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We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of PKR (Protein Kinase RNA-activated). PKR's pro-apoptotic role through eIF2α phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR's activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival NF-κB pathway. Thus, PKR plays a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886's role in the PKR pathway during tumorigenesis.
We have recently characterized a novel type of non-coding RNA, nc886 (pre-miR-886 or vtRNA2-1) (1). As its aliases indicate, it was originally known as a precursor microRNA (2) or as an RNA component of the vault complex (3, 4). However, we have shown that it is neither a precursor microRNA nor a vtRNA (1); so we have renamed it nc886 (non-coding RNA-886) and this term will be used throughout this paper and in the future. A brief summary of nc886 is: 1) its expression is ubiquitous in non-malignant human tissues but suppressed in a wide range of cancer cells, and 2) its artificial suppression leads to the activation of PKR (Protein Kinase RNA-activated) and impaired cell proliferation (1). Recently, nc886's significance in the biology and the prognosis of acute myeloid leukemia has been reported (5).
PKR is an interferon-inducible and double-stranded RNA (dsRNA)-dependent kinase, whose role has been well established in the host defense against viral infection. In the canonical PKR pathway [reviewed in (6)], virus-derived dsRNA binds to PKR and mediates its autophosphorylation. Phosphorylated PKR (P-PKR) is an active kinase that phosphorylates eIF2α (eukaryotic initiation factor 2 α subunit) at Ser51. Phosphorylated eIF2α (P-eIF2α) blocks global protein synthesis by preventing its efficient recycling by the guanine nucleotide exchange factor eIF2B (eukaryotic translation initiation factor 2B), and subsequently leads to cell death (7-9). PKR also has the dual role of activating the NF-κB pathway which promotes cell proliferation [reviewed in (10)].
In addition to its established role in the interferon response, PKR is involved in many pathways and exerts various functions on cell growth independently of viral infection [reviewed in (11)]. Thus, not surprisingly, PKR is implicated in tumorigenesis. However, PKR's exact role in cancer biology remains controversial. Initially, PKR was thought to be a tumor suppressor. Early studies indicated that malignant transformation was induced by dominant-negative mutants of PKR (and thus its loss of function). This corroborated PKR's pro-apoptotic role. It has been also reported that PKR was suppressed or inactivated in some malignancies (12-16). However, several observations have challenged the proposed role of PKR as a tumor suppressor. In some cancers, PKR and its downstream eIF2α are induced in expression and activity (1, 5, 17-29). In addition, NF-κB, a pro-proliferative factor which is the downstream of PKR, is also activated in many cancers [reviewed in (10)].
What is the biological significance of activated PKR in cancer? Our data about nc886, together with the previous knowledge of PKR, led us to propose a tumor surveillance model for the nc886/PKR role in cancer biology. In this model, decrease of nc886 during early stages of tumorigenesis activates the classical PKR/eIF2α pathway which leads to cell death and eliminate most precancerous and/or cancerous cells. Cancer cells that have survived must have developed bypass mechanisms to proliferate in the presence of elevated P-PKR and P-eIF2α. Once cancer cells have overcome the PKR/eIF2α cell death pathway, P-PKR could play the pro-proliferative role necessary for further tumor progression through its downstream NF-κB pathway.
In this paper, we have investigated our tumor surveillance model in cholangiocarcinoma (CCA), a cancer from the hepatobiliary epithelium. This study is the first step to elucidate the roles of nc886/PKR in cell death/proliferation during tumorigenesis.
We measured nc886 in a battery of bile duct cells (Table 1). Like other non-malignant human cells in our previous studies (1), nc886 was expressed in immortalized but not malignant cholangiocyte MMNK1 cells (Fig 1). nc886 was also expressed in three CCA cells (M156, M214, and M055), whereas it was not expressed in M213, its derivative lines M213L0H and M213L5H, and M139 cells. For comparison, all of the bile duct cells constitutively expressed vtRNA1-1, a canonical vtRNA (Fig 1).
