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Published as doi: 10.1096/fj.06-7799com.
 (The FASEB Journal. 2007;21:2247-2256.)
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Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS

Jie Liang, Yanglin Pan, Dexin Zhang, Changcun Guo, Yongquan Shi, Jingbo Wang, Yu Chen, Xin Wang, Jie Liu, Xuegang Guo, Zheng Chen, Taidong Qiao and Daiming Fan1

State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an, China

1Correspondence: State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, 15 West Chang-Le Rd., Xi’an, Shaanxi Province, China. E-mail: fandaim{at}fmmu.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The function of cellular prion protein (PrPC), the essential protein for the pathogenesis and transmission of prion diseases, is still largely unknown. The putative roles of PrPC are thought to be related to cell signaling, survival, and differentiation. In a previous study, we showed that PrPC was overexpressed in gastric cancer tissues. In the present report, we show that ectopic expression of PrPC could promote tumorigenesis, proliferation, and G1/S transition in gastric cancer cells. Furthermore, CyclinD1, a protein related to cell cycle, was shown to be significantly up-regulated by PrPC at both mRNA and protein levels. PI3K/Akt pathway mediated above PrPC signal since PrPC increased the expression of phosphorylated Akt, and the specific inhibitor of Akt, LY294002, could markedly suppress growth of SGC7901 and transactivation of CyclinD1 induced by PrPC. Octapeptide repeat region played a vital role in this function, as deletion of this region abolished or reduced these effects. Collectively, this study demonstrates that overexpression of PrPC might promote the tumorigenesis and proliferation of gastric cancer cells at least partially through activation of PI3K/Akt pathway and subsequent transcriptional activation of CyclinD1 to regulate the G1/S phase transition, in which octapeptide repeat region might be an indispensable region.—Liang, J., Pan, Y., Zhang, D., Guo, C., Shi, Y., Wang, J., Chen, Y., Wang, X., Liu, J., Guo, X., Chen, Z., Qiao, T., Fan, D. Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS.


Key Words: PrPC proliferation • cyclinD1 • PI3K/Akt • octapeptide repeat region


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WHILE A ß-SHEET-RICH FORM of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive (1) . Over the past years, many investigations have focused on PrPC functions in various physiological and pathological processes (2 , 3) . However, despite the abundant information available on the function of the misfolding prion protein PrPSc, relatively little is known about the characteristics of PrPC. Perhaps one of the well-studied functions of PrPC is its ability to selectively bind to copper ions through the octapeptide region localized in its N-terminal. This binding confers redox properties to PrPC (4) . Another emerging function of PrPC is its protective role for cell survival, as regards protection against oxidative stress, serum deprivation, and TNF-{alpha}-induced apoptosis (5 , 6) . Besides the nervous system, PrPC is also expressed in many normal peripheral tissues (7) and has been shown to play an important role in neurogenesis and differentiation (8) . Pammer et al. (9) found that the expression of PrPC was negative or weak in the neck region of gastric mucosa but was up-regulated in the mucosa of patients infected with Helicobacter pylori (Hp). Recent work (10) confirmed the result and further proved that up-regulation of gastric PrPC expression by Hp infection was linked to Hp-induced hypergastrinemia. Since Hp infection is thought to play an important role in carcinogenesis of gastric mucosa, it is interesting to investigate the relationship between PrPC and gastric cancer.

Our previous studies demonstrated that human prion protein gene (PRNP) was up-regulated in multidrug-resistant gastric cancer cell lines (11) and that forced expression of PrPC affected drug accumulation (12) . PrPC was found to be widely expressed in several gastric cancer cell lines, which could be digested by proteinase K. Overexpression of PrPC was found in gastric cancers and correlated with histopathological differentiation parameters (13) . Forced PrPC overexpression could inhibit apoptosis and promote metastasis (14 , 15) . All of these evidences indicated that PrPC might be involved in gastric cancer cell proliferation and progression.

