s2fieldsofstudy: Biology Chemistry

text:
The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M

Background Emerging data have suggested that cell surface GRP78 is a multifunctional receptor and has been linked to proliferative and antiapoptotic signaling cascades. Activated α2−macroglobin (α2M*) is a natural circulating ligand of cell surface GRP78. Association of cell surface GRP78 with α2M* is involved in the regulation of cell proliferation, survival and apoptosis in human cancers. Methods The invasion and metastasis of HCC cells were examined using transwell and wound healing assay; Cell surface expression of GRP78 was detected by in cell western assay. Translocation of GRP78 from cytosol to cell surface was observed by transfection of GRP78-EGFP plus TRIRC-WGA staining. The levels of Src, phosphor-Src, FAK, phospho-FAK, EGFR, phospho-EGFR, phospho-Cortactin, phospho-Paxillin were determined by western blot. Cell surface expression of GRP78 in HCC tissue samples was observed by immunofluorescence. The distribution of Paxillin and Cortactin in HCC cells was also observed by immunofluorescence. The interaction between GRP78 and Src were detected by far-western blot, co-immunoprecipitation and GST pulldown. GRP78 mRNA was detected by RT-PCR. Results In the current study, we showed that association of cell surface GRP78 with α2M* stimulated the invasion and metastasis of HCC. Cell surface GRP78 could interact directly with c-Src, promoted the phosphorylation of c-Src at Y416. Inhibition of the tyrosine kinase activity of c-Src with PP2 reverted the stimulatory effect caused by association of cell surface GRP78 with α2M*. Moreover, association of cell surface GRP78 with α2M* facilitates the interaction between EGFR and c-Src and consequently phosphorylated EGFR at Y1101 and Y845, promoting the invasion and metastasis of HCCs. However, inhibition of the tyrosine kinase of c-Src do not affect the interaction between EGFR and Src. Conclusion c-Src plays a critical role in the invasion and metastasis of HCC induced by association of cell surface GRP78 with α2M*. Cell surface GRP78 directly binds and phosphorylates c-Src. As a consequence, c-Src phosphorylated EGFR, promoting the invasion and metastasis of HCCs.

Background
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide [1]. Invasion and metastasis contributed largely to the high mortality of HCC [2]. Therefore, exploring the mechanisms regulating the invasion and metastasis is critical for searching new strategies to improve the outcome of HCC.
Human ɑ 2 -macroglobulin (α 2 M) is a typical member of the pan-proteinase inhibitors of the α2M family, which is mainly synthesized by the liver [3]. Many data have reported that α 2 M is overexpressed in HCC with the background of hepatitis B infection and the increased serological α 2 M is associated with HCC in humans, identifying α 2 M as a cytochemical marker for the diagnosis of HCC [4]. α2M is activated by intracellular proteinases. When activated, α2M binds directly with corresponding cell surface receptors and functions as a regulator of many signaling pathways and plays a growth factor-like role in many human cancers. So far, two cell surface receptors that specifically bind with activated α2M (α2M*) have been identified, namely cell surface glucose-regulated protein 78 (GRP78) and LDL receptor related protein (LRP) [5]. Upon most occasions, GRP78 is regarded as an endoplasmic reticulum chaperone, whose major function is to fold and process the unfolded or malfolded proteins [6]. However, it is also presented on the cell surface under stress condition [7]. Cell surface GRP78 acts as a multifunctional receptor which plays critical role in the proliferation, viability and apoptosis [8,9]. For example, association of cell-surface GRP78 with α2M* triggers MAPK and Akt signaling cascades, promoting cellular proliferation of 1-LN prostate cancer cells [3,10,11]. Ligation of cell surface GRP78 with α2M* activates the NF-kappaB signaling pathway, decreases p53 level and plays a stimulatory role in the proliferation and viability of prostate cancer cells [11,12].
Although a large body of evidence has linked cell surface GRP78 to proliferative and antiapoptotic signaling cascades, little is known about the role of cell surface GRP78 in the invasion and metastasis of human cancer cells. Cellular Src (c-Src), a non-receptor protein tyrosine kinase, is overexpressed and hyperactivated in many human cancers [13,14]. Increasing evidence has demonstrated that c-Src is implicated in the regulation of a variety of cellular functions, such as tumor invasion and metastasis, by interacting with and phosphorylating a wide range of intracellular proteins including epithelial growth factor receptor (EGFR) [15]. EGFR is a member of the ErbB family of receptor tyrosine kinases and is overexpressed in many types of human cancers including HCC [16]. Accumulating evidence has suggested that Src could form a complex and mediated EGFR phosphorylation, regulating the tumor progression [17].
In this article, we found that GRP78 is overexpressed on the cell surface in HCC tissue samples. Association of cell surface GRP78 with α2M* promotes the invasion and metastasis of HCC in hepatocellular carcinoma cell lines QGY-7703 and PLC by stimulating the translocation of GRP78 from the cytosol to plasma. Downstream, cell surface GRP78 interacts directly with c-Src and promotes the phosphorylation of c-Src at Y416. Moreover, association of cell surface GRP78 with α2M* facillitates the interaction between EGFR and c-Src. As a consequence, c-Src phosphorylated EGFR at Y1101 and Y845, promoting the invasion and metastasis of HCCs.

Antibodies and other reagents
The following antibodies were used: anti-Src, anti-pSrcY416, anti-FAKpY397, anti-pPaxillin Y118, anti-Paxillin, anti-pCortactin Y486, anti-pCortactin Y466, anti-Cortactin were all from life technologies; anti-GRP78 N20, antiGRP78 C20 (for antibody blocking), anti-β-actin were from Santa Cruz. Anti-GRP78 (for immunoprecipitation and in cell western analysis), anti-pEGFR Y1101, anti-pEGFR Y1068, anti-pEGFR Y845, anti-EGFR, anti-p-Tyr and rabbit isotype IgG were obtained from Abcam. Anti-EGFP was obtained from Origen. All the secondary antibodies except for IRDYE680RDconjugated antibody (LI-COR) were all from abcam. α2M was purchased from Sigma-Aldrich and activated as previously reported [18]. PP2, lipofectamine 2000, TRITC-WGA and fibronectin were from life technologies. Protein A/G agarose beads and G-sepharose beads were purchased from GE healthcare. RT-PCR kit was from Takara. Plasma protein isolation kit was purchased from Pierce. GRP78-EGFP recombinant and corresponding pEGFP-N1 were kindly given by the Cell Biology Department of China Medical University.

Cell culture and treatment
Human hepatocellular carcinoma cell line QGY-7703 and PLC were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM supplemented with 10 % fetal bovine serum, 1 mM L-glutamine and 1 % penicillin/ streptomycin antibiotics. For experimental purpose, cells were serum starved for 4 h. PP2 (10 μM for 30 min) or blocking antibodies (4 μg/ml for 1 h) were added for 1 h, following α2M* stimulation (50pM for 30 min), cells were harvested and subjected to subsequent analysis.
Human tissue specimens and ethics statement All 10 cases of paired HCC tissue samples were obtained from the Department of Gastroenterology of the General Hospital of Chinese Liberation Army. The Ethics Committee of Liaoning Medical College approved and supervised specimen collection procedures (No. 20130023). All the experimental performances related to the tissue samples were in compliance with Helsinki Declaration. We have got the permissions of all the patients before specimen collection. The differentiation extents were re-evaluated by two pathologists according to Edmondson-Steiner grading system. None of the patients has received chemotherapy or irradiation before surgery.

Transwell, wound healing assay
In vitro cell invasion and migration were analyzed using transwell assay and wound healing assay as previously described [19]. The experiments were repeated for 3 times and the data were represented as X AE SD.

In cell western assay
In cell western were carried out as previously reported [10]. Briefly, confluent cells were harvested, diluted to 4 × 10 5 cells/mL with complete culture medium and dispensed to 96-well culture plate (100 μl per well). After 24 h, cells were rinsed for three times with cold PBS and serum starved for 4 h. following α2M* stimulation (50pM for 30 min). Cells were fixed with 2 % formaldehyde in PBS, washed for three times with PBS with or without 0.05 % Tween 20, blocked with 3 % BSA for 2 h with gentle agitation. After blocking, cells were incubated with anti-GRP78 (1:50 dilution) in blocking solution overnight with gentle agitation, stained with IRDYE 800 D-conjugated secondary antibody in PBS for 60 min at RT and washed for three times with PBS and imaged by LICOOR Odyssey®. Cell surface GRP78 were quantified according to the manufacturer's instructions.

Cell adhesion assay
The binding ability of tumor cells with fibronectin was detected using cell adhesion assay. Briefly, pretreated cells (10 4 each well) were trypsinized, re-plated on fibronectin-coated coverslips (10 μg/ml) and incubated at 37°C for 1 h. Following incubation, non-adherent cells were removed by washing for 3 times with PBS, adherent cells were fixed with 4 % paraformaldehyde solution, stained with crystal violet (1 %) and dissolved by 1 % SDS. Absorbances at 595 nm were determined using a microplate reader.

Transfection and plasma staining
Cells were cultured in a six-well culture plate and grown to 90 % confluent. Before transfection, Cells were cultured in complete medium without antibiotics overnight and transfected with 4 μg GRP78-EGFP or pEGFP-N1 for 16 μl lipofectamine 2000 in 500 μl Opti-MEM (Gibco). After 48 h, cells were serum starved for 4 h, simulated with α2M* for 30 min stained with TRITC conjugated WGA (1:200 dilution) for 1 h under normal culture condition. Following WGA staining, cells were observed using inverted fluorescence microscope (DMI-4000B, Leica, Germany).

Immunofluorescence
The cellular distribution of Cortactin, Paxillin and GRP78 was observed using immunofluorescence [21,22]. Immunofluorescence was performed as previously reported and observed using laser confocal microscope (SP5II, Leica, Germany).

Cell surface protein biotinylation
Confluence cells were rinsed with ice-cold PBS twice and serum starved for 4 h. PP2 was added for 30 min. following α 2 M* stimulation, EZ-link Sulfo-NHS-LC-Biotin (Pierce, USA) was added to cover the surface of the cell layer and the flasks were gently shaken at 4°C for 30 min. The biotinylation reaction was stopped by adding Tris-HCl (pH 7.5) to a final concentration to 100 nM. Then, the cells were rinsed with ice-cold PBS twice and then lysed with RIPA buffer and Neutravidin-agarose beads (Pierce, USA) were added in whole cell lysate overnight and incubated at 4°C with agitation. The beads were washed for 5 times with PBS buffer. The cell surface proteins were released by 1× SDS-PAGE sample loading buffer and heating at 100°C for 5 min. The cell surface proteins were subjected to western blotting analysis.