While PKR was constitutively expressed in cholangiocyte and CCA cells (total PKR in Fig 1), it was activated in all the CCA cells except for M156, as indicated by P-PKR Western blot (Fig 1). Similarly to P-PKR, P-eIF2α was elevated in all the CCA cell lines, but again not in the non-malignant MMNK1 (Fig 1). In our experiments in Fig 1, it should be noted that all assays were performed in a normally growing condition without exposure to dsRNA, a natural activator of PKR that leads to apoptosis.
In general, CCA cells tended to have decreased nc886 but elevated P-PKR/P-eIF2α relative to non-malignant MMNK1 cells (Fig 1 and Table 1). Certainly, there were exceptional cell lines; nc886 was not silenced in all the CCA cells, and nc886 was co-expressed with P-PKR in M214 and M055 cells. This is explained by the genetic diversity of CCA cells. PKR has been known to be regulated by a number of intra-/extra-cellular factors besides dsRNA [reviewed in (6)]. For example, natural PKR regulators such as PACT or p58IPK may have contributed to the activation of PKR in M214 and M055 cells. We speculate that nc886 is also under complex regulation, as a number of transcription factors are predicted to bind to the vicinity of the nc886 locus.
Taken together with our previous result that suppression of nc886 activates PKR to P-PKR (1), the anti-correlation between nc886 and P-PKR in a battery of cholangiocyte/CCA cells (Fig 1) was intriguing and raised these questions: 1) is the expression of nc886, P-PKR, and P-eIF2α related causally during tumorigenesis? and 2) how do CCA cells overcome P-PKR/P-eIF2α's apoptotic effect and proliferate? We addressed these issues in the following experiments. As we used several cell lines with different expression of nc886, P-PKR, and P-eIF2α, we designated their expression by +/− signs in a parenthesis, for easy reading. For example, MMNK1 cells are (nc886+, P-PKR−, P-eIF2α-; see also Table 1).
A number of papers have reported PKR activation in malignancies; however, most of them did not elucidate a mechanism for that activation. In one report (18), a PKR inhibitor protein p58IPK (30) was suggested to be the cause, because p58IPK was decreased in breast cancer cells whereas PKR was activated. However, this observation was not reproducible in other papers, and thus p58IPK's universal involvement in PKR regulation seemed unlikely (17, 20). In this study we found that the expression of p58IPK was consistent among the cell lines (Fig 2A), and therefore ruled out its role in PKR regulation in CCA.
Based on our data in Fig 1 and our previous study (1), we hypothesized that suppression of nc886 activated PKR in CCA cells. To investigate the nc886/PKR relationship, we suppressed nc886 by transfecting antisense oligonucleotides (anti-oligos) into MMNK1, M156, and M214 cells (all nc886+ cells) and then measured P-PKR. Compared to an anti-oligo against vtRNA1-1 (“anti-vt21_2”) as a negative control, anti-oligos against nc886 (“anti886s”) successfully negated nc886 and induced P-PKR in all the three cell lines examined (Fig 2B-D). Interestingly, there were two distinct mechanisms for anti-oligos to induce P-PKR. In the case of non-malignant MMNK1 cells, the anti-oligos depleted nc886's intracellular level (Northern blot in Fig 2B). In M156 and M214 CCA cells, the anti-oligo interfered with nc886/PKR interaction (pull-down assay in Fig 2C).
Our cellular data were reinforced by in vitro experiments. Addition of the anti-oligo against nc886 in MMNK1 lysate (nc886+) increased PKR activity, as measured by in vitro kinase assays (Fig 2E). Conversely, addition of in vitro transcribed synthetic nc886 in M213 lysate (nc886−) inhibited PKR activity (Fig 2F). We also showed their direct physical interaction by electrophoretic mobility shift assays (EMSA) with synthetic nc886 and purified PKR from E. coli (Fig 2G).