Here we report that ectopic expression of PrPC could promote tumorigenesis and cell proliferation of gastric cancers, and we further prove that PI3K/Akt pathway might be responsible for this function. Down-regulation of PrPC by RNAi showed opposite effect. Further studies showed PrPC promoted cell progression by promoting G1/S phase transition. The target molecules in this biological process were selected by gene array and then proved by RT-PCR and Western blot. CyclinD1 was found to be up-regulated at both mRNA and protein levels, and its promoter could be transcriptionally activated by PrPC in gene reporter assays. Octapeptide repeat region, a highly conserved region among different species, played a vital role in this function. Deletion of this region abolished or reduced the effect of PrPC on cell proliferation. PI3K/Akt pathway also mediated PrPC signal since PrPC increased the expression of phosphorylated Akt, and the specific inhibitor of Akt, LY294002, could markedly suppress growth of SGC7901 and transactivation of CyclinD1 induced by PrPC. Collectively, this study demonstrate that overexpression of PrPC might promote the tumorigenesis and proliferation of gastric cancer cells, at least in part, via activation of PI3K/Akt pathway and consequent transcriptional activation of CyclinD1 to regulate the G1/S phase transition, in which octapeptide repeat region might be an indispensable region.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and animals
Human gastric cancer cell lines SGC7901 and AGS were obtained from Beijing Institute of Oncology. Cells were cultured in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal calf serum in a 37°C humidified CO2 incubator. BALB/c nude mice (4–6 weeks old) were purchased from Shanghai Cancer Institute. The animal experiments were performed in accordance to the institutional animal welfare guidelines.

Plasmid construction and transfection
Target sequences were aligned to the human genome database in a BLAST search to ensure that the chosen sequences were not highly homologous with other genes. The primers were designed with Primer.5 software or https://www.genscript.com/ssl-bin/app/rnai as in Table 1 (13 , 15) . PGL3-CCND1 promoter vector was a gift from Dr. Richard G. Pestell and Chenguang Wang (Georgetown University, Washington, DC, USA). CCND2 and CCND3 promoters were cloned with the primers reported before (16) . Mutant PrPC ({Delta}22–47, {Delta}51–90, {Delta}24–90, {Delta}96–230, {Delta}231–253) duplexes were obtained through recombinant PCR. The G418-resistant multiple combined clones were selected, and expression levels of PrPC were evaluated by Western blot analysis. Gastric cancer cell line SGC7901 transfected with PrPC, PrPC ({Delta}22–47), PrPC ({Delta}51–90), PrPC ({Delta}24–90), PrPC ({Delta}96–230), PrPC ({Delta}231–253), and pcDNA3.1B were designated as SGC7901/ PrPC, SGC7901/ PrPC ({Delta}22–47), SGC7901/ PrPC ({Delta}51–90), SGC7901/ PrPC ({Delta}24–90), SGC7901/ PrPC ({Delta}96–230), SGC7901/ PrPC ({Delta}231–253) and SGC7901/pcDNA3.1B, respectively. AGS transfected with PrPC(RNAi), pSilencer were designated as AGS/PrPC(RNAi), AGS/pSilencer.


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  		Table 1. Primers for plasmids construction

MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate cell proliferation as previously (17) . The absorbance at 490 nm (A490) of each well was read on a microplate reader BP800 (Biohit, Helsinki, Finland). Each experiment was performed in quadruplicates and was repeated 3 times.

Plate colony formation assay
Log phase cells were trypsinized into single cell suspension and passaged into 90 mm2 plates at a density of 1 x 103 cells/well. The colonies were stained with Giemsa, and the total number of colonies was counted. Each assay was performed in triplicate.

Soft agar colony formation assay
Gum agar (2%) was melted in a microwave and cooled to 50–60°C in a water bath. The top agar was prepared with 2% agar, RPMI 1640, and FCS to give 0.3% agar and 10% FCS. Cells were harvested, washed, and mixed with the top-agarose suspension at a final concentration of 0.3%, which was then layered onto the bottom agar. The dish was overlaid with 1 ml of RPMI 1640 containing supplements. Cells were incubated for 2 wk at 37°C in 5% CO2 before colony counting. Each assay was performed in triplicate.

Tumor growth in nude mice
Mice were handled using best humane practice and were cared for in accordance with NIH Animal Care and Use Committee guidelines. The logarithmically growing cells were harvested from tissue culture flasks using trypsin and resuspended in PBS. Mice were injected subcutaneously with 2 x 106 cells in 0.1 ml into the right upper back. The mice were then monitored for tumor volume, overall health, and total body weight. The size of the tumor was determined by measurement of the subcutaneous tumor mass with a caliper. Tumor volume was calculated according to the formula 0.5x (lengthxwidth)2. Two independent experiments were performed and gave similar results.

Cell cycle analysis
Subconfluent cells were washed with ice-cold PBS, suspended in 0.5 ml ethanol, and then kept at 4°C for 30 min. The suspension was filtered through a 50 µm nylon mesh, and the DNA content of stained nuclei was analyzed by a flow cytometer (EPICS XL, Coulter, Miami, FL, USA). The cell cycle was analyzed using Multicycle-DNA Cell Cycle Analyzed Software. The proliferative indexes (PI) was calculated as follows: PI = (S+G2)/(S+G2+G1).