RT-PCR
Conventional RT-PCR was performed as previously reported [23].

Co-immunoprecipitation assay
One thousand micrograms of plasma membrane protein extract from each sample was pre-cleared with 50 μl of protein A/G-Sepharose beads for 1 h at 4°C and incubated with 5 μg of anti-GRP78 or anti-EGFR (Abcam) overnight at 4°C on a rotator. Following antibody incubation, 50 μl of protein A/G-Sepharose beads (50 % slurry) were added and rotated at 4°C for 3 h. The beads were then centrifuged at 12,000 g for 5 min and washed for 5 times with the lysis buffer. The precipitates were eluted by adding of 20 μl of 1× SDS-PAGE sample loading buffer (50 mm Tris-HCl, pH 6.8, 100 mm DTT, 2 % SDS, 0.1 % bromphenol blue, 10 % glycerol), followed by heating at 100°C for 5 min. The supernatant obtained after centrifugation was resolved by SDS-PAGE and subjected to Western blot analysis.

GST pulldown assay
Plasma membrane extract was isolated as described previously. The clarified plasma membrane extract was incubated with 50 μl of a 50 % slurry of glutathione-Sepharose 4B (GE Healthcare, USA) and 25 μg GST for 1 h at 4°C. GST-Grp78-bound resin, GST-bound resin was incubated with cell lysates containing 1 mg protein extract overnight at 4°C on a rotator. The resin was then washed 5 times with ice-cold lysis buffer. Proteins were eluted by adding 25 μl of 2 × Laemmli sample buffer at 100°C for 5 min and centrifuged for 5 min at 12,000 g. The supernatant was resolved by SDS-PAGE and subjected to Western blot analysis using anti-c-Src (Abcam).

Statistical analysis
Comparison of the data was performed using one way ANOVA, student t-test and chi-square test. A p-value less than 0.05 was considered to be statistically significant.

Results
Association of cell surface GRP78 with α2M* facilitated the invasion and metastasis of HCC Increasing data have linked α2M* to pro-proliferative and anti-apoptotic role. However, whether it plays a role in the invasiveness and metastasis of HCC remains unknown. To investigate the role of α2M* in tumor invasion, QGY-7703 and PLC cells were serum starved for 4 h, followed by stimulation with α2M* for 30 min (50 pM) [10]. Using transwell assay, we found that treatment of QGY-7703 and PLC cells with α2M* caused significant increase in the invasiveness relative to that in cells treated with vehicle, indicating that α2M* stimulation facilitates the invasion of QGY-7703 (Fig. 1a) and PLC cells (Fig. 1b). We next assessed whether α2M* modulate the metastasis of cancer cells using wound healing assay and found that stimulation with α2M* significantly facilitated the metastasis of QGY-7703 (Fig. 1c) and PLC cells (Fig. 1d).
To investigate whether cell surface GRP78 is the surrogate of α2M* in this process, serum starved QGY-7703 and PLC cells were pretreated with the antibody directed against the NH2-termnial domain (NTD), COOHterminal domain (CTD) of GRP78 or isotype IgG for 1 h followed by α2M* stimulation [25,26]. Transwell assay revealed that pretreatment with the GRP78 NTD antibody caused a marked decrease in the invasion potential as compared with vehicle upon α2M* stimulation. However, pretreatment with CTD had no effect on the invasion potential as compared with pretreatment with isotype IgG or vehicle upon α2M* stimulation ( Fig. 1e-g). Using cell adhesion assay, we found that blockade of cell surface GRP78 with the NTD antibody decreased the binding ability of tumor cells to fibronectin in QGY-7703 cells (Fig. 1h). These data suggested that cell surface is the surrogate of α2M* and cell surface GRP78 promotes the invasion and metastasis by its NH2-terminal domain.
α2M* induced GRP78 translocation from cytosol to cell surface in HCC We next determined whether α2M* affected the cell surface expression of GRP78 in QGY-7703 and PLC cells. Serum starved QGY-7703 and PLC cells were treated with α2M* and vehicle. Using in cell western assay, cell surface and total GRP78 were determined with or without Tween 20 permeabilization. We found that α2M* stimulation significantly elevated the cell surface faction of GRP78, whereas did not affect total GRP78 in QGY-7703 and PLC cells (Fig. 2a). In QGY-7703 cells, α2M* stimulation caused a~2.7 fold increase in the cell surface expression of GRP78 as compared with vehicle. In PLC cells, a~2.1 fold increase was observed upon α2M* stimulation (Fig. 2b). Using RT-PCR, we found that GRP78 mRNA levels in cells treated with α2M* were similar with that in QGY-7703 and PLC cells treated with vehicle (Fig. 2c). These data suggested that the elevated expression of cell surface GRP78 is not regulated at transcriptional and translational level and raised the possibility that α2M* could induce the translocation of GRP78 from cytosol to plasma membrane. To answer this question, we transiently transfected GRP78-EGFP into QGY-7703 cells [27]. After 48 h of transfection, exogenous and endogenous GRP78 were examined using western blot (Fig. 2d). QGY-7703 cells transfected with GRP78-EGFP were serum starved for 4 h, followed by α2M* stimulation, stained with TRITC-conjugated wheat germ All these experiments were repeated for 3 times in triplicate. These data were presented as X AE SD and analyzed using student's t-test and one-way ANOVA. The difference is regarded to be statistically significant when p < 0.05. *represented that the difference is statistically significant agglutinin (TRITC-WGA), a specific plasma membrane dye. Using confocal microscopy, we found that GRP78 colocalized with TRITC-WGA on the cell surface (yellow on the cell surface) in α2M* treated cells. By contrast, the colocalization of GRP78 and TRITC-WGA was not observed in cells treated with vehicle (Fig. 2e). These data demonstrated that α2M* induces the translocation of GRP78 from cytosol to cell surface.

c-Src interacts directly with GRP78 on the cell surface of HCCs
We have previously reported that cell surface GRP78 facilitates the invasion of HCC [28]. To further extend this finding, we sought to determine the downstream target of cell surface GRP78. For this purpose, we compared tyrosine-phosphorylated proteins in serum starved QGY-7703 cells treated with α2M* or vehicle. As determined by western blot using anti-p-Tyr antibody. A tyrosine phosphorylated band migrating at~60 KDa was significantly induced in QGY-7703 cells treated with α2M*, suggesting that cell surface GRP78 may regulate the phosphorylation of a unknown tyrosine kinase whose molecular weight is~60 KDa (Fig. 3a). Based on this result, we tried to determine whether cell surface GRP78 regulated the activity of c-Src in HCCs. We first identified GRP78 binding proteins in the whole cell lysates from QGY-7703 and PLC cells by far-western blotting using the biotin-conjugated rhGRP78 as the bait. For this purpose, we isolated plasma membrane proteins using cell surface protein biotinylation assay and performed co-immunoprecipitation experiment using the anti-GRP78 antibody (Abcam). As shown in Fig. 3b, a protein, migrating at~60 kDa, could interact specifically with the biotin-conjugated rhGRP78. Moreover, we subjected the precipitated proteins to SDS-PAGE and found a~60 kDa band in the precipitated proteins. Western blotting showed that c-Src is presented in the proteins co-precipitated with GRP78, suggesting that the cell surface GRP78 modulates the activity of Src by direct interaction (Fig. 3c). Finally, we performed GST-pulldown assay to verify the interaction between cell surface GRP78 and c-Src using GST-GRP78 as the bait. We found that a~60 kDa band by SDS-PAGE and identified that this protein was c-Src by western blot (Fig. 3d). Using western blot, we examined p-Src phosphorylation at Y416 in cell lysates from serum starved QGY-7703 and PLC cells treated with α2M* or vehicle. We found that α2M* stimulation significantly increased p-c-Src levels as compared with vehicle in both QGY-7703 and PLC cells, indicating that cell surface GRP78 facilitates c-Src phosphorylation in HCC (Fig. 4a). We next determined whether the tyrosine kinase activity of c-Src is involved in the regulation of adhesion and invasion of HCC cells in QGY-7703 cells. Serum starved QGY-7703 cells were pretreated with PP2 at a concentration of 10 μM for 30 min followed by α2M* stimulation. Cell adhesion assay revealed that pretreatment of serum starved QGY-7703 cells with PP2 caused a significant decrease in binding ability to fibronectin as compared with vehicle upon α2M* stimulation. No difference was observed between cells treated with PP2 alone and cells treated with PP2 followed by α2M* stimulation (Fig. 4b). The invasion potentials were analyzed using transwell assay. As shown in Fig. 4c, pretreatment of serum starved QGY-7703 cells with PP2 caused a marked decrease in the invasion potential as compared with vehicle upon α2M* stimulation. It is worth to note that QGY-7703 cells treated with PP2 alone share similar inhibitory extent on tumor invasion as compared with cells pretreated with PP2 followed by α2M* stimulation. Similar results were obtained in PLC cells (Fig. 4d-e). These data suggested that c-Src is the downstream signaling molecule of α2M*. We also examined whether cell surface GRP78 could activate c-Src downstream signaling pathways [29]. As shown in Fig. 4f, treatment of serum starved QGY-7703 and PLC cells with α2M* caused significant increase in the phosphorylation levels of FAK (Y397), Cortactin (Y486 and 466) and Paxillin (Y118) as compared with vehicle treated cells.
Although many data have demonstrated that α2M* could bind with cell surface GRP78 and stimulate the signaling pathways downstream of cell surface expression of GRP78, we still need to preclude the possibility that α2M* binds with other cell surface protein and facilitates c-Src phosphorylation. To obtain this goal, serum starved QGY-7703 and PLC cells were incubated with the antibody directed against the NH2-termnial domain (NTD) The data were presented as X AE SD and analyzed using student's t-test. The difference is regarded to be statistically significant when p < 0.05. *represented that the difference is statistically significant Fig. 5 Association of Cell surface GRP78 with α2M* induces invadopodia formation. QGY-7703 cells were treated with vehicle, α2M*, PP2 or PP2 in combination with α2M* and co-stained with TRITC-conjugated Phalloidin and anti-Cortactin antibody. The distribution of F-actin (red) and cortactin (green) was observed using a confocal microscope. The invadopodia was indicated as yellow patches. Scale Bar 25 μm or COOH-terminal domain (CTD) of GRP78 for 1 h prior to α2M* stimulation. Many reports by other groups have demonstrated that the antibodies we used could block the binding of cell surface GRP78 with α2M*. Western blot analysis showed significantly lower pY416-Src and pY397-FAK levels in cells pretreated with NTD antibody as compared with cells pretreated with isotype IgG upon α2M* stimulation. However, pretreatment with CTD antibody did not affect pY416-Src and pY397-FAK levels (Fig. 4g). These data suggested that cell surface GRP78 is the surrogate of α2M* induced c-Src phosphorylation and activates c-Src via its NH2-terminal domain.

