transformation, which passed via crisis (50–100 passages), HCV NS3-transfectants demonstrated morphological and proliferative changes that were typical for transformation by 5–10 passages after transfection. These changes were stable during our experiment (100 passages) (Fig. 2a; Table 1). Both transfectants – HCV core by 50–100 passages and HCV NS3 by 5–10 passages – exhibited focus-forming morphology, decreased attachment to the substratum, higher saturation density and decrease of doubling time (Fig. 2a; Table 1). Despite rapid morphological and proliferative changes observed in NS3-transfectants, the ability to grow in semisolid agar was delayed by 30–50 population doublings after transfection (Fig. 2b).


Fig. 2

(a) Morphological changes of stable transfectants. The cells are presented at 100th passage. The NS3-transfectants achieved these changes by 5–10 passages and retained them during our study. (b) Colony formation in soft agar. The cells are presented at 100th passage.


Table 1.

Growth propertiesa of NIH 3T3, core and NS3 cells

Cell linesDoubling time (h)Saturation density (cells/cm2, 104)Growth in soft agar (%)
NIH 3T322 ± 1.527 ± 0.70
Core19 ± 1.748.5 ± 8.55.1 ± 1.8
NS316 ± 0.565 ± 27.7 ± 0.7
a The cells were used at 100th passage. The NS3-transfectants achieved indicated doubling time and saturation density by 5–10 passages and did not change them during our study.


In addition to described properties NS3, as well as core- transfectants, also acquired one of the hallmarks of fibroblast transformation – the ability to proliferate in the absence of serum (Fig. 3a). The parental NIH 3T3 cells in the serum free media died quickly (within 4–6 days), whilst the transfectants survived for 2 weeks (Fig. 3b).


Fig. 3

Serum-independent growth of core and NS3 transfectants. (a) Cell cycle analysis of cells incubated in serum free medium for 48h. (b) Cells incubated in serum free media for 7 days. The cells are presented at 100th passage.


The p53 tumor suppressor gene product is central to the cellular response to stress that can be induced by expression of core and NS3 genes. To see if that is the case, we investigated stable core- and NS3-transfectants as well as NIH 3T3 transiently transfected with core and NS3 genes for the status of endogenous p53 activity using a reporter plasmid pG13-luc (Fig. 4). In the presence of transiently expressed core, the endogenous p53 activity increased nearly twofold. In contrast, when pG13 was transiently co-transfected with NS3 gene, the p53 activity decreased more than eightfold. The same inhibitory effect was also exerted by the transient transfection of pG13-luc into both stable transfectants – core and NS3 alike. To study whether this effect is dose-dependent we compared different amounts of both proteins (Fig. 4b). As well as was shown by Kao et al. (2004) the low level of HCV core (0.25μg) enhanced p53 transcription activity, but high levels (1.75μg) inhibited it. NS3 does not influence p53 transcription activity in a dose-dependent manner; NS3 inhibits it at all concentrations.


Fig. 4

(a) Effect of HCV core and HCV NS3 on transcriptional activation of p53 in stable clones (at 100th passage) and in NIH 3T3 during temporary transfection with pCMVcore or pCMV NS3. (b) Effect of HCV core and HCV NS3 concentration on transcription activation of p53. Transcriptional activation of p53 in NIH 3T3 was taken for 100%.


To determine if the disability of p53 reflects on the checkpoints we compared cell cycle of stable transfectants and parental cell line after gamma-irradiation. For G0/G1 and G2/M blocks estimation we used G0/G1 to S and G2/M to S ratios, respectively. An increase in the G0/G1 to S ratio from 2.4 to 10.6 for NIH 3T3 cells (Fig. 5a) is consistent with the arrest in the G0/G1 phase of the cell cycle. On the contrary all transfected cells demonstrated no increase of this value, which implies the absence of G0/G1 cell cycle arrest. An increase in the G2/M to S ratio from 0.8–1.2 to 3.1–4.1 for all cell lines (Fig. 5b) is consistent with an arrest in the G2/M phase of the cell cycle at 8h after irradiation. After 8h the cells demonstrate a significant difference in G2/M block release. NIH 3T3 begins to release from G2/M block after 16h, whilst the transfectants take just over 8h. By 40h all the transfectants demonstrate complete release from G2/M block while the control cells remain in G2/M block.