Cell cycle synchronization
Transfected cells were synchronized by double thymidine block as previous described (17) . Briefly, cells that were ~50% confluent were treated with 2 mM thymidine for 16 h. Cells were then washed two times and incubated for an additional 8 h in the absence of thymidine. Cells were then incubated a second time with 2 mM thymidine for 16 h to arrest cells at the G1/S boundary of the cell cycle. Cells were harvested at 4 h intervals for 16 h. Then cells at each time point were washed twice with PBS and fixed with 70% ethanol, washed again, and resuspended at 37°C for 15 min in freshly prepared PI staining solution [PBS containing 0.1% (v/v) Triton X-100 (Sigma, St. Louis, MO, USA), 0.2 mg/ml DNase-free RNase A (Roche, Mannheim, Germany), and 20 µg/ml PI]. Cells (1x104) were then analyzed for fluorescence intensity by a flow cytometer (EPICS XL, Coulter) using Multicycle Software.

Gene array
The total RNA was extracted from all transfected cells using Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. DNase was used to decrease the contamination of genomic DNA. The quantity and purity of the RNA prepared from each sample was determined by electrophoresis and the ratio of the optical density at 260 nm to that at 280 nm. Compared samples SGC7901/PrPC and SGC7901/pcDNA3.1 B were hybridized into a 14 K human cDNA chip (Shanghai Biochip, Shanghai, China) and labeled with cy3 and cy5. Scanning with the Agilent Scanner (Scan resolution 10 µm, PMT 100%) and normalized with Genespring. The differently expressed genes were standardized with cy3/cy5 ratio ≥ 2 as up-regulated genes and cy3/cy5 ≤ 0.5 as down-regulated genes.

RNA extraction and semiquantitative RT-PCR
Total RNA was extracted, and DNase was used to decrease the contamination of genomic DNA. The PCR primers and reaction parameters used for Cyclin and CDK family genes amplification are listed in Table 2 . The reaction condition of PCR (e.g., CyclinD1) was as follows: initial denaturation at 94°C for 10 min; 35 cycles of denaturation at 94°C for 45 s, annealing at 59°C for 30 s, and extension at 72°C for 45 s on a Touchgene Gradient thermal cycler (Techne, Cambridge, UK). Appropriate cycles were chosen to ensure the termination of PCR amplification before reaching stable stage in each reaction. Gene expression was presented by the relative yield of the PCR product from target sequences to that from the ß-actin gene. PCR products were loaded onto a 1.5% agarose gel and electrophoretically separated. The gel was then visualized under ultraviolet light following ethidium bromide staining.


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  		Table 2. PCR primers and reaction parameters that were used for Cyclin and CDK family genes

Western blot analysis
Cells grown to 70–90% confluence were collected by scraping and washed with ice-cold PBS for two times. The cell pellets or tissues were homogenized in extraction buffer (50 mM Tris-HCl, 0.1% SDS, 150 mM NaCl, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium orthovanadate), then incubated at 4°C for 30 min and centrifuged 20 min at 12,000 g/min. Concentration of total protein in the supernatant was quantified by Bradford assay. The total proteins (50–100 µg/lane) were resolved in 10–15% SDS-polycrylamide gels, and then transferred onto nitrocellulose membrane (0.45 µm, Millipore, Bedford, MA, USA) in 25 mM Tris-base, 190 mM glycine, and 20% methanol using a semidry blotter. Following blocking with 10% nonfat milk and 0.1% Tween20 in TBS for 2 h, the membranes were incubated with anti-PrPC (3F4, Sigma, 1:1000), anti-His(Tian Gen, 1:500), anti- Cyclin D1 (Upstate Biotechnology, Lake Placid, NY, USA; 1:500), anti- CDK4 (Upstate Biotechnology, 1:500), anti-Total Akt (Cell Signaling Technology, Danvers, MA, USA; 1:500), phospho-Akt- (pSer 473) (Cell Signaling; 1:500) and anti-ß-actin (Sigma; 1:2000), respectively, at 4°C overnight. After binding of horseradish peroxidase (HRP)-coupled goat anti-mouse or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:5000) at room temperature for 2 h, antigens were visualized by enhanced chemiluminescence (ECL-kit, Santa Cruz Biotechnology). All results are representatives of three independent experiments.