Collectively, our data demonstrated that nc886 alone is necessary and sufficient for suppression of PKR through direct physical interaction. nc886 keeps PKR repressed in MMNK1 cells, and PKR is released from that repression when nc886 is suppressed in CCA cells. However, nc886 levels could not explain P-PKR in all the CCA cells, for example, in M214 and M055 (nc886+, P-PKR+). As mentioned earlier, their intrinsic level of P-PKR could have been activated by a factor other than nc886 or p58IPK, or in these malignancies there seems to be a higher threshold level of nc886 necessary for suppression of P-PKR.
One pre-requisite for our tumor surveillance model is that the canonical PKR/eIF2α pathway operates normally and leads to apoptosis in most cells, because the suppression of nc886 would eliminate such cells through this pathway. We attempted to recapitulate such a situation in non-malignant MMNK1 cells (nc886+, P-PKR−, P-eIF2α-). Upon nc886 depletion, P-PKR phosphorylated eIF2α, decreased global protein synthesis and inhibited cell proliferation (Fig 3A-B).
P-PKR is known to induce apoptosis mainly through two pathways involving FADD/caspase-8 and APAF/caspase-9, both of which merge onto caspase-3 [reviewed in (6)]. Consistently, we detected the active form of cleaved caspase-3 and consequent cleavage of PARP (poly(ADP-ribose) polymerase) upon nc886 knockdown in MMNK1 cells (lane 1-2 in Fig 3C). In contrast, neither caspase-3 nor PARP cleavage was seen in M156 and M214 CCA cells (lane 3-6 in Fig 3C), although PKR was indeed activated by nc886 depletion (Fig 2D). Hence, there were two distinct consequences of P-PKR activity between MMNK1 cells and CCA cells. It is worth noting that M156 and M214 CCA cells were both P-eIF2α+ (Fig 1).
To interrogate which step of the PKR pathway is abrogated in these two cell lines, we measured P-eIF2α and global protein synthesis upon nc886 knockdown (Fig 3D). In the case of M156 cells (nc886+, P-PKR−, P-eIF2α+), P-eIF2α was induced but global protein synthesis was not significantly decreased. In the other case of M214 cells (nc886+, P-PKR+, P-eIF2α+), the induction of P-eIF2α was not seen and consistently global protein synthesis was unaffected.
So far, we have shown that the canonical PKR/eIF2α pathway operated normally in non-malignant MMNK1 cells, but not in CCA cells. Phosphorylation of eIF2α (M214) or inhibition of global protein synthesis (M156) malfunctioned so that the two CCA cells escaped from apoptosis upon nc886 suppression.
Next, we expanded our investigation to CCA cells lacking nc886. To activate the PKR/eIF2α pathway, we transfected a dsRNA mimic Poly(I:C) into M139 cells (nc886-, P-PKR+, P-eIF2α+). For comparison, we included two cell lines, MMNK1 (nc886+, P-PKR−, P-eIF2α-) and M214 (nc886+, P-PKR+, P-eIF2α+) in these experiments.
Poly(I:C) activated PKR in all the cell lines tested (Fig 4A). The intact PKR/eIF2α pathway was again confirmed in non-malignant MMNK1 cells in which Poly(I:C) inhibited global protein synthesis via P-eIF2α (Fig 4A). In contrast, P-PKR did not further phosphorylate eIF2α nor decrease global protein synthesis in M214 and M139 cell lines (Fig 4A). In M214 cells, Poly(I:C) treatment and nc886 knockdown yielded the same result (compare Fig 4A and and3D).3D). So, P-PKR failed to phosphorylate eIF2α in these two cells.