Association of cell surface GRP78 with α2M* induces invadopodia formation and Paxillin redistribution
Invadopodia is a specialized invasive organelle for tumor cells undergoing invasion and metastasis [30]. To investigate whether cell surface GRP78 regulates the formation of invadopodia, the distribution of Cortactin in serum starved QGY-7703 cells treated with α2M* or vehicle was observed using immunofluorescence [21]. By co-staining of Cortactin and F-actin, we observed that treatment with α2M* caused a marked increase in the number of speckles in cell cortex as compared with that treated with vehicle, while pretreatment with PP2 significantly decreased the number of speckles on cell cortex. Furthermore, α2M* stimulation caused a subtle increase the number of speckles in cell cortex in PP2 pretreated cells, indicating that c-Src is essential for the formation of invadopodia induced by association of cell surface GRP78 with α2M* (Fig. 5).
We also observed whether association of cell surface GRP78 with α2M* could cause the redistribution of Paxillin. Immunofluorescence microscopy revealed that Paxillin exhibited a dense punctate distribution on the cell periphery in serum starved QGY-7703 cells treated with α2M*as compared with that treated with vehicle, Fig. 6 Association of Cell surface GRP78 with α2M* induces paxillin formation. QGY-7703 cells were treated with vehicle, α2M*, PP2 or PP2 in combination with α2M* and stained with anti-Paxillin antibody. The distribution Paxillin (green) was observed using a confocal microscope. Scale Bar 25 μm indicating that cell surface GRP78 induced the redistribution of Paxillin. Pretreatment with PP2 decreased the cell periphery distribution. Moreover, α2M* stimulation caused a moderate increase in the cell periphery distribution of Paxillin in PP2 pretreated cells, indicating the critical role of c-Src in Paxillin redistribution caused by association of cell surface GRP78 with α2M* (Fig. 6).
Association of cell surface GRP78 with α2M* facillitated the maximal activation of EGFR in a c-Src dependent manner For the critical role of EGFR in the invasion and metastasis of HCC, we wonder whether cell surface GRP78 could regulate EGFR phosphorylation. For this purpose, we examined EGFR phosphorylation levels at Y1068, Y845 and Y1101 in serum starved QGY-7703 cells treated with α2M* or vehicle. Using western blot, we found that α2M* stimulation significantly increased the phosphorylation level of EGFR at Y1068, Y845 and Y1101 as compared with vehicle, while did not affect EGFR expression. Pretreatment of serum starved QGY-7703 cells with PP2 caused a marked decrease in the phosphorylation of EGFR at Y845 and Y1101 as compared with cells pretreatment with vehicle upon α2M* stimulation. However, PP2 treatment did not affected EGFR phosphorylation at Y1068 (Fig. 7a, b).
Accumulating evidence has shown that EGFR is a downstream signaling molecule of c-Src and c-Src could directly bind with EGFR and activate EGFR [31,32,17]. We next determined whether cell surface GRP78 could regulate the interaction between EGFR and c-Src. For this purpose, whole cell lysates from serum starved QGY-7703 cells treated with α2M* or vehicle were immuonprecipitated with excessive anti-EGFR antibody (~5 μg in a 1 ml volume containing 1 mg of total protein). The precipitations were subjected to western blot analysis and we found that c-Src level in the whole cell lysate from α2M* treated cells was higher than that from vehicle treated cells. It is worth to note that c-Src was not detected in isotope IgG precipitation. These data indicated that cell surface GRP78 facilitates the interaction between EGFR and c-Src (Fig. 7c). These data raised the question whether the tyrosine kinase activity of c-Src is essential for the interaction between EGFR and c-Src induced by cell surface GRP78. Using Co-immuoprecipitation in the lysates from serum starved QGY-7703 cells pretreated with PP2 or vehicle prior to α2M* stimulation, we found that the amount of c-Src co-immunoprecipitated with EGFR in the whole cell lysate from PP2 pretreated cells were paralleled with that from cells pretreated with vehicle, indicating that the tyrosine kinase activity of c-Src is not essential for the interaction between EGFR and c-Src (Fig. 7c, d). GRP78 is highly expressed on the cell surface in tissue samples of HCC Using immunofluorescence, we observed the cellular localization of GRP78 in ten cases of paired surgically resected HCC tissue samples. We found that GRP78 is expressed both in the cytosol and on the cell surface in 9 of 10 cases HCC tissue samples (Fig. 8c, d). By contrast, cell surface staining of GRP78 was not observed (7 of 10 cases) or very weak (3 of 10 cases) of paired peri-cancer tissue samples (Fig. 8a, b). Statistical chi-square test revealed that the difference is significant (p < 0.01). These data demonstrated that GRP78 is highly expressed on the cell surface of HCC.

Discussion
Cell surface GRP78 has been implicated in the progression of many human cancers including melanoma, breast, prostate cancer and hepatocellular carcinoma [9]. Lots of data have linked cell surface GRP78 to pro-proliferative and anti-apoptotic signaling pathway [33]. However, the role of cell surface GRP78 in tumor invasion and metastasis remains poor documented. In this article, we stimulated hepatocellular carcinoma cell lines PLC and QGY-7703 with α2M* and demonstrated that c-Src is the surrogate in cell surface GRP78 induced tumor invasion and metastasis. Cell surface GRP78 facilitated the phosphorylation of c-Src at Y416 via direct interaction.
Although many data have identified α2M* as a natural ligand of cell surface GRP78, the mechanism by which α2M* activates cell surface GRP78 remains unclear. Here, we found that α2M* did not augment GRP78 expression at transcriptional and translational levels. Based on these results, we hypothesized that elevated presence of GRP78 on the cell surface may be caused by GRP78 redistribution. In consistent with our hypothesis, we demonstrated that α2M* could induce GRP78 translocation from cytosol to plasma. Furthermore, we demonstrated that cell surface GRP78 is the surrogate of α2M* induced invasion and metastasis in HCC. It is the NH2terminal domain of GRP78 but not the COOH-terminal domain played critical role in tumor invasion and metastasis. Other reports have demonstrated the critical roles of COOH-terminal domain of GRP78 in the regulation of proliferation and apoptosis in many types of human cancers including melanoma, breast and prostate cancers [3,10,34]. For example, ligation of cell surface GRP78 with α2M* promotes the proliferation of cancer cells in prostate cancer cells, blockade of cell surface GRP78 using the antibody against the COOH-terminal domain of GRP78 reverted the pro-proliferative effect of α2M* [10,11,35,36]. Taken together, these data suggested that cell surface GRP78 regulated the invasion, metastasis, proliferation and apoptosis with different mechanism. That is the NH2-terminal domain of GRP78 is involved in the regulation of tumor invasion and metastasis. However, the COOH-terminal domain of GRP78 is involved in the regulation of proliferation and apoptosis of tumor cells.
Further investigation of potential downstream molecules of cell surface GRP78 revealed that cell surface GRP78 could interact directly with a~60KDa protein and augment its phosphorylation in QGY-7703 and PLC cells. Furthermore, we identified the~60KDa protein as c-Src. These raised the question of how cell surface GRP78 and c-Src interaction affected the invasion and metastasis of HCC. To answer this question, we analyzed whether pretreatment with PP2 could inhibit α2M* induced adhesion and invasion in QGY-7703 cells. We found that inhibition of the tyrosine kinase activity of c-Src inhibited α2M* induced adhesion and invasion. The inhibitory extents were paralleled in cells treated with PP2 with or without α2M* stimulation. We further found that blockade of cell surface GRP78 using the antibody against the NH2 terminal domain of GRP78 inhibited the phosphorylation of Src at Y416 and FAK at Y397. However, the antibody against the COOH terminal domain of GRP78 did not affect Src and FAK phosphorylation. These data further demonstrated that the NH2-domain of cell surface GRP78 is involved in the regulation of tumor invasion and metastasis upon α2M* stimulation. Conflicting results have been described regarding the role of the NH2-terminal domain of GRP78 in the regulation of cell proliferation and apoptosis. In NCCIT cells, blockade of Cripto binding to cell surface GRP78 using the antibody against the NH2terminal domain inhibited tumor cell proliferation, suggesting that the NH2-terminal domain of cell surface GRP78 also plays a pro-proliferative role in NCCIT cells [26]. This could be explained by the difference in ligands that bind with GRP78 [37,38]. This opinion was supported by the fact that binding of cell surface GRP78 with Par-4 played an pro-apoptotic role in prostate cancer [25,39].
We also found that ligation of cell surface GRP78 with α2M* triggered EGFR signaling pathway in a Src-dependent manner. Inhibition of the tyrosine kinase activity inhibited EGFR phosphorylation at Y1101 and Y845 induced by α2M* stimulation. However, treatment of PP2 did not inhibit the phosphorylation at Y1068, indicating that Src plays critical role in the maximal activation of EGFR in HCC. This raised the question of how c-Src may regulate EGFR activity induced by the association of cell surface GRP78 with α2M*. Other reports have demonstrated that Src could regulate EGFR activity by direct interaction. We found that association of cell surface GRP78 with α2M* facilitated the interaction between c-Src and EGFR. Furthermore, inhibition of the tyrosine kinase did not affect the interaction between c-Src and EGFR. However, inhibition of Src tyrosine kinase activity inhibiting the maximal activation of EGFR by preventing the phosphorylation of EGFR at Y845 and Y1101. These data were consistent with the role of c-Src in EGFR activation and suggested that both tyrosine kinase activity and protein binding function of Src are essential for cell surface GRP78 induced EGFR activation.

Conclusion
Our research revealed that c-Src plays a critical role in the invasion and metastasis of HCC induced by association of cell surface GRP78 with α2M*. Cell surface GRP78 directly binds and phosphorylates c-Src. As a consequence, c-Src phosphorylated EGFR, promoting the invasion and metastasis of HCCs.


extfieldsofstudy: Biology Medicine

text:
Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.