Reporter assay
Sequences of CCND2 and CCND3 promoters were amplified by PCR from genomic DNA of peripheral blood mononuclear cells (16) . CCND1 promoter sequence was given by Dr. Richard G. Pestell and Chenguang Wang (Georgetown). These promoter sequences were then cloned into pGL3 enhancer vector (Promega, Madison, WI, USA) to give the reporter vectors (designated pGL-CCND1, pGL-CCND2, and pGL-CCND3, respectively). SGC7901 cells were seeded into 24-well plates at a density of 5 x 105 cells/well and were used at 60–70% confluence. PrPC, PrPC ({Delta}51–90), or empty pcDNA3.1 plasmids were cotransfected into SGC7901 cells with pGL-CCND1 or pGL-CCND2, pGL-CCND3, or pRL-TK was used as a control for transfection efficiency. LipofectAmine 2000 was used for transfection following the manufacturer’s instructions (Invitrogen). Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega), following the vendor’s manual. Each experiment was performed in triplicate and repeated twice.

Statistical analysis
All the values of the in vitro assays were expressed as means ± SD. ANOVA analysis was performed using statistics package SPSS (version 10.0; SPSS, Chicago, IL, USA). Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of PrPC enhances tumor cell proliferation, cellular transformation, and tumorigenesis
Our previous work has shown that PrPC could be detected in several human gastric cancer cell lines. Relatively lower PrPC expression was found in SGC7901 cells, but higher expression of PrPC was noted in AGS (12) . To show the differential effects of PrPC expression on cell proliferation, PRNP gene was stably transfected into human gastric cancer cell SGC7901 or was blocked by RNAi in AGS cells, respectively. After cell transfection and antibiotic selection for more than 2 mo, multiple drug-resistant clones were selected and the expression of PrPC in the cells was confirmed by Western blot (Fig. 1 A).


Figure 1
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  		Figure 1. Effects of PrPC on cell proliferation and tumorigenesis of gastric cancer. A) Western blot analysis of the vector transfectants and PrPC transfectants. ß-actin was used as a loading control. B) Detection of the cell growth rate in vitro. Cell number was evaluated by the absorbance at 490 nm in MTT assay at the indicated time. The value shown is the mean of three determinations. C) Detection of the colony formation. Cells were placed in media containing soft agar or plate and incubated for 20 d. The number of foci >100 µm was counted. Vertical bars represent mean ± SD from at least three separate experiments, each conducted in triplicate. D) In vivo tumorigenicity was evaluated by nude mice assay. Mice were injected subcutaneously with 2 x 106 transfected cells. One month after cell injection, the volume of tumor was calculated according to the formula 0.5 x length x width2. Two independent experiments were performed. *P < 0.05 versa control vector transfected cells.

MTT assay, plate, and soft agar colony formation assays were done to examine the effect of PrPC on cell proliferation and cell transformation in vitro. Compared with empty vector-transfected cells and parental cells, PrPC-transfected cells showed significantly increased rate of cell proliferation as shown by MTT assay (Fig. 1B ). Anchorage-independent growth is one of the important characteristics of in vitro tumor growth; therefore, we examined whether up-regulation of PrPC expression could promote SGC7901 cell growth in soft agar and plate. As shown in Fig. 1C , PrPC transfection resulted in a marked accumulation of growth of SGC7901 in colony formation assay, with average increase rates of 65.1% and 58.6% in soft agar and plate assay, respectively. Down-regulation of PrPC by RNAi inhibited the colony formation both in soft agar and in plate.

To confirm this effect in vivo, we injected subcutaneously PrPC-expressing SGC7901 cells into the right back limb of nude mice. On day 25, the average tumor volume in SGC7901-PrPC group was 1.86 ± 0.29 cm3, compared with 0.84 ± 0.15 cm3 of the control group and 1.04 ± 0.21 cm3 of the vector group (Fig. 1D ).

PrPC promotes G0/G1 to S-phase transition in the cell cycle
To explore the possible roles of PrPC in controlling cell proliferation, we examined the cell cycles of transfected cells by FACS for three times. The mean proliferative indexes (PI) of SGC7901/PrPC (0.449±0.017) were significantly higher than those of SGC7901/pcDNA3.1 (0.373±0.023) and SGC7901 (0.360±0.009; P<0.05). The proliferative index of AGS/PrPC(RNAi) (0.489± 0.038) was lower than that of AGS/pSilencer (0.537±0.049) and AGS (0.562±0.074). These data suggest that PrPC promoted G0/G1 to S transition in PrPC-transfected cells and, therefore, might have contributed to the enhanced proliferation rate (Fig. 2 A).