This raised a question as to whether P-PKR also failed to activate its other downstream events. As eIF2α and NF-κB are two representative downstream branches in the PKR pathway, we examined the NF-κB pathway (Fig 4B). Poly(I:C) treatment activated the NF-κB pathway in all the three CCA cell lines tested, including M214 and M139 where P-PKR failed to phosphorylate eIF2α. This NF-κB activation was PKR-dependent, as it was abrogated by 2-aminopurine (2-AP), a PKR inhibitor (Fig 4B). Thus, M214 and M139 CCA cells selectively blocked the eIF2α branch, but not the NF-κB branch in the PKR pathway.
As the activation of NF-κB by P-PKR was operative in the presence of dsRNA, we questioned if the intrinsic NF-κB activity was elevated in CCA cells (P-PKR+) relative to MMNK1 cells (P-PKR−). First, we wanted to ensure that PKR activity was indeed higher in CCA cells than in MMNK1 cells by employing in vitro kinase assays. Consistent with our earlier P-PKR Western data (Fig 1), M214 and M213 cells (P-PKR+) had a robust PKR kinase activity, compared to the very low activity of the MMNK1 cell line (P-PKR−) (Fig 5A). Accordingly, the NF-κB activity of these two CCA cells was higher than that of MMNK1, as assessed by luciferase assays measuring the NF-κB-responsive promoter activity (Fig 5B) and by qRT-PCR measurement of NF-κB target mRNAs (Fig 5C).
The dependence of NF-κB activity on P-PKR (thus PKR's kinase activity) was reinforced by knockdown experiments. In M156 and M214 (both nc886+), nc886 knockdown resulted in PKR activation in our earlier data (Fig 2D). Consistently, NF-κB was activated, as shown by qRT-PCR of NF-κB target genes (Fig 5D). This activation was significantly decreased by simultaneous knockdown of PKR by siRNA, demonstrating the requirement of PKR for the NF-κB activation by nc886 depletion.
Finally, we examined if the elevated PKR/NF-κB activity played a role in the proliferation of CCA cells. For this, we treated cells with caffeic acid phenethyl ester (CAPE), a chemical inhibitor for NF-κB, and measured cell proliferation. CAPE treatment suppressed proliferation of M214 and M213 (both P-PKR+) CCA cells (Fig 5E), demonstrating NF-κB's pro-proliferative role therein. A similar effect of CAPE on CCA growth has been reported previously (31). In agreement, inhibition of PKR activity by 2-AP treatment also attenuated the proliferation of these CCA cells (Fig 5F).
The intrinsic expression of P-eIF2α in CCA cells was still intriguing but not addressed yet. An important obvious question was what caused eIF2α to be phosphorylated. The primary candidate was certainly P-PKR, an eIF2α kinase, because its expression was positively correlated with P-eIF2α (Fig 1). However, this was questionable, because eIF2α was not phosphorylated by P-PKR in CCA cells upon nc886 knockdown or dsRNA treatment (Fig 3--4).4). To test this, we suppressed PKR in M214 and M213 cell lines (both P-PKR+, P-eIF2α+). siRNAs against PKR successfully decreased total PKR and P-PKR, but not P-eIF2α (Fig 6A). Thus, it is unlikely that P-eIF2α was induced by P-PKR in these cell lines.
Another candidate was MetAP2(p67), as it is known to bind eIF2 and protect eIF2α from phosphorylation by P-PKR [reviewed in (32)]. We found its expression to be higher in CCA cells than in MMNK1 (Fig 6B). These data were against our scenario that eIF2α would be released from its protection and hyper-phosphorylated when MetAP2(p67) decreased in CCA. Our data suggested that P-eIF2α was induced in CCA cells by some factors other than P-PKR or MetAP2(p67).
An equally important question was how CCA cells could proliferate in the presence of P-eIF2α. Obviously, their protein translation normally occurred in spite of P-eIF2α's intrinsic expression (Fig 1), as well as its induction by nc886 knockdown (M156 in Fig 3D). P-eIF2α's mechanism for inhibition of protein translation is to bind to eIF2B with an excessively high affinity and consequently deplete productive eIF2B, an essential factor for protein translation (33). Accordingly, overexpression of eIF2B has been shown to negate P-eIF2α's inhibitory effect on protein translation (34, 35). In addition, high expression of eIF2B in malignancies has been reported (18, 36). We measured eIF2B-ε, the catalytic subunit of the penta-peptide eIF2B complex (37), and found it to be elevated in CCA cell lines relative to MMNK1 (Fig 6B). Thus it is likely that compensating high levels of eIF2B was one mechanism by which CCA cells overcame P-eIF2α.