Introduction
Non-coding repetitive DNA includes diverse types of sequences which comprise a considerable portion of most eukaryotic genomes and their function is intensively investigated. In particular, mobile transposable elements are predicted to be a source of noncoding material and to influence evolution of gene regulatory networks [1]. Effects on gene regulation of another abundant class of non-coding repetitive DNA, tandemly repeated satellite DNA, was proposed based on the presence of promoter elements and transcription factor binding sites within some satellite sequences, as well as due to their transcriptional activity [2][3][4]. However, experimental studies revealing the influence of satellite DNA on the activity of genes are still lacking. This is mostly due to the specific organization of satellite DNAs which in the form of long arrays constitute gene-poor heterochromatin located in pericentromeric, centromeric and/or telomeric regions of chromosomes [5,6]. There are however several examples of satellite DNAs present not exclusively within pericentromeric heterochromatin but also dispersed as single repeats or short arrays in the vicinity of genes within euchromatin, such as the 1.688 Drosophila melanogaster satellite DNA [7] and two satellite DNAs from the beetle Tribolium castaneum [8,9]. Such dispersed organizational patterns might be formed due to intrastrand homologous recombination within satellite arrays [10], resulting in the formation of extrachromosomal circles [11] and their subsequent insertion throughout the genome [8,9]. Although satellite DNAs are mainly embedded in tightly packed heterochromatin which is perceived as transcriptionally silent, low expression of these sequences was reported in vertebrates, invertebrates and plants [2,12]. Satellite DNA transcripts were shown to be processed into small interfering RNAs (siRNAs), 20-25 nt in length, which are involved in the epigenetic process of heterochromatin formation in diverse organisms such as fission yeast Schizosaccharomyces pombe, nematodes, flys and plants [13][14][15][16]. The transcription of satellite DNAs is usually gender or stage specific and is often associated with differentiation and development or with certain stress conditions, particularly heat stress. In Arabidopsis, after exposure to prolonged heat stress, transcription of satellite DNA is significantly increased and is accompanied by heterochromatin decondensation [17,18]. Expression of human satellite III which is not detected under standard conditions is also induced by heat stress [19,20]. Increasing evidence on the reorganization of heterochromatin elicited by heat stress and other stress treatments could indicate a possible role for satellite DNA in a general stress response activated in cells to cope with harmful conditions [4].
The insect Tribolium castaneum has a major satellite DNA TCAST1 which comprises 35% of the genome and encompasses the pericentromeric regions of all chromosomes [21], but is also dispersed within euchromatin [8]. The expression of the satellite DNA TCAST1 is strongly induced by heat shock and proceeds in the form of long double-strand transcripts which are rapidly processed into 21-30 nt siRNAs [22]. Enhanced TCAST1 satellite DNA expression is accompanied by an increase in repressive epigenetic modifications of histones at satellite DNA regions in heterochromatin [22]. Now we propose that single repeats or short stretches of TCAST1 satellite DNA which are dispersed in the close vicinity of protein-coding genes within euchromatin are also subjected to such epigenetic changes induced by TCAST1 siRNAs. Since heterochromatin influences genes located in the vicinity [23,24], we expect that heterochromatic marks which might be established on dispersed TCAST1 repeats could affect nearby genes, most probably by inducing their silencing. The aim of the present study was to test the proposed hypothesis of a potential gene-regulatory role of TCAST1 satellite DNA. Our approach was to analyse insertion and copy number polymorphism of dispersed TCAST1 satellite repeats among T. castaneum strains and to examine if the presence/absence of TCAST1 repeats is correlated with the activity of adjacent genes under standard and heat stress conditions. In addition, we examined the epigenetic state of regions flanking dispersed TCAST1 satellite elements and correlated it with the expression level of nearby genes. Our results reveal for the first time a role for satellite DNA in the modulation of gene expression under the specific condition of long-term heat stress. In addition, it was shown that transient RNAi based establishment of heterochromatin at dispersed satellite elements and its spreading into proximal regions represents a mechanism responsible for the satellite DNA-mediated silencing of nearby genes.

Detection of dispersed TCAST1 elements polymorphic among strains
Bioinformatic analysis of T. castaneum genomic data revealed 68 elements homologous to the major TCAST1 satellite DNA, which are dispersed in the vicinity of protein coding genes [8]. There are two types of TCAST1-like elements: TCAST1 satellite-like elements in the form of partial or tandemly arranged monomers and TCAST1 transposon-like elements with a complex unit that resembles a non autonomous DNA transposon. In order to test the potential gene-regulatory role of dispersed TCAST1 elements we first analysed their insertion polymorphism among ten T. castaneum strains which originate from diverse geographic locations characterized by distinct environments. Polymorphism analysis was performed for 32 out of a total of 68 dispersed TCAST1 elements, 19 transposon-like and 13 satellite-like elements. For the remaining elements, flanking sequences were either not well characterized in GenBank or contained other repetitive sequences which made construction of single locus-specific primers impossible. The polymorphisms were associated with the following TCAST1 satellite-like elements: element 2 located within the first intron of a probable Ser/Thr kinase gene (GenBank accession number: 661947), element 12 located within the fifth intron of a putative uncharacterized protein gene (GenBank accession number: 100141521), element 21 in the second intron of WD repeat containing protein 47 gene (GenBank accession number: 657535) and element 46 located at an intergenic region: 5820 bp from 3' end of a putative uncharacterized protein gene (GenBank accession number 657017) and 7000 bp from 5' end of ribonucleoside-diphosphate reductase gene (GenBank accession number 657098). Schematic representation of genes associated with polymorphic TCAST1 elements 2, 12, 21 and 46 is shown on S1 Fig. Polymorphism of the TCAST1 element 2 is due to a difference in copy number of satellite repeats among T. castaneum strains: GA1, 61, 55, VT and GA2 containing a homozygous trimer, 43, 50, 52 and Zg contain a homozygous monomer, while strain 57 has mostly homozygous dimers but in rare individuals a trimer is also detected (Fig 1). Sequence analysis reveals that trimers and dimers are composed of two related repeats which belong to TCAST1 subfamilies Tcast1a and Tcast1b [20], respectively. TCAST1 element 12 is present as a homozygous dimer of two partial Tcast1a and Tcast1b monomers in the genome of GA2 strain and is completely absent from strains GA1 and 43 (Fig 1). TCAST1 element 21 in the form of a homozygous dimer is present in GA2 strain while strains GA1, 61 and 55 reveal its complete absence (Fig 1). TCAST1 element 46 in the form of a homozygous dimer is found in strains GA2, 55, 57, VT, 61, GA1, while it is completely absent in strain 43. Strains 50, 52 and Zg, due to heterozygosity, have both forms present (Fig 1).