Figure 2
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  		Figure 2. Effects of PrPC on cell cycle distribution in gastric cancer cells SGC7901 and AGS. A) Cell cycle distribution in transfected cells. The cells were detergent extracted, stained with propidium iodide, and analyzed by flow cytometry. B) Cell cycle synchronization. Effect of PrPC on cell cycles by arresting cells at G1/S boundary by adding thymidine (2 mM) at 0 h. Four hours after releasing, the cells began entering into S phase. The average releasing rate of cells from G1 to S phase in SGC7901/PrPC was 45.7%, which was significantly higher than those of vector control cells (34.6%). Eight and twelve hours later after releasing, most cells had gone into G2 phase. By 16 h, cells entered a new cell cycle. There was no significant difference in cell cycle profile between PrPC-transfected cells and the control cells at later times. *P < 0.05 versa SGC7901/pcDNA3.1.

We further analyzed the effect of PrPC on cell cycles by synchronization. It was found that after cell cycle blocking by addition of thymidine(2 mM), cells were mostly arrested in G1 phase. Four hours after releasing, the cells began entering into S phase. The average releasing rate of cells from G1 to S phase in SGC7901/PrPC was 45.7%, which was significantly higher than those of vector control cells (34.6%). Twelve hours later after releasing, most cells had gone into S phase. By 16 h, cells entered a new cell cycle. As seen from the Fig. 2B, no significant difference was found in cell cycle profile between PrPC-transfected cells and control cells at later times. Taken together, these data strongly indicate that PrPC might play an important role in promoting the growth of gastric cancer cells through accelerating G1 to S phase transition in the cell cycle.

Octapeptide repeat region plays a vital role in promoting PrPC-transfected gastric cancer cell proliferation
To investigate which region(s) in PrPC were critical for the function of PrPC in promoting gastric cancer cell proliferation, we’ve constructed deletion mutant plasmids of PRNP gene according to the reported functional and structural regions of PrPC (18) . N-terminal flexible region ({Delta}22–47), lipid targeting region ({Delta}24–90), octapeptide repeat region ({Delta}51–90), C-terminal globular region ({Delta}90–230), and GPI attachment sequence ({Delta}230–253) were deleted, respectively, with recombinant PCR techniques (Fig. 3 A). We checked the expression of deletion mutants of PrPC in these stable cell lines by Western blot. Considering the epitope of anti-PrP antibody (aa109–112) was deleted in certain kinds of the mutants, the expression of external PrPC deletion mutants were detected by anti-6His antibody. As shown in Fig. 3B , all the 6xHis-tagged mutants of PrPC could be detected, whereas no signal was found in control cell transfected with empty vector.


Figure 3
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  		Figure 3. Effects of different deletion mutants of PrPC on proliferation of gastric cancer cells SGC7901. A) Schematic presentation of full length, ({Delta}22–47), ({Delta}51–90), ({Delta}24–90), ({Delta}90–230), and ({Delta}231–253) of PrPC. B) SGC7901 cells were stably transfected with full-length and deletion mutants of PrPC, respectively. The expression of PrPC in cells was evaluated by Western blot with anti-6His antibody. ß-Actin was used as a loading control. Line 1 SGC7901/PrPC, Line 2 SGC7901/ PrPC ({Delta}22–47), Line 3 SGC7901/PrPC ({Delta}51–90), Line 4 SGC7901/PrPC ({Delta}24–90), Line 5 SGC7901/PrPC ({Delta}90–230), Line 6 SGC7901/PrPC ({Delta}231–253), and Line 7 SGC7901/pcDNA3.1. C) Growth rate of the cells’ detection by MTT assay. Cell number was evaluated by the absorbance at 490 nm in MTT assay at the indicated time. The values shown are the means of three determinations. D) Cell cycle distribution and proliferative indexes (PI) in PrPC, PrPC mutants, and control cells. The cells were detergent extracted, stained with propidium iodiols, and analyzed by flow cytometry. *P < 0.05 versa SGC7901/PrPC.

MTT assay and FACS were performed to examine the roles of the different regions of PrPC in regulating cell proliferation and cell cycle. Compared with PrPC-transfected SGC7901cells, deletion of octapeptide repeat region almost completely abolished the cell proliferation promoting the effect of PrPC (Fig. 3C ). Cell cycle analysis by FACS showed similar results. The average PI of cells transfected with octapeptide repeat region-deficient vector(0.367±0.017) was similar to that of pcDNA3.1-transfected cells (0.373±0.023), which was significantly less than that of PrPC-transfected cells (0.424±0.038) (P<0.05) (Fig. 3D ).