Fig. 5

(a) The dependence of G0/G1 to S (G0/G1 block) and (b) G2/M to S (G2/M block) ratio on time after X-ray irradiation. The cells are presented at 100th passage.


4 Discussion

There is some convincing evidence of transforming potential of both HCV core and HCV NS3 proteins (Ray et al., 2000; Moriya et al., 1998; Sakamuro et al., 1995; Zemel et al., 2001). Our results support this contention. But these two HCV genes induced transformation of NIH 3T3 via different pathways. As NS3 transfection induced rapid phenotypic changes, core transfection was followed by a long period of crisis. It is known that carcinogenesis is a multistep process and the most commonly occurring step involves functional inactivation of p53 tumor suppressor gene product (Halazonetis, 2004). We assume that different ways of transformation of HCV core and HCV NS3 can be explained by various means of their interaction with p53. The ability of HCV NS3 to interact with p53 and thereby deregulate p53 normal functions was demonstrated by Ishido and Hotta (1998). Later the study of Kwun et al. (2001) revealed that NS3-induced repression of the p21waf/cip1 was mediated through modification of p53 activity. On the contrary, Lu et al. (1999) have demonstrated that a direct physical interaction between HCV core protein and p53 led to enhanced expression of downstream effector genes of p53 in particular of gene p21waf/cip1 and suppression of cell growth. Furthermore, the core protein can act as a transcriptional co-activator of p53 with dose-dependent p21waf/cip1 induction (Otsuka et al., 2000). Studies of Kao et al. (2004) indicated that HCV core protein has dual effects on p53-mediated transactivation depending on viral protein concentration: inhibition at high levels of expression and coactivation by low levels of HCV core protein. Our work confirms this biphasic nature of dependence. At the same time we show that HCV NS3 inhibits p53 transactivation at all concentrations.

We propose that HCV NS3-induced repression of p53 transactivation creates transformation advantage for NS3-transfectants. In the case of core gene the transfected cells underwent a crisis period, perhaps induced by overexpression of p21. During this crisis many cells die before permanently establishing highly transformed cell lines. Their recovery is correlated with inactivation of p53. In view of the fact that the p53 transactivation function is dependent on the concentration of HCV core protein we cannot rule out the selection of inactivated p53 through the selection of the cells with high levels of expression of HCV core. Whatever is the cause, the result showed that surviving core-transformants contain functionally inactivated p53.

Loss of p53 leads to a reduction of checkpoint controls (Laiho and Latonen, 2003; Fei and el-Deiry, 2003; Iliakis et al., 2003; Eastman, 2004; Halazonetis, 2004) that facilitates the development of the tumor. NS3 as well as core-transfectants completely lose their G1/S checkpoint and G2/M checkpoint is reduced to a short delay.

NS3-transfectants are characterized by early morphological and proliferative changes but delayed growth in semisolid agar. These observations suggest that multiple cellular changes are required for the acquisition of the ability for anchorage-independent growth of NS3-transfectants. This implies that NS3-induced neoplastic transformation also represents a progressive process through qualitatively different stages.

Recently Takamatsu et al. (2001) has shown suppression of serum starvation-induced apoptosis by HCV core protein. But core as well as NS3-transfectants not only survive but also continue to proliferate in the absence of serum. This finding suggests that these cells can synthesize their own growth factors through an autocrine loop or may bypass the need for growth factor stimulation through constitutive activation of mitogenic pathways downstream of growth factor receptors (Sporn and Roberts, 1992).

In spite of the fact that both proteins induce transformation via different pathways, by the 100th passage both core and NS3-transfectants become highly transformed cell lines.

Acknowledgements

This work was funded by the grants of the New Visby programme of the Swedish Institute. Drs. Olga Petukhova and Elena Kornilova are gratefully acknowledged for valuable comments and criticisms.

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Received 25 April 2005/14 April 2006; accepted 8 June 2006

doi:10.1016/j.cellbi.2006.06.020


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)