Influence of TCAST1 elements on gene expression under standard conditions
To test the potential role of dispersed TCAST1 elements in the regulation of nearby genes we analysed expression of genes associated with TCAST1 elements that showed insertion polymorphism among T. castaneum strains. The purpose of the analysis was to determine if there is any difference in the expression of genes with respect to the presence or absence of TCAST1 elements in their neighbourhood (genes associated with elements 12, 21 and 46) or with respect to different copy number of repeats within particular TCAST1 element (gene associated with element 2). The gene associated with element 12 was expressed at an extremely low level in all strains and was eliminated from further analysis.
The expression of the WD repeat containing protein 47 gene was analysed by qPCR using primers designed for exons 4 and 5 (S1 Fig). Expression was analysed in strains with (GA2) and without (GA1 and 61) the gene-associated TCAST1 element 21 (Fig 2a). The mean No value (see Materials and Methods, section Quantitative real-time PCR analysis) of the gene in GA2 strain was 0.3205 ± 0.0145 relative to the mean No value of 0.3350 ± 0.0303 in the strains GA1 and 61 (P = 0.2671), as determined by the unpaired t-test, indicating no significant difference in expression between the two groups of strains. However, analysis of strain variation using the one-way ANOVA statistical test showed that genetic background plays a significant role in gene expression (P = 0.0448). Moreover, it seems that genetic background is dominating over the effect of the repetitive insert and at our level of statistical power this enables a firm conclusion regarding the influence of TCAST1 inserts on gene expression. Expression analysis of the ribonucleoside-diphosphate reductase gene in strains with (GA2, GA1 and 55) and Analysis of expression of a probable Ser/Thr kinase gene was performed in strains which contained the gene-associated TCAST1 element 2 as a trimer (GA1, 61, 55, GA2 and VT) and as a monomer (43, 52, 50, Zg), using primers designed for exons 1 and 2 as shown in S1 Fig. There was substantial variation in gene expression within strains containing a trimer and to a lesser extent within strains containing a monomer (Fig 2c). The mean No values for the gene associated with trimer and monomer were 0. 2345 ± 0.03197 and 0.2655 ± 0.01703, respectively, with a P value of 0.403, as determined by the unpaired t-test. The result shows no significant difference in the average expression of gene associated with trimer and monomer however using the one-way ANOVA statistical test again reveals that strain variation has a significant impact on gene expression (P = 0.0004).
These results reveal that under standard physiological conditions, when only influence of insert is considered, TCAST1 elements do not exhibit a significant effect on the expression of neighbouring genes. However, as strain variation is a significant and dominant factor in gene expression, we cannot make any firm conclusion about the influence of TCAST1 on gene expression.
Influence of TCAST1 elements on gene expression after heat stress a) Expression of genes associated with polymorphic TCAST1 elements. Heat stress (HS) influences expression of TCAST1 satellite DNA in T. castaneum and affects heterochromatin structure [22]. To check if dispersed TCAST1 elements affect the expression of neighbouring genes under heat stress conditions, we analysed expression of genes associated with elements 2, 21 and 46 in different T. castaneum strains after temperature stress at 40°C. A short heat stress of 2 h, as well as prolonged heat stress of up to 8 hrs, resulted in no significant change in the expression of tested genes (S2 Fig). To test if long-term heat stress has any influence on gene expression, we subjected adults of T. castaneum to 40°C for 24 hours, followed by recovery at 25°C. Under such conditions survival of adult beetles of all tested populations was almost 100%. The expression of TCAST1 satellite DNA was also checked after long-term heat We first assessed expression of WD repeat containing protein 47 gene in strains GA2 and 61, with and without the associated TCAST1 element 21, respectively. Gene expression was measured immediately after heat stress and after 30 min, 1 hr, 1.5 hrs and 3 hrs of recovery ( Fig  3a). In both strains, gene expression was slightly reduced immediately after heat stress and continued to decrease reaching approximately 60% and 50% of control activity in 61 and GA2 strain, respectively, after 30 min of recovery. However, after 1 hr of recovery, gene expression in strain GA2 continued to decrease being 3 times lower relative to the control, while expression in strain 61 returned to a level almost identical to the control. Approximately 70-80% recovery of gene expression in strain GA2 was observed after 1.5 hrs and almost complete recovery is observed after 3 hrs (Fig 3a). The difference in the expression of WD repeat containing protein 47 gene in strains 61 and GA2 at 1 hr of recovery after long-term heat stress was confirmed using qPCR with a gene TaqMan probe (Fig 3b). The gene expression at 1 hr of recovery after HS was also checked in strains 55 and GA1 which, like strain 61, lack element 21 (Fig 3b). Strains 61, GA1 and 55 exhibit no significant difference in gene expression at 1 hr of recovery after HS relative to the control without HS (P values >0.4), as determined by the unpaired t-test. In contrast, in the GA2 strain the level of expression at 1 hr of recovery after HS is 3.1 times lower relative to the control (P<0.0001). The results indicate an influence of TCAST1 element 21 on the expression of an associated gene during heat stress response: the satellite element suppresses activity and slows down the kinetics of gene expression recovery after heat stress. In strains without TCAST1 element 21 such as 55, GA1 and 61, the suppression of gene activity is lower and complete recovery of gene expression occurs 1 hr after HS, while expression of the same gene with inserted TCAST1 satellite element in GA2 strain is completely recovered 3 hrs after HS.
We analysed the activity of the ribonucleoside-diphosphate reductase gene in strains GA2, GA1 and 55 which have the TCAST1 element 46 located at the 5' end of the gene as well as in strain 43 where element 46 is completely absent. After long-term heat stress and a recovery period of 1 hr, expression of the tested gene revealed no significant downregulation in strain 43 (P = 0.0856), while in strains GA2, GA1 and 55 the downregulation is 3.4 x (P = 0.0001), 2.8 x (P = 0.0041) and 3.0 x (P = 0.0089), respectively, relative to the control without HS (Fig 3c), and reaches control level after 3 hrs. Therefore, the results were very similar to those for the gene associated with element 21: in both cases TCAST1 elements influence gene expression after long-term heat stress by repressing gene activity and slowing down the kinetics of gene expression recovery.
Next we analysed the activity of the gene associated with TCAST1 element 2 in strains GA2, 57 and 43. In strain GA2 element 2 is in the form of a trimer while in strains 57 and 43 it is in the form of a dimer and monomer, respectively. After a long-term heat stress of 24 hrs and recovery period of 1 hr, the gene associated with element 2 was downregulated in all strains (Fig 3d). The downregulation was 3.8 x (P = 0.0055) relative to the level before HS for the gene associated with satellite trimer, 3.1 x (P = 0.0075) and 2.4 x (P = 0.0047) for the same gene associated with dimer and monomer, respectively. The P values in all cases were calculated using the unpaired t-test.
b) Expression of genes associated with nonpolymorphic TCAST1 elements. To validate gene regulatory effect of TCAST1 satellite elements revealed by polymorphic elements 2, 21 and 46, we expanded our analysis to genes associated with TCAST1 elements which exhibited no insertion polymorphism (S1 Table). After exclusion of pseudogenes and genes with a very low level of expression, 17 genes were suitable for expression analysis. Of these, six genes were associated with TCAST1 satellite-like elements, carrying them in introns (elements 41, 59 and 67) or at 3' sites (elements 14, 15 and 30) while eleven genes were associated with complex TCAST1 transposon-like elements which were either located within introns (elements 11,13,23,27,52,57) or at intergenic regions (10,25,39,42). Expression of genes was examined at 1 hr of recovery after long-term HS at 40°C in the GA2 strain and the results revealed significant downregulation relative to the control without HS for all 17 genes (Fig 4). The level of downregulation with statistical significance expressed in P values, calculated by the unpaired t-test, is listed in Table 1 for all tested genes associated with TCAST1 elements, either polymorphic or nonpolymorphic. TCAST1 elements are classified as satellite-like and transposon-like elements and for each of them the position relative to the associated gene and distance from the transcription start site (TSS) of the associated gene is indicated as well as the number of TCAST1 satellite monomers contained within each dispersed element (Table 1). T. castaneum strains used in expression analyses are also indicated in Table 1.
The data reveal an average 1.90 ± 0.28 x downregulation of genes associated with TCAST1 satellite-like elements composed of a full or partial monomer. The downregulation occurs irrespective of the position of TCAST1 satellite element relative to the gene, either at intergenic regions or within introns. The level of downregulation shows statistically insignificant, negative correlation (Pearson correlation coefficient = -0.5076, P = 0.3826) for the distance of elements from the TSS within the tested range from 1.5 kb up to 39 kb. TCAST1 satellite-like elements which contain a dimer exhibit a stronger effect on associated genes relative to the previous group, with an average 3.00 ±0.32 x downregulation, while the effect of a trimer is even stronger (Table 1), indicating a positive correlation between TCAST1 copy number and the level of gene downregulation (correlation coefficient = 0.7487, P = 0.0127).
TCAST1 transposon-like elements contain approximately a single TCAST1 monomer and genes associated with them show an average downregulation of 2.30 ± 0.69 which is not significantly different from the downregulation exhibited by a satellite monomer (P = 0.1681) or dimer (P = 0.0586), as determined by the unpaired t-test. The influence is similar on genes located 5' or 3' relative to the TCAST1 transposon-like element, as shown for the two genes flanking element 10 ( Table 1). TCAST transposon-like elements are located within 7-35 kb from the TSS with the exception of a single element at 80 kb distance. The level of gene expression downregulation does not seem to be significantly affected by the distance of the elements from the TSS (correlation coefficient = -0.1984, P = 0.557). However, the lowest level of 1.5 x gene expression downregulation is observed for the most distant element (80 kb from TSS) indicating that at such distances effect of TCAST elements on gene downregulation might be reduced.
In conclusion, both TCAST1 satellite-like and TCAST1 transposon-like elements affect expression of associated genes during the recovery period of HS. The level of gene downregulation is positively correlated with the number of TCAST1 repeats within the elements, but it is not influenced by the position of TCAST1 element relative to the gene (5' or 3' end, intron) or by the distance from the associated gene, at least up to 35-40 kb from the TSS.

Mechanism of TCAST1-mediated gene regulation
Previous investigation revealed a transient increase of TCAST1 satellite DNA transcription after heat stress which correlated with increased di-and tri-methylation of H3K9 at TCAST1 satellite DNA regions [22]. Now we propose that TCAST1-derived siRNAs target, via sequence identity, not only tandemly repeated TCAST1 satellite DNA within heterochromatin but also dispersed TCAST1 elements within euchromatin. Therefore, an increased level of TCAST1 siR-NAs might possibly affect H3K9 methylation not only at tandemly repeated TCAST1 arrays within heterochromatin but also at dispersed TCAST1 elements and their flanking regions. Such "heterochromatinization" at dispersed elements and the spreading of heterochromatin marks to the neighbouring regions might consequently influence expression of nearby genes (Fig 5). In order to test this model we performed analysis of H3K9me2 and H3K9me3 levels within regions flanking dispersed polymorphic TCAST elements 2, 21 and 46 in different T. castaneum strains subjected to heat stress, using a chromatin immunoprecipitation (ChIP) assay. The H3K9me2/3 was analysed within regions 100-150 bp long, positioned within a few kb either 5' or 3' from the insertion site of TCAST1 elements. The position of primers used in ChIP experiments is indicated on S1 Fig. ChIP assay was performed on chromatin isolated from adult beetles subjected to long-term heat shock of 24 hr at 40°C, and the level of H3K9me3 and H3K9me2 was measured after 45 min and 1.5 hrs of recovery at 25°C and compared to the level of control (no HS) using the unpaired t-test. The level of H3K9me2/3 was also examined at TCAST1 repeats after long-term heat stress revealing a 100% and 80% increase of H3K9me3 after 45 min and 1.5 hrs of recovery, respectively, while in the same recovery periods the increase of H3K9me2 was lower, 30% and 50% respectively (S4a Fig). At a region 6.5 kb distant from the 3' end of TCAST1 element 46 in strain GA2 the level of H3K9me2 was increased by approximately 50% at 45 min after heat stress and remained constant during the recovery period of up to 1.5 hrs (Fig 6a; P = 0.0260). The level of H3K9me3 was almost 5 times higher relative to the control (no HS) after 45 min of recovery (P<0.001) and dropped to the level of control after 1.5 hrs (Fig 6a). The corresponding region in strain 43 which does not have an inserted TCAST element 46, was characterized with no significant change in the level of H3K9me2 (P = 0.4374) after HS and with only slight increase in H3K9me3 (P = 0.0709) after 45 min of recovery (Fig 6a). In strain GA2, within a region at the 5' end of TCAST1 element 46, a pattern of change of H3K9me2 and H3K9me3 after heat stress was very similar to the pattern observed at the 3' site, while the same region in population 43 revealed no considerable change (S4b Fig). At a region located 400 bp from the 5' end of TCAST1 element 21 in strain GA2 the level of H3K9me2 was increased 45 min after HS, reaching a maximal two-fold increase relative to the control (no HS) after 1.5 h of recovery (Fig 6b; P = 0.0053). The level of H3K9me3 was also increased but with different dynamics reaching a maximal two-fold increase after 45 min of recovery and 50% increase after 1.5 hrs (Fig 6b; P = 0.0018). The corresponding region in strain 61 which does not contain a TCAST1 element, revealed no significant increase in H3K9me2 (P = 0.1474) as well as in H3K9me3 (P = 0.3844) relative to the control (Fig 6b). A region 800 bp distant from the 5' end of TCAST1 element 2 in strain GA2 revealed an increase of 70% in the level of H3K9me2 at 45 min of recovery which remained constant up to 1.5 hrs of recovery (Fig 6c; P = 0.009). The same region in strain 43 where element 2 is in the form of a monomer showed a lower increase of 40% which was also constant during the recovery period (Fig 6c;  P = 0.9359). The level of H3K9me3 at a region proximal to element 2 in both strains was similar and reached a maximal increase of 80% relative to the control after 45 min of recovery (P = 0.009 for trimer, P = 0.0144 for monomer) and after 1.5 hrs dropped to the level close to the control (Fig 6c).
The results reveal that exposure of adult beetles to heat shock is characterized by the enrichment of H3K9me2 and H3K9me3 at regions flanking both 5' and 3' to dispersed TCAST elements. The H3K9me2/3 increase was detected at regions within 1 kb distance from dispersed TCAST1 elements as well as at a region distant 6.5 kb. However, the same regions in strains which do not have TCAST1 elements inserted in the vicinity, do not exhibit a significant change in the two histone marks after long-term heat stress treatment. This reveals an essential role of dispersed TCAST1 elements in the accumulation of H3K9me2/3 marks and their spreading to proximal regions after heat stress. The peak of H3K9me3 was observed after approximately 45 min of recovery period and dropped down after 1.5 hrs of recovery to a level similar to that of the control, while the level of H3K9me2 remained increased after 1.5 hr of recovery. The dynamics of change of the specific heterochromatin marks H3K9me2 and H3K9me3 within regions proximal to dispersed TCAST elements is consistent with the observed change in the expression of a neighbouring gene: maximal increase of both histone marks is detected within the first hour after heat stress which coincides with the maximal repression of a neighbouring gene after 1 hr of recovery. After 1.5 hrs of recovery H3K9me3 is restored close to the control level, while H3K9me2 still remains increased resulting in approximately 80% of recovery of gene expression. The results indicate that satellite DNA-associated siRNAs, transiently activated after heat shock, affect the epigenetic state of dispersed TCAST1 satellite elements inducing their "heterochromatinization". In addition, heterochromatin marks are spread to the regions flanking dispersed satellite DNA elements. The transient increase in H3K9me3/2 levels induces partial repression of genes located in the vicinity and confirms the proposed model of gene regulation mediated by satellite DNA (Fig 5).