Cyclin D1 is involved in proliferation and G1/S transition of gastric cancer cells regulated by PrPC
To identify molecules that are regulated by PrPC and are responsible for the effect caused by PrPC in gastric cancer cells, gene array was used to screen for target molecules. Of the 14,000 genes in the human cDNA chip, 84.6% could be detected with our samples and coefficient of variation (CV) was 12.4%. Together, 736 genes were found to be up-regulated in PrPC-transfected cells and 218 genes were down-regulated. Among them, 120 genes related to cell progression, especially five genes (CDC2L5, CCND1, TUBE1, E2F3, and ASNS) closely related to cell cycle were found to be up-regulated in PrPC-transfected cells.

Confirmed by the gene array results of skin fibroblast cell lines established from PrP-deficient (PrP–/–) mice and PrP+/+ mice (19) , CyclinD1 was found to be up-regulated in PrPC transfected cells, which is a key molecular in G1/S phase transition regulation. Therefore, we examined the expression of CyclinD1 in gastric cancer cells after PrPC transfection. Increased transcription of CyclinD1 and CDK4 was confirmed by RT-PCR in PrPC-transfected cells. Similarly, overexpression of PrPC was found to increase protein expression of CyclinD1 in SGC7901 cells by Western blotting. Deletion of the octapeptide repeat region of PrPC partially abolished the up-regulation of CyclinD1 and CDK4 (Fig. 4 A). Interestingly, in SGC7901/PrPC transfectant, only RNA transcription but not protein expression of CDK4 was up-regulated compared with that of empty vector controls (Fig. 4B ).


Figure 4
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  		Figure 4. The inducible effect of PrPC on CyclinD1. A) Detection mRNA expression of Cyclin and CDK family molecules by RT-PCR. B) Detection of protein expression of Cyclin D1 and CDK4. ß-actin was used as a loading control. Compared with empty vector pcDNA3.1 transfected cells, cells transfected with PrPC would increase the expression of Cyclin D1 at both mRNA and protein levels, while deletion of the octapeptide repeat region inhibited the effect. C) Relative luciferase activity of CyclinD promoters in SGC7901 cells cotransfected with PrPC, PrPC ({Delta}51–90) or empty vector were evaluated by dual luciferase reporter assay. *P < 0.05 vs. SGC7901/pcDNA3.1.

To investigate the possible mechanisms involved in the regulation of CyclinD by PrPC, we performed dual-luciferase-reporter assay. Luciferase reporter plasmids pGL3-CCND1, pGL3-CCND2, and pGL3-CCND3, containing CyclinD1, CyclinD2, and CyclinD3 promoters, respectively, were transiently transfected into SGC7901/PrPC, SGC7901/PrPC ({Delta}51–90), and SGC7901/pcDNA3.1 together with the pRL-TK. As seen in Fig. 4C , the intensity of luciferase luminescence in SGC7901/PrPC cells cotransfected with either pGL3-CCND1, pGL3-CCND2, or pGL3-CCND3 was 4.94-, 3.36-, and 3.65-fold higher than that of SGC7901/pcDNA3.1 control cells, respectively, indicating that PrPC triggered transactivation of CyclinD. Whereas, deletion of the octapeptide repeat region partially inhibited the transactivation of CyclinD (Fig. 4C ).

PI3K/Akt signal is involved in gastric cancer cell proliferation and PrPC-induced CyclinD1 transactivation
It has been demonstrated that phosphatidylinositol 3-kinase (PI3K) signal is an important downstream effector of PrPC (6 , 20 , 21) . Thus, we determined to examine whether PI3K pathway might be involved in PrPC-related proliferation of gastric cancer cells. Firstly, the phosphorylation status of the member of PI3K, Akt, in SGC7901/PrPC, SGC7901/pcDNA3.1, and SGC7901 cells was evaluated by Western blot. The results showed that phosphorylated Akt was markedly up-regulated by PrPC transfection, while the expression levels of total Akt was not altered (Fig. 5 A). We then investigated the influence of LY294002, an Akt specific inhibitor, on the proliferation and expression of CyclinD1 in gastric cancer cells. LY294002 could inhibit the proliferation both in PrPC-expressing or non-PrPC-expressing cells. However, treatment with LY294002 at the concentration of 10 µM for 48 h wouldn’t greatly inhibit the proliferation of SGC7901 cells (Fig. 5B ). The inhibition occurred mostly on G1/S phase (Fig. 5C ). The inhibition rate of colonies formation by LY294002 10 uM for 48 h in soft agar of SGC7901/PrPc was 51.19%, higher than that of SGC7901/pcDNA3.1 (29.39%) (P<0.05) (Fig. 5D ). Both protein and RNA expression of CyclinD1 was significantly down-regulated in gastric cancer cells treated with LY294002, especially in SGC7901 cells transfected with PrPC (Fig. 5E ). The inhibition of CyclinD was more significant in PrPC-transfected cells than in control cells (Fig. 5F ). All of these results suggested that PI3K/Akt signal is involved in PrPC-related proliferation and mediates transactivation of CyclinD1 induced by PrPC.