Thus far, we have shown the significance of nc886 in the PKR pathway in the cholangiocyte/CCA cell culture. To corroborate our in vitro findings, we measured nc886 in clinical specimens from CCA patients. Among 16 pairs of CCA tumors and adjacent normal tissues, nc886 decreased in tumor in eight cases, increased in four cases, and did not change significantly in four cases (Fig 7A). This pattern was very similar to that observed in the seven CCA cell lines (Fig 1). Compared to MMNK1, nc886 decreased in four CCA cell lines and increased in one CCA cell line.
Next, we measured P-PKR in another set of eight CCA samples with matched normal liver tissue samples by immunohistochemistry (IHC). P-PKR was strongly expressed in CCA but not at all in surrounding fibrous tissues (Fig 7B). In six of eight patients, the P-PKR expression in CCA was stronger than in matched bile ducts or liver tissues (Fig 7B-C). Thus, we have provided evidence here that the nc886/P-PKR expression in CCA patients was similarly altered to that observed in the CCA cell lines (Fig 1). Although accumulation of more clinical data will be needed to assess nc886/P-PKR's in vivo significance more accurately, we suggest that the nc886/PKR tumor surveillance mechanism could operate in vivo.
In this report, we substantiated that a non-coding RNA, nc886, represses PKR. We have provided clear evidence that nc886 directly binds to PKR and inhibits its phosphorylation and kinase activity (Fig 2). Their molecular interaction is being characterized in detail (SHJ and YSL; manuscript in preparation). The high P-PKR (thus elevated PKR activity) in CCA cells, albeit not all of them, is attributed to suppression of nc886 therein. This causal relationship is probably true also in breast cancer cells, as we found an anti-correlation between nc886 and P-PKR by comparing published data (18) to our data (1).
Our data presented here in CCA support our tumor surveillance model, which is described in the Introduction and illustrated in Fig 8. In non-malignant MMNK1 cells, artificial suppression of nc886 led to apoptosis through the classical PKR cell death pathway via P-eIF2α (Fig 3). We presume that the canonical PKR/eIF2α pathway is intact in most pre-malignant cells and eliminates these cells when nc886 is suppressed during certain stages of tumorigenesis, similarly to MMNK1 cells.
However, cancer is a complex genetic disease involving an accumulation of mutations. By chance, these mutations could confer upon cells mechanisms to subvert the PKR/eIF2α cell death pathway. Such malignant cells would survive a high expression of P-PKR and/or P-eIF2α, as exemplified by the CCA cells in this study. Such bypass mechanisms seem to be diverse. Herein we have identified two distinct survival mechanisms: 1) the failure of eIF2α phosphorylation by P-PKR (M214 and M139) and 2) sustained global protein synthesis despite P-eIF2α (M156). At the molecular level, there may be myriads of such mechanisms which await further study.
Regarding the modified PKR pathways that CCA cells have developed, several points should be discussed. First, the P-eIF2α branch ceased to function, but P-PKR and its NF-κB branch remained intact in all the CCA cells tested here. Although CCA cells were P-PKR+, PPKR was further induced by nc886 depletion (Fig 2) or introduction of dsRNA (Fig 4), and the P-PKR activated NF-κB (Fig 5). Second, the elevated intrinsic expression of P-eIF2α in CCA cells was independent of P-PKR (Fig 6A). This was surprising, given their established relationship. However, PKR is not the only kinase for eIF2α. Besides PKR, there are at least three kinases that phosphorylate eIF2α [reviewed in (38)]. P-eIF2α is also regulated by its de-phosphorylation (39, 40). Currently, we do not know what causes the intrinsic expression of P-eIF2α. In any case, we have shown that one mechanism for CCA cells to tolerate P-eIF2α was overexpression of eIF2B (Fig 6B).
