¹Department of Surgery, New Jersey Medical School/University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey, NJ 07103, USA

²Surgical Services, VA New Jersey Healthcare System, 385 Tremont Avenue, East Orange, New Jersey, NJ 07018, USA

³Department of Biochemistry and Molecular Biology, New Jersey Medical School/University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey, NJ 07103, USA

⁴Pathology and Laboratory Medicine Services, VA New Jersey Healthcare System, 385 Tremont Avenue, East Orange, New Jersey, NJ 07018, USA

Correspondence to: S H Kim, E-mail: steve.kim@umdnj.edu

The interferon-inducible, double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays key roles in regulation of cell growth and differentiation, and has been postulated as a tumor suppressor. Downstream effectors of PKR include the translation initiation factor, eIF2, and the transcription factor, NF-B. We found elevated levels of PKR protein, dsRNA-dependent PKR autophosphorylation activity, and phosphorylated eIF2 in melanoma cells compared to nontransformed melanocytes in culture. Treatment with interferon-2b further induced PKR expression and activity. Immunohistochemical analysis of primary melanomas demonstrated minimal PKR immunoreactivity, but melanoma lymph node metastases expressed a high level of PKR protein. Furthermore, analysis of colon cancer specimens revealed that transformation from normal mucosa to adenomas and carcinomas was coincident with an increase in PKR expression. These data do not support the concept of PKR as a classic tumor suppressor but instead suggest that PKR upregulation occurs at defined steps in cancer progression, probably as a cellular response to neoplasia.

Oncogene (2002) 21, 8741-8748. doi:10.1038/sj.onc.1205987

PKR; interferon; melanoma; colon; cancer

Introduction

The double-stranded (ds) RNA-activated protein kinase, PKR, is a critical element of the eukaryotic response to viral infection. It has also been associated with a wide range of regulatory and pathologic processes including differentiation, apoptosis, and transformation. PKR controls gene expression at both translational and transcriptional levels. Induction of PKR expression by interferons and its activation by dsRNAs (which are by-products of viral replication) results in phosphorylation of the translation factor eIF2, leading to inhibition of protein synthesis (Barber, 2001; Barber et al., 1995; Clemens and Bommer, 1999; Gil and Esteban, 2000; Jagus et al., 1999; Jaramillo et al., 1995; Mathews and Pe'ery, 2001; Proud, 1995; Samuel, 2001; Sudhakar et al., 2000; Williams, 1999, 2001). By its interaction with IKK-beta, PKR may also regulate the activity of transcriptional activator NF-B. The IKK complex then phosphorylates IKB, the inhibitory subunit of NF-B. This event leads to IB degradation with subsequent release and nuclear translocation of NF-B (Bonnet et al., 2000; Gil et al., 2000, 2001; Ishii et al., 2001; Offermann et al., 1995; Zamanian-Daryoush et al., 2000). It has been postulated that increasing PKR activity has a net tumor suppressive effect within the cell. Early evidence for this came in experiments in which expression of mutant PKR in mouse 3T3 cells led to cellular transformation (Koromilas et al., 1992; Meurs et al., 1993; Romano et al., 1998), whereas upregulation of wild-type PKR activity in M1 myeloid leukemia cells resulted in reversal of the transformed phenotype or apoptosis (Raveh et al., 1996). Furthermore, transformation also resulted from transfection of 3T3 cells with eIF2 mutants that were no longer effective substrates for PKR (Donze et al., 1995).

Despite the above data, the role of PKR as a tumor suppressor is far from clear. Two lines of mice with germline deletions of PKR did not demonstrate any increased propensity to tumorigenesis (Abraham et al., 1999; Yang et al., 1995). Furthermore, fibroblasts from these animals actually had slower proliferation rates in culture than normal controls, and also demonstrated a stall at G1/S (Zamanian-Daryoush et al., 1999). Data from human tumors also is equivocal as to PKR's effect on neoplastic progression. Experiments examining cancers of the colon, breast, liver, and head and neck have shown a positive correlation between increasing PKR expression and more well-differentiated tumors (Haines et al., 1992, 1993b, 1996, 1998; Shimada et al., 1998; Singh et al., 1995; Terada et al., 2000b). Increased expression of PKR has also been shown to correlate with better patient prognosis for certain tumors (Haines et al., 1993a; Zhou et al., 1998). Paradoxically, however, normal tissues tend to demonstrate lower PKR levels than their neoplastic counterparts (Singh et al., 1995; Haines et al., 1996; Shimada et al., 1998; Terada et al., 2000a,b).