Discussion
Our present study shows that increased transcription of abundant TCAST1 satellite DNA in the beetle T. castaneum, which occurs after long-term heat stress, correlates with an increased level of repressive heterochromatin marks H3K9me2 and H3K9me3 not only on satellite repeats in constitutive heterochromatin but also on dispersed TCAST1 elements located in euchromatin. A temporary increase of heterochromatic marks on dispersed TCAST1 elements and their flanking regions correlates with the suppression of activity of nearby genes. This is the first report on satellite DNA elements influencing expression of nearby genes and confirms our previous hypothesis regarding the gene-regulatory role of satellite DNAs [4]. Aside from satellite DNAs, there are many studies on transposons which affect expression of neighbouring genes by spreading heterochromatin marks, as reported for diverse plants [25][26][27] and mammals [28]. In Drosophila, transposable elements dispersed within euchromatin were shown to trigger formation of H3K9me3 islands which are established through the action of the piRNA pathway, and influence expression of neighbouring genes [29]. In T. castaneum, the effect of dispersed TCAST1 satellite elements on nearby genes was not detected under standard physiological conditions as well as after short-term heat stress, although the short-term heat stress induces increase of TCAST1 siRNA levels [22]. However, restoration of the constitutive level of TCAST1 transcripts occurs at 30 min after short-term heat stress while after long-term heat stress the restoration takes approximately 3 hours. Long-term heat stress also results in complete cytosine demethylation of TCAST1 satellite DNA, while demethylation after short-term heat shock is only partial [30]. It could be proposed that increased TCAST1 satellite DNA expression and accumulation of TCAST1 siRNAs which occurs during a few hours after longterm heat stress is a prerequisite for the establishment of epigenetic changes on dispersed TCAST1 elements and their flanking regions in the form of H3K9me2/3 enrichment. This results in the repression of a proximal gene which occurs regardless of TCAST1 insertion upstream, downstream or within the intron and the level of repression seems to be constant up to a distance of 40 kb from the transcription start site (TSS). However, it cannot be excluded that in addition to H3K9me2/3 enrichment some other epigenetic changes occur within the same chromatin regions after long-term heat stress and also affect activity of nearby genes. TCAST1 satellite DNA repeats dispersed within euchromatin are distributed equally on all chromosomes [8]. Analysis of distances of TCAST1 elements from TSS of associated genes based on published data [8], shows that 70% of them are within range of 40 kb, while only 10% are above 100 kb (S5 Fig). This indicates that most of these TCAST1 elements are located within distances at which their effect on the expression of neighbouring genes is maximal. Such a distribution of elements with the majority of them influencing genes may also indicate that the effect of TCAST1 on genes is probably not deleterious, but may be either neutral or beneficial. Polymorphism analysis of 32 TCAST1 elements, 19 transposon-like and 13 satellite-like among ten populations revealed insertion polymorphism in four satellite-like elements while transposon-like elements were fixed at 100%. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum populations and might have some impact on adaptation of populations to different environmental conditions. Geographical populations of D. melanogaster exhibit variation in thermal sensitivity which is partially due to regulatory variation exerted by repetitive elements abundant in the Y chromosome [31].
What might be the physiological consequence of the temporary change in activity of TCAS-T1-associated genes after long-term heat stress? In order to answer this question, it would be necessary to understand the function of TCAST1-associated genes and the biological processes in which they are involved. Although many genes in T. castaneum are not well annotated, previous analysis of TCAST1-associated genes showed a statistically significant overrepresentation of immunoglobulin-like genes within this gene set [8]. The molecular function of most of these immunoglobulin-like genes is unknown and they might be involved in different biological processes such as cell adhesion, protein phosphorylation and axon guidance. Based on iBeetle-Base (http://ibeetle-base.uni-goettingen.de/) [32] which contains RNAi phenotypes in T. castaneum, knock down of almost 45% of TCAST1-associated genes causes severe developmental defects either in embryogenesis or metamorphosis and affects survival. It is therefore very probable that the modulation of activity of TCAST1-associated genes induced by environmental factors such as long-term heat stress might influence development of T. castaneum. It is also important to mention that since different TCAST1-associated genes are modulated in parallel, their effect on biological processes could be cumulative.
It can be also hypothesized that the temporary suppression of TCAST1-associated genes after long-term heat shock might influence stress recovery process. Heat shock is a substantial stress to cells which immediately respond by activation and expression of heat shock proteins (HSPs) that act as molecular chaperons involved in restoration and maintenance of proteins [33]. Another possible response to heat stress might be downregulation of expression of most of genes except HSPs which is observed after heat stress, presumably leading to the reduction of damaged proteins in heat stressed cells [34]. Since heat stress provokes a number of remarkable changes inside the cell including massive destruction and reorganization of cellular components [35], the process of cell recovery after heat stress is complex and includes sequential restoration of gene expression. We propose that dispersed TCAST1 satellite DNA elements, by modulating the activity of the neighbouring protein-coding genes within a short time frame after long-term heat shock, might contribute to the recovery of cells and organism from harmful heat-stress conditions.
Further studies are necessary to see if transient suppression of genes by dispersed satellite DNA elements is a general phenomenon or it is only limited to TCAST1 elements which are very abundant within pericentromeric heterochromatin and whose expression within heterochromatin is significantly affected by heat stress. To answer this question other minor satellites in T. castaneum will be examined and their effect on gene expression checked as well as satellite DNAs in other model organisms.

Beetle strains
The following strains of T. castaneum were used: GA2 strain, originally used in the genome sequencing project and deriving from North American wild-type strain collected in

DNA extraction and PCR analysis
DNA was extracted from 50 to 100 mg of adult insects, which corresponds to 10-20 individuals, using the DNeasy Blood & Tissue Kit (Qiagen). DNA concentration was measured using the Quant-iT dsDNA assay kit and the Qubit fluorometer (Invitrogen) and the average amount of DNA isolated using the DNeasy kit ranged from 30 ng/μl to 60 ng/μl. Primers used in the analysis of polymorphism of 32 TCAST1 elements where designed in a unique sequence around the insert (upstream and downstream from insert) using Primer3Plus software and are listed in S1 Table. It was not possible to design primers for 36 TCAST1 inserts due to poor annotation in GenBank (unique sequence necessary for designing primers was missing) or enrichment of neighbouring sequence with repetitive elements other than TCAST1 elements. PCR reactions were performed using premade 2 x DreamTaq Green PCR Master Mix (Fermentas) in a final reaction volume of 20 μl containing 0.2 μM of each primer and 50 ng of genomic DNA. The following reaction conditions were used for the amplification: 94°C for 1 min, 10 cycles at 94°C for 30 s, 60°C for 30 (with annealing temperature declining in each cycle for 0.25°C) and 72°C for 1 min followed by 20 cycle at 94°C for 30 s, 55°C for 30 s and 72°C for 90 s. PCR amplification products were subjected to 1.2% (w/v) agarose gel electrophoresis and if PCR product size differed from the expected one (insert was absent or present in different copy number), a band was cut from the gel, DNA isolated using QIAquick gel extraction kit and sequenced using an ABI Prism 310 (Applied Biosystems).

RNA isolation and reverse transcription
RNA used in the analysis of gene expression was isolated from Tribolium adult beetles (approximate weight of sample 20 mg) using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. In strain 57, due to individual polymorphism of TCAST1 element 2, RNA was isolated from single individuals. RNA was additionally digested with Turbo DNase (Ambion), quantified with the Quant-IT RNA assay kit using Qubit fluorometer (Invitrogen), quality-checked on gel, and PCR-checked for the presence of TCAST DNA. RNA (approximately 1 μg) was reverse transcribed using PrimeScript RT-PCR Kit (Takara) in 10 μl reaction using random primers. Negative controls without reverse transcriptase were used for all samples.
Quantitative real-time PCR (qPCR) analysis cDNA samples were amplified using Applied Biosystem ABI7300 Real-Time PCR System. The qPCR reactions were done in triplicate, in 50 μl reaction volume with 0.5 μM specific primers, 2X Power SYBR Green PCR Master mix (Applied Biosystems) and 30 ng of cDNA. Primers for expression analysis of TCAST1 associated genes are listed in S1 Table, while primers for expression analysis of TCAST1 satellite DNA are: F CCATAAGCGAGTTATAGAGTTGG and R CTTTAGTGACTTTTATGTCTTCTCC. To correct for the differences in sample composition and in the yield of the reverse transcription reaction, ribosomal protein S18 (RPS18) was used for normalization [36,37]. The thermal cycling conditions were as follows: 50°C 2 min, 95°C 7 min, 95°C 15 s, 60°C 1 min for 40 cycles followed by dissociation stage: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s. TaqMan probe for gene associated with element 21 as well as TaqMan Gene Expression Assay were obtained from Applied Biosystems. Amplification specificity was confirmed by dissociation curve analysis. Specificity of amplified product was additionally tested on agarose gel. Control without template (NTC) was included in each run. Post-run data were analysed using LinRegPCR software v.11.1. [38,39]. The software enables calculation of the starting concentration of amplicon ("No value") which is expressed in arbitrary fluorescence units and is calculated by taking into account PCR efficiency and baseline fluorescence. "No value" determined for each technical replicate was averaged. Averaged "No values" for gene of interest were divided by "No values" of endogenous control for normalization. Statistical analysis of qPCR data between populations was done using GraphPad v.6.01. Normalized No values were compared using unpaired t-test which compares the mean of two unmatched groups. Strain variations were analysed using one-way ANOVA test.