Collectively from our data obtained from CCA cells, we propose an updated tumor surveillance model. First, cells develop a mechanism (for example, eIF2B overexpression) by which they survive in spite of the classical P-eIF2α effects. Perhaps, this process is stochastic by random mutations. In any case, these cells are able to survive, even if nc886 is suppressed and thereby PKR is activated during tumorigenesis. Once these cells bypass the nc886/PKR/eIF2α action, they take advantage of P-PKR's oncogenic role by virtue of its ability to activate NF-κB. Consistently, we and others have shown that NF-κB activity is higher in CCA and required for CCA cell proliferation [Fig 5 and (31)].
Given that nc886 (1) and PKR [reviewed in (6)] are expressed ubiquitously, we expect that the tumor surveillance mechanism operates in a wide range of cancers besides CCA. Additionally, the suppression of nc886 may be used for therapeutic purposes or as prognosis of cancer progression. We will expand our study from CCA to other types of cancer, such as breast, lung, and head-and-neck cancers (1). Obviously, there are many unanswered questions. For example, what triggers the suppression of nc886, the first critical event in the nc886-PKR pathway? What is the molecular mechanism for the modified P-eIF2α branch? Most importantly, an animal model system needs to be developed in order to evaluate the in vivo contribution of the nc886/PKR tumor surveillance mechanism to cancer incident rates.
MMNK1 cholangiocyte and CCA cells (summarized Table 1) were a kind gift from Dr. Sopit Wongkham (Khon Khan University, Thailand). Antibodies were obtained as follows: p58IPK, caspase-3 (cleaved), AIF, PARP (cleaved), eIF2B-ε from Cell Signaling Technology (Danvers, MA); MetAP2(p67) from Invitrogen (Carlsbad, CA). All other antibodies were described in (1). Basic chemicals and reagents, including 2-AP and Fumagillin, were purchased from Sigma-Aldrich (St. Louis, MO). CAPE was purchased from Tocris Bioscience (Minneapolis, MN)
pNF-κB-Luc (Stratagene, La Jolla, CA ), pRL-SV40 (Promega, Madison, WI), and pcDNA3.1-Zeo(+)-Pp (1) were used for luciferase assays. siRNAs against PKR were Stealth RNAi™ siRNA (Invitrogen) whose sequences are available upon request. Yeast tRNA was purchased from Applied Biosystems/Ambion (Carlsbad, CA). Anti-oligos (against nc886 and vtRNA1-1), synthetic nc886, and Poly(I:C)-LMW (0.2-1 kb) were purchased or prepared as described in (1). siRNA and anti-oligos were transfected with Lipofectamine™ RNAiMAX reagent (Invitrogen). Poly(I:C), yeast tRNA, and plasmid DNAs were transfected with Lipofectamine™ 2000 reagent (Invitrogen),
All these assays were performed as previously described in (1), except that BrdU incorporation assays were performed by using Cell Proliferation ELISA BrdU (Roche Applied Science, Indianapolis, IN).
PKR immunoprecipitation was performed as described in (41), except that all the buffers contained 80 units/ml of RNase inhibitor (New England Biolabs, Ipswich, MA) and 1 mM Ribonucleoside Vanadyl Complex (New England Biolabs). Washed immunoprecipitates (bound to beads) were subjected to different further manipulations per experimental purposes. To test nc886 association with PKR (Fig 2C experiments), the beads were resuspended with Trizol reagents (Invitrogen) for RNA isolation. During RNA isolation, 5 μg of yeast tRNA was added as a carrier for isopropanol precipitation. To measure in vitro kinase activity of PKR (Fig 2E-F and Fig 5A experiments), the beads were resuspended in 20 μl of kinase buffer (41) containing 2 μCi of [γ-32P]-ATP and incubated at 30°C for 20 min. The reaction was quenched by adding 2X SDS-PAGE loading buffer and resolved on a 10% SDS-polyacrylamide gel.