Figure 5
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  		Figure 5. Akt signal in regulating promotion of gastric cancer cells induced by PrPC. A) The expression of phospho-Akt and total Akt were determined in gastric cancer cells by Western blot. ß-actin was used as an internal control. B) Effect of Akt specific inhibitor LY294002 on the cell growth of SGC7901. C) Cell cycle distribution and proliferative indexes (PI) of cells treated with or without LY294002 (10 µM 48 h). D) Cell number in soft agar of PrPc transfected and control cells treated with or without LY294002. E) After cells were treated with or without 10 µM of LY294002 for 48 h, the expression of CyclinD1 was examined by Western blot and RT-PCR. ß-actin was used as an internal control. F) After cells were treated with or without 10 µM of LY294002 for 48 h, the relative luciferase activity of CyclinD promoters in cells cotransfected with PrPC or empty vector was evaluated by dual-luciferase-reporter assay. *P < 0.05 vs. cells not treated LY294002.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we present the first evidence that forced overexpression of PrPC could promote the proliferation of gastric cancer cells and facilitate G1 to S transition in the cell cycle. Down-regulation of PrPC by RNAi showed the opposite effect. Further experiment indicates PrPC would trigger the transactivation of CyclinD1, and the N-terminal octapeptide repeat region of PrPC might be indispensible in this process. PI3K/Akt pathway was found to be involved in PrPC-related proliferation and mediated transactivation of CyclinD1 induced by PrPC. To our knowledge, this is the first report that reveals a link between PrPC and cancer cells proliferation.

Cellular prion protein PrPC is mostly present in hippocampus, hypothalamus, and olfactory bulb, where rapid cell renewing is common (22) . Recent studies show PrP is expressed immediately adjacent to the proliferative region but not in mitotic cells in the brain (8) . Role of PrPC in against oxidative stress and serum deprivation have also been intensively studied (5 , 6) . In addition to the nervous system, PrPC is also expressed in the mucosa of gastrointestinal tract (23) , especially in the case of Hp infection (10) . Our previous studies showed that PrPC was overexpressed in gastric cancer tissues and correlated with tumor histological types and TNM stages (12 , 13) . In line with our data, Comincini et al. reported that the prion-like protein doppel gene (PRND) was differentially expressed in human gliomas (24) . As far as we know, limitless replicative potential is one of the characteristic of tumor cells. All above data pointed to the fact that PrPC is involved in the proliferation of certain types of cells, including gastric cancer cells.

In the present study, the results of MTT assay, colony formation assay, and in vivo tumor formation assay in nude mice all suggested that forced PrPC overexpression could promote tumorigenesis, proliferation, and transformation of gastric cancer cells SGC7901. Down-regulation of PrPC expression by RNAi showed the opposite effect in gastric cancer cells AGS. PrPC synthesis in T98G cells was previously demonstrated to be dependent on G1 phase of the cell cycle (25) . Gougoumas et al. confirmed the similar results in the viral-transformed mouse spleen hematopoietic cells and other types of inducible cells. They showed that the housekeeping gene PRNP was transcriptionally activated in G1 phase in confluent and terminally differentiated cells (26) . FACS and synchronization with thymidine (2 mM) in our experiment proved that PrPC might promote gastric cancer cells progression by regulating the G1/S phase cell cycle.