Chromatin immunoprecipitation
Adult beetles were frozen and homogenized in Nuclear Isolation buffer (10 mM MOPS; 5 mM KCl; 10 mM EDTA; 0.6% Triton X-100) containing protease inhibitor cocktail Complete Mini (Roche). Un-homogenized tissue was removed by centrifugation at low rpm for 1 min at 4°C. Formaldehyde in a final concentration of 1% was added to supernatant, followed by incubation for 10 min at RT and addition of 0.136 M glycine. Nuclei were pelleted by centrifugation, resuspended in Nucler Lysis buffer (50 mM Tris-HCl pH 7.7; 1% SDS; 20 mM EDTA) and sonicated 8 times for 20 s on ice, centrifuged at max speed for 3 min at 4°C. 10% of chromatin was stored for input fraction. The remaining supernatant was incubated for 3 h at 4°C with pre-prepared solution of Protein A-coated paramagnetic beads (Dynabeads 1 Protein A, Invitrogen) and antibodies in ChIP dilution buffer (1% Triton X-100; 10 mM EDTA; 15 mM Tris-HCl pH 7.5; 300 mM NaCl). The immune complex was subsequently washed five times in ChIP dilution buffer, beads were captured by magnet and the immuno-complex was eluted by centrifugation for 10 min at 65°C in elution buffer (1% SDS; 10 mM NaHCO 3 ). The reversal of cross link was performed by addition of 20 mM EDTA and 0.2 M NaCl and incubation overnight at 65°C. The material was digested with proteinase K (200 μg/ml) and DNA was extracted by phenol: chloroform: isoamyl alcohol (25:24:1). The following antibodies were used: H3K9me3 (Abcam, ab8898) and H3K9me2 (Abcam, ab1220). Binding of precipitated target was determined by qPCR using the SYBR Green PCR Master mix, primers listed in supplementary S1 Table and reaction conditions described above. The No value was normalized using No value of input fraction.  Table. List of primers used for the analysis of polymorphism of dispersed TCAST1 elements, for the expression analysis of TCAST1-associated genes and for the analysis of histone methylation within regions flanking polymorphic TCAST1 elements. (DOCX)


A Novel Cell Substrate Candidate for Rabies Virus Vaccine Propagation and Production

Rabies is worldwide endemic fatal viral encephalitis, which continues to be a major public health and veterinary problem in many parts of the world [1,2]. Rabies is an under-reported, neglected and deadly disease estimated to cause more than 50000 human deaths annually and millions of animal bites each year [3]. Domestic animals, mainly dogs and cats, or wild animals (bats, monkeys, and foxes) are important virus reservoirs that transmit this disease to human and animals of great economical interest, such as cattle and horses [4].

Introduction
Rabies is worldwide endemic fatal viral encephalitis, which continues to be a major public health and veterinary problem in many parts of the world [1,2]. Rabies is an under-reported, neglected and deadly disease estimated to cause more than 50000 human deaths annually and millions of animal bites each year [3]. Domestic animals, mainly dogs and cats, or wild animals (bats, monkeys, and foxes) are important virus reservoirs that transmit this disease to human and animals of great economical interest, such as cattle and horses [4].
Once clinical rabies symptoms are evident, the prognosis for survival is poor and death is almost inevitable. The population at risk includes 2.5 billion people currently living in regions in which rabies is endemic. Half of the victims of dog bites and subsequently rabies and death are children younger than 15 years of age [5]. Clearly, greater efforts should be made to improve the control of rabies, a zoonotic disease with the highest rate of fatality cases known to humans. One of the most important elements in the effective control of human rabies is the use of efficacious vaccines.
Since the introduction of the first vaccine by Pasteur in 1885, many rabies vaccines were developed using various cell substrates including animal brains, avian embryos, primary mammalian cells, human diploid cell and Vero cells. Although rabies vaccine was also produced in mammalian neural tissues over 100 years ago, the vaccines that were produced in cell culture and embryonated eggs have dramatically decreased human deaths throughout the world [6][7][8][9].
Using neural tissue vaccine (NTV) was very common in the past and this type of crude vaccine was improved over the years, but remained associated with serious adverse events and severe neurological reactions, because of the production of antibodies against myelin or other substances of the animal nervous tissue present in the product. Hence, the use of NTV was phased out slowly as recommended by the World Health Organization (WHO) [10].
Many different cell culture systems have been used extensively in research, diagnostics and the manufacturing of biological products including vaccines. The use of cell lines allowed largescale production of vaccines using micro-carriers and cultures in bioreactors [11]. Despite supporting studies reporting the safety of Vero cell as a cell substrate for vaccine production [12][13][14], other reports claim contamination of some cell lines including Vero cell used for vaccine production with adventitious agents [15]. Therefore, a continuous search for an appropriate cell substrate for the production of vaccines remains useful.
Currently, stem cells have gained increasing attention in research because of their promising applications in medicalclinical as well as developmental biology. Stem cells have selfrenewal capacity and the ability to provide differentiated and undifferentiated cells that allow a diverse range of specialized cells and tissues. They can be obtained from different tissues including the dental pulp [16][17][18], and the cultivation these cells under different conditions has been established [19][20][21]. So far no report to date has documented the use of stem cells in research or for the production of rabies vaccine. In this paper we describe a novel cell substrate, stem cell line derived from dental pulp (RHDP), for the production and quality control of rabies virus vaccine. We show that rabies virus could be adapted to these cells and harvested at high titers.

Rabies virus strain
Rabies Pasteur strain (PV) adapted to Vero cells was provided by WHO collaborating center for references and research on rabies, Pasteur Institute, Tehran, Iran.

Cell culture
MRC-5 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco BRL) supplemented with 8% fetal bovine serum (FBS, Gibco BRL) and kanamycin-neomycin (Biosera, United Kingdom) as previously described [22]. Tissue culture flasks (175 or 25 cm 2 ) were seeded by MRC-5 at 1×10 5 cells/ ml and incubated at 37°C. The flasks were sub cultured when confluency was reached, normally every 6-7 days. For further subculture, the cell monolayer was washed with phosphatebuffered saline (PBS) followed by 0.025% trypsin to suspend cells and subculture at appropriate concentration. The RHDP cells were cultured according to the same protocol, except that we used 5% fetal calf serum instead of 8% and sub cultured every 4 days.

Cell counting by hemocytometer
Cell counting was performed by hemocytometer [22]. Briefly, a flask containing a monolayer cell culture was trypsinized and suspended by pipetting to obtain a homogenized cell suspension. 50 µl of cell suspension was mixed carefully with 50 µl of 0.4% Trypan blue in a clean micro tube. A sample of stained cells was added to the counting chamber of a hemocytometer slide (Improved Neubauer, HBG, Germany). The cells were counted on a microscope and the cell concentration was calculated.

Infection of cell monolayer with rabies virus:
Virus inoculation was performed according to the modified Koprowski method [9]. Briefly, the culture medium was removed and the cell monolayer was washed with PBS, then the virus seed was applied on a monolayer in a minimal volume of DMEM to cover the monolayer at a multiplicity of infection (MOI) of 0.1 and incubated at 37°C for 45 min. Finally, DMEM media supplemented by 0.2% human albumin was added and incubated at 32-33°C. The virus was harvested at different times post infection according to the experimental design.

Infection of cell suspension with rabies virus
Virus inoculation was performed according to the modified Koprowski method [9]. Briefly, freshly trypsinized cells suspension were inoculated with virus seed at a MOI of 0.1 and incubated at 37 °C for 30 min. Then the virus propagation DMEM media supplemented by 5% FBS was added and incubation was continued at 37°C for 4 and 2 days for MRC-5 and RHDP cell lines, respectively. After washing the cell monolayer twice with PBS, sufficient virus propagation media was added to the flask and incubated at 32-33°C. The virus was harvested at different times according to the experimental design.

Rabies Virus titration
Virus titer in the infected cultures was determined according to the modified Fluorescent antibody staining technique described by Kaplan [9] and expressed in Fluorescent Focus Unit per ml (FFU/ml). Briefly, serial dilutions of virus were prepared and an aliquot of each dilution (100µl) was transferred to each chamber of a Slide Chamber (Lab Tech) followed by 200 µl of media and 100 µl of cell suspension. Slide chamber was incubated at 36-37°C for 48 hours then subjected to staining. All chambers were washed twice with PBS and after fixation of cells in acetone, the chambers were stained with anti rabies nucleocapsid conjugated antibody. Slides were examined by fluorescent microscope for presence of the Negri bodies.

Cell growth kinetics
The cell growth kinetics was calculated according to the methods used by Samia Rourou [23,24].
The Specific Cell Growth rate, μ (h-1) was estimated using equation 1: Where X represents the viable cell density per ml, t represents the time points of sampling expressed in hour, the subscripts n and n-1 stand for two successive sampling points.
Cell Division Number, Cd was calculated according to the equation 2: The virus growth kinetics was calculated according to the methods used by Samia Rourou [23,24].

Virus growth kinetics
Specific Virus Production Rate, P was calculated according to equation 3: Where C n+1 and C n represent virus titer in FFU/mL at time t n and t n+1 , respectively. X n and X n+1 represent cell density level at time t n and t n+1 , respectively.
The Overall Virus Productivity was calculated according to equation 4: Where X infection and X end represent the cell density level at the time of infection and at the end of culture, respectively. C stands for the virus titer in FFU/ml obtained at the end of the culture. Infection period corresponds to the duration of the virus production phase in hours.

Cell infection monitoring
Cell lines were seeded in a standard Slide Chamber and infected by 100 µl of virus seed and incubated at 36°C for 48 hrs. The slide was then fixed with 80% cold acetone and stained with fluorescein-labelled anti-rabies nucleocapsid antibody.