Synthetic nc886 for an EMSA template was prepared by in vitro transcription with [α-32P]-CTP. Purified PKR was the N-terminal segment (amino acid coordinates 1-184 of NP_002750.1) containing two dsRNA binding domains (dsRBM1-2). dsRBM1-2 was overexpressed from pET-23a plasmid (Novagen, Gibbstown, NJ) in E. coli and was purified on a Ni-NTA column (Qiagen, Valencia, CA). Purified nc886 and dsRBM1-2 were mixed in 20 μl EMSA buffer (200 mM Tris pH7.4, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 40 units RNase inhibitor, 2.5 μg tRNA, 2 μg BSA) and incubated for 15 min on ice. The samples were resolved on a 10% non-denaturing polyacrylamide gel.
Subconfluent cells (~106) in a 6-cm dish were treated with 4 μCi of [35S]-methionine in 2 ml of Dulbecco's Modified Eagle Medium (without L-glutamine, L-methionine, and L-cystine) containing dialyzed fetal bovine serum. After 1 hr incubation, cells were harvested and lysed. Cell lysates were precipitated with 10% trichloroacetic acid, resuspended in 1X SDS-PAGE loading buffer, and loaded onto a 10% SDS-polyacrylamide gel. The gel was stained with Coomassie brilliant blue, dried, and visualized by autoradiography. Aliquots of lysates were also subjected to scintillation counting using Beckman LS 6500 Scintillation Counter to quantify incorporated [35S]-methionine.
The frozen liver tissues of 16 CCA patients admitted to Srinakarin Hospital (an affiliated hospital to Khon Kaen University) were obtained from the specimen bank of the Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine (Khon Kaen University, Thailand). Informed consent was obtained from each subject before surgery and the research protocol (#HE521209) was approved by the Human Research Ethics Committee (Khon Kaen University). Normal bile duct epithelia were examined and taken from non-cancerous portions of CCA liver tissues.
Eight formalin-fixed, paraffin-embedded intrahepatic CCA samples with matched normal liver tissue samples were obtained from University of Texas, MD Anderson Cancer Center. All samples were prepared in one tissue microarray block and then cut in 5 μm-thick sections. Using the rabbit antibody against P-PKR at Thr446 (Abcam, Cambridge, MA), the immunostaining was performed by the avidin–biotin–peroxidase complex method. Antigen retrieval was performed by using citrate buffer (pH 6.0). The section was incubated with the the primary antibody (1:50 dilution) for 45 minute and biotinylated anti-rabbit IgG for 30 minutes at room temperature. Finally, 3,3′-diaminobenzidine and Mayer's hematoxylin were used as a chromogen and counter staining, respectively. The P-PKR expression was interpreted by a pathologist.
We thank Dr. Sopit Wongkham, Dr. Kanlayanee Sawanyawisuth, and Ms. Sirinapa Sribenja (Khon Khan University, Thailand) for CCA cells and helpful discussion; Dr. Inhan Lee (miRcore, MI) for helpful advice; Research Histology Core Laboratory Facility, University of Texas, MD Anderson Cancer Center (funded by the National Cancer Institute Grant # CA16672) for tissue preparation and IHC. This work was supported by a Research Scholar Grant, RSG-12-187-01 – RMC from the American Cancer Society to YSL, by start-up funding from the Sealy Center for Cancer Biology at the University of Texas Medical Branch to YSL, by the Royal Golden Jubilee (RGJ) scholarship (PHD/0105/2550) of Thailand Research Fund (TRF) to NK, and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0022022) to SHJ.
Conflict of Interest
The authors declare no conflict of interest.