In mammalian cells, there are two classes of cyclin-dependent kinase (CDK) that function at the G1/S phase transition. G1 progression depends on the sustained expression of D-type cyclins, which, in turn, depends on continuous mitogenic stimulation and provides a link between mitogen signaling and the cell-cycle machinery (27 , 28) . CyclinD1 was found to be reduced in a PrP-deficient (PrP–/–) mice derived skin fibroblast cell line by a cDNA expression array (19) . In our study, five cell-cycle-related genes were found to be up-regulated in cells transfected with PrPC by gene microarray analysis. Among them CyclinD1 and E2F are closely related to G1/S phase transition. Another gene, CDC2L5 was also reported to affect cells splicing in vivo (29) . RT-PCR and Western blot were used to further confirm that PrPC could increase both the RNA and protein expression of Cyclin D1 in SGC7901 cells, while CDK4 was only up-regulated at RNA level. Whether CDK4 was activated in cells transfected with PrPC awaits our further studies. Gene reporter assay suggested PrPC could stimulate the promoter activity of CyclinD. Taken together, PrPC might stimulate cells proliferation by accelerating the transcription of CyclinD1 and thus, facilitating cells cycle transition from G1 to S phase.

The N-terminal fragment of PrPC is a flexible region, which is highly conserved among different species, indicating this region might be a functional domain of PrPC (30) . The octapeptide repeat region aa51–90 of PrPC is a copper binding site, which is composed of five copies of the fundamental sequence PHGGGWGQ. Prion proteins with insertion mutations have altered N-terminal conformation and increased ligand binding activity and are more susceptible to oxidative attack. The motif is also believed to mediate PrP-dependent activation of superoxide dismutase and play a role in apoptosis and cell survival (31) . Our preliminary study showed that complete deletion of the octapeptide repeat region could substantially abolish the increased cell proliferation induced by ectopic PrPC expression, partially through the inhibition of transcriptional activation of CyclinD1. An interesting finding was that cells transfected with the plasmids containing PrPC deletion mutant ({Delta}24–90), in which the octapeptide repeat region (51–90) was also deleted, couldn’t significantly inhibit the cell proliferation-promoting effect of PrPC, which was somewhat hard to interpret. The same phenomenon also occurred when all of the deletion mutants were transfected into another gastric cancer cell line MKN28. It could be postulated the region aa24–50 might act against the octapeptide repeat region in cell proliferation. However, the oncogenesis function of the PrPC and the precise region(s), which mediate(s) the malignant transformation of gastric cancer still requires further investigation.

The PI3K/Akt pathway has been demonstrated to be an important mediator of tumor progression and cell survival (32) . Previous studies proposed that PI3K/Akt is a target of PrPC-mediated signal. Overexpression of PrPC prevented human breast carcinoma cell line from tumor necrosis factor {alpha}-induced cell death, which was reported to be related to the PI3K/Akt pathway (20) . PrP could also induce phosphorylation of ERK1, 2 and Akt kinase in monocytes (21) . Recruitment of PI3K by PrPC is shown to contribute to cellular survival toward oxidative stress (6) . Here we demonstrated that PrPC could increase the expression of phosphorylated Akt in gastric cancer cells. The model of PrPC/PI3K signal could also be applied to explain the findings on prion protein function in several other studies. It was found that Fyn governed a lot of PrPC-induced pathways that converge to the PI3K module in neurons (33) . PI3K is known to physically associate with Fyn in transducing differentiation signals (34) . Grb2, another PrPC-interacting protein (35) , is also a known upstream activator of PI3K (36) . It is thus likely that Fyn and Grb2 mediate the activation of PI3K/Akt by PrPC in gastric cancer cells. The results from our study showed that the proliferation promoting effects of PrPC could be inhibited by pharmacological blockade of Akt by LY294002, indicating PI3K/Akt pathway might play an important role in transducing proliferation-promoting signal of PrPC in gastric cancer cells. However, mechanisms other than PI3K/Akt pathway activation and Cyclin D1 up-regulation could also exist which are responsible for PrPC proproliferative actions.

In conclusion, this study strongly demonstrates that overexpression of PrPC might promote the proliferation of gastric cancer cells at least partially through activation of PI3K/Akt pathway and subsequent transcriptional activation of CyclinD1 to accelerate the G1/S phase transition. The octapeptide repeat region might be an indispensable region for this function. To our knowledge, this is the first report that reveals a link between PrPC and cancer cell proliferation. The present study sheds new sights on the function of PrPC and suggests that down-regulation of PrPC by RNAi, deletion of octapeptide repeat region of PrPC or suppression of PrPC signals may be another possible approach in the management of human gastric cancer.


   ACKNOWLEDGMENTS
 
We thank Richard G. Pestell and Chenguang Wang (Lombardi Comprehensive Cancer Center and Department of Oncology, Georgetown University Medical Center, Washington, DC, USA) for the CyclinD1 promoter plasmid. We are also grateful to Bo Huang for help in luciferase activity analysis and technicians Baojun Chen and Baohua Song for their excellent technical assistance.

Received for publication November 20, 2006. Accepted for publication February 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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