Statistical evaluation
The statistical analyses were done by Excel software.

Cell growth kinetic
To find out the kinetic of cell growth, we investigated the cell growth rate, measuring the total cell number in 25 cm 2 flasks within a defined time schedule. 24 flasks were cultured by either MRC-5 or RHDP cell line according to the above mentioned methods and measured the total cell count in three flasks separately at 24-hour intervals.
The results showed that MRC-5 had a higher growth in terms of total number of cell in each flask compared with RHDP. The total cell number per 25cm 2 flask, when the monolayer was completed were 4.2x10 6 ±1.5x10 5 and 2.1x10 6 ± 2.6x10 5 for MRC-5 and RHDP, respectively (Table 1).
However, the RHDP reached its maximum density sooner than MRC, as shown in Figure 1 (at day four in RHDP compared with day 8 in MRC-5).
Different parameters such as specific growth rate as well as cell division number can give us much more important information on cell behavior in culture system. So we calculated these parameters for both cell lines based on data obtained from the cell growth curve.
Specific growth rate in RHDP (which reflected the cell growth per time unit) was higher than MRC5 at all sampling time during the 8 days of culture. The specific growth rate in RHDP was at its maximum level (0.034±0.011 μ [h-1]) on the first day of culture, whereas this parameter reached its maximum level (0.010 ± 0.001 μ [h-1]) 3-4 days after starting culture in MRC cell. It means that the RHDP grew more rapidly than MRC5 (Figures 2a  and 2b).
The cell division number (Cd) on the first day of culture for MRC and RHDP cells were 0.37±0.04 and 0.70±0.19, respectively. Similarly, the Cd on the last day of culture was 1.16±0.04 and 1.53±0.11 for MRC and RHDP cells, respectively. Furthermore, the Cd in RHDP significantly increased on day three of culture and with no significant changes for the rest of the culture period. For MRC-5, the Cd increased significantly until the sixth culture day and with no any changes then after ( Table 1, Figures 2c and  2d).

Cell infection monitoring
The rabies virus (PV strain) underwent passage on monolayers of RHDP cell line in 25 cm 2 tissue culture flask. One slide chamber was prepared in parallel at each passage and stained for microscopic investigation. The slide chambers were stained with fluorescein-labelled antirabies nucleocapsid antibody.
In the first virus passage on the cell substrate, limited focuses were seen but the number of these focuses increased in subsequent passages and massive focuses were observed in the sixth passage (Figure 3).

Virus growth kinetic
To study the effect of the cell substrate on virus productivity, a sufficient number of cell culture flasks were provided and inoculated with virus seed according to the monolayer method. Samples were withdrawn at 24-hour intervals and cell concentrations as well as virus titer were measured in the samples. The specific virus production rate and the virus productivity were calculated using data obtained from the above mentioned methods (Table 2).
MRC-5 cell densities were 4.0*10 5 ±1.0*10 4 and 4.3*10 5 ± 2.1*10 4 per ml at 24 and 144 hours after initiating the virus inoculation, respectively. An almost constant cell concentration was seen and no significant changes in cell density were observed during the 6 days of cell infection (P˃0.05) (Figure 4a). However, a constant increase in virus titer was noted during the incubation period. The highest virus titer was reached on the sixth day. The specific virus production rate was 0.0028 FFU/ cell/h and 3.625 FFU/cell/h on day one and five, respectively (Table 2 and Figure 4b).
In contrast, the RHDP cell densities were 1.8*10 5 ± 2.1*10 4 and 0.47*10 5 ±1.5*10 4 per ml, 24 and 144 hours after initiating the virus inoculation, respectively ( Table 2). We found a significant decrease in cell density during the six days of virus propagation, starting from day 4 (P˂0.05). At the end of the infection phase, almost all cells had experienced lysis or were rounded and detached, because of the cytopathy effect (CPE) of rabies virus on RHDP cell substrate.
After cell infection, a sharp decrease of cell density with a concomitant secretion of the virus into the culture medium was observed in the RHDP cell line. The maximum virus titer was reached 4 days post infection. The specific virus production rates were 0.0092 FFU/cell/h and 5.2431 FFU/cell/h on day one and four, respectively (Table 2 and Figure 4c, 4d).
The Mean virus productivities were 0.0372±0.0231 and 0.1326±0.0375 for MRC and RHDP cell respectively, 48 hrs after inoculation. This parameter reached its maximum level on the fourth day of culture then decreased dramatically in both cell substrate as shown in figures 4b and 4d. The maximum level of virus productivity was 3.4826±2.9216 and 2.4682±0.0351 for MRC and RHDP cells, respectively ( Table 2).

Discussion
Vaccination as a deliberate attempt to protect humans against infectious diseases has a long history; although only in the 20th century did the practice flourish into the routine vaccination of large population. Rabies is an acute viral-encephalitis that is transmitted from animal to animal or from animal to human by exposure to saliva. All mammalian species are believed to be susceptible to rabies. In an area in which animal control programs are not extensively developed, dogs and cats account for most of the reported rabid animals and cause most (90%) of the human rabies exposures. Wildlife is the main reservoir of the rabies virus [25,26]. Human infection with the virus occurs often after a bite from a rabid animal. However, exposure through the inhalation of the virus or through the transplantation of infected corneas have also occurred [27].
One of the most important elements to effectively control human rabies is using an efficacious vaccine. Intensive efforts were exerted, world-wide, to produce cost-effective vaccines that meet or improve the levels of safety and efficacy achieved with human diploid cells [6,25,28].
Animal cell culture was established as a manufacturing technology for the preparation of viral vaccines since the late 1940s [23]. However, the use of cell substrate presents several disadvantages such as the potential to induce hypersensitivity, batch variability, and possibility of contaminants with adventitious agents, heterogenic proteins, bovine viruses, prions, mycoplasma, DNA, and other components. Therefore, the requirements for the use of animal cells as in vitro substrates for the production of biologicals were published by the WHO [29].
However, there are some safety issues using cell lines including Vero cell [15]. Moreover, because of absence of rabies CPE on cell substrate, different techniques such as titration in mice and staining with fluorescence conjugated rabies virusnucleocapsid-antibodies are applied for virus titration. Some of these methods are expensive and/or time consuming.
In this study, we investigated the characteristic of the RHDP cell line (a cell line derived from human dental pulp stem cell) in terms of growth in stationary condition and use as a substrate for the propagation of rabies virus, compared with MRC-5 cell substrate. Also we evaluated the potential application of this cell line for vaccine production as well as using it in developmental research.
Both MRC-5 and RHDP cell lines were evaluate according to their growth parameters as well as virus propagation characteristics. We found that MRC-5 had a higher growth rate in terms of total cell number in each flask compared with RHDP. The total cells per a 25cm 2 flask with a confluent monolayer, were 4.2*10 6 ±1.5*10 5 and 2.1*10 6 ±2.6*10 5 for MRC-5 and RHDP, respectively, but the RHDP reached its maximum density sooner than MRC-5.
The specific growth rates were 0.034±0.011 μ (h-1) and 0.010±0.001 μ (h-1) in RHDP and MRC-5, respectively. Maximum specific growth rate in RHDP was on day one of subculture and therefore, the lag time was very short. Meanwhile, the maximum growth rate in MRC-5 was observed on days 3 and 4 of culture, showing a relatively long lag phase. It can be concluded that RHDP grows more rapidly than MRC-5. However, the specific growth rate in RHDP was higher than those reported in other studies on Vero cell [23].
The Cd plot was also consistent with these findings. Cd was 0.7±0.19 on day one after starting the culture and reached 1.53±0.19 on day 3 and fixed at this level. Therefore, for expanding the RHDP cell in an optimum condition, sub culture should be made every 3-4 days to obtain the maximum cell in a defined period of time.
RHDP is highly sensitive to rabies virus and as shown in figure 3, the virus was well adapted to this cell substrate in only 6 passages. Rabies virus can cause cell lysis and develop a CPE in RHDP cell monolayer which can be easily observed by light inverted microscope. The CPE is characterized by cells granulation and rounding, cell lysis and finally cell detachment.
This ability can make the RHDP as a cell substrate which can potentially be used for virus titration.
Recent evidence suggest that the dental pulp stem cells are thought to be mesenchymal stem cells, and able to differentiate into a wide variety of other cell and tissue types including neural cells, adipocytes, osteoblasts, chondrocytes [30]. Therefore, special attention should be for using RHDP as a cell substrate for vaccine production.
A significant decrease in cell density after virus inoculation was observed in RHDP cell line and it was due to cell lysis which is concomitant with virus release in media. The RHDP cell densities 24 and 144 hours after initiating the virus inoculation were 1.8*10 5 ±2.1*10 4 and 0.47*10 5 ±1.5*10 4 per ml, respectively. No significant changes in cell density were observed during the virus propagation on MRC-5 cell substrate. Only a slight change included limited and small granule in some cells or cell inflation in some cases at sixth inoculation day were observed.
The maximum specific virus production rate in RHDP was higher than MRC-5 and was 5.243 FFU/cell/h for RHDP compared with 3.625 FFU/cell/h for MRC-5 and in both cases was less than those reported for Vero cell cultured in suspension condition [23].
Maximum virus productivities were 3.4826±2.9216 and 2.4682±0.0351 for MRC-5 and RHDP cells, respectively, both observed on day four. Cell lysis in RHDP had caused such decrease in virus productivity compared with MRC cell line. The ability to amplify and support a virus replication in a cell substrate is a valuable indicator for evaluating the cell, to select it as a suitable substrate for vaccine production or further study. Virus Productivity can reflect this ability in cell substrate which can easily be calculated base on cell and virus growth kinetics. Higher Virus Productivity means more virus particle produced by a single cell.
Using micro carrier for mass production of Vero cell as well as stem cell by fermentor has been reviewed by many researchers and well documented [19][20][21] and can be used for RHDP cultivation in large scales to increase the virus titer.
In conclusion, in this study a new cell substrate (RHDP) was presented which can be used for propagation of rabies virus and vaccine researches. All essential elements such us cell kinetic as well as virus growth properties on both, RHDP and MRC-5 cell substrates were investigated. To the best of our knowledge, this is the first report on a stem cell derived cell line (RHDP), used for rabies virus propagation. Nevertheless, further studies are still needed to improve the culture conditions and increase production using a bioreactor. Also more studies are essential to introduce a rabies virus titration method base on RHDP cell culture.