In our previous report (Kim et al., 2000), we demonstrated an elevation in PKR level and activity in breast cancer cells when compared to nontransformed mammary cells from patients with fibrocystic disease. Furthermore, a PKR inhibitor found in the non-cancerous cells resulted in a profound decrease in their PKR activity. Overexpression of a second translation initiation factor, eIF2B, spared the breast cancer cells from the growth-inhibitory effect of PKR on protein synthesis. These observations strongly suggested that PKR did not have a tumor suppressor function in these cells, and might even have tumor-promoting properties. Results of the present study indicate that this phenomenon is not limited to breast cancer.

Melanomas are rapidly rising in incidence, and their prognosis is often dismal when they are deeply invasive or have spread to lymph nodes (Kim and Coit, 1998). Systemic treatment with interferon-2b has been shown to improve survival in patients with advanced melanoma (Kirkwood et al., 1996; Pehamberger et al., 1998). Since PKR is a crucial protein mediating cellular response to interferon, we wished to examine what role it may play in these tumors. Our results show that melanomas in cell culture had higher levels of PKR expression and autophosphorylation activity than nontransformed melanocytes, and this was inducible with interferon treatment. Immunohistochemistry for PKR confirmed the higher levels of PKR in melanomas compared to melanocytes. Examination of human tumors revealed a much higher expression level in melanoma lymph node metastases than in primary melanomas. Extension of the immunohistochemical analysis to human colon cancer specimens demonstrated a significantly greater PKR immunoreactivity in adenomas and carcinomas than in adjacent non-neoplastic mucosa. Given these observations, it is clear that PKR cannot be considered a tumor suppressor gene in the classic sense.

Results

PKR protein levels are higher in melanomas than in nontransformed melanocytes

Immunohistochemical examination of two melanoma cell lines, Mel 5 and Mel 24, revealed a higher level of PKR expression than in the NHEM nontransformed melanocyte line (Figure 1a, top panel). The PKR immunoreactivity of the melanoma cells was somewhat less than that of MCF7 breast cancer cells that overexpress PKR (Figure 1a, top panel) (Kim et al., 2000). Similar results were obtained when cell lysates from melanomas and melanocytes were analysed by Western blotting. When normalized to actin expression loading controls, both melanoma lines demonstrated higher levels of expression than the melanocytes (P<0.05) (Figure 1a, bottom panels and Figure 2c). PKR was found mostly in the cytoplasm in the malignant as well as the benign cells, but there was also evidence of nucleolar staining (Figure 1b). Treatment with 500 U/ml of interferon-2b for 18 h increased the level of PKR expression in both melanoma cells and melanocytes to comparable degrees (Figure 2a). These results demonstrate that, as in mammary cells (Kim et al., 2000), progression from the normal to the transformed state in melanocytes is associated with increasing PKR protein levels. The inducibility of PKR by interferon suggests that PKR overexpression in the tumor cells retains some aspects of normal cellular regulation.

PKR kinase activity is much higher in melanomas than in melanocytes

Western blotting for eIF2 detected a higher amount of the phosphorylated form of the initiation factor in the melanoma cells than in melanocyte line, although interferon did not cause significant increase (Figure 2a). This observation suggested that PKR kinase activity is increased in the tumor cells. To determine directly whether the increase in PKR protein level in melanomas is accompanied by an increase in kinase activity, we conducted PKR autophosphorylation assays. Significantly higher PKR activity was observed with extracts of melanoma cells as compared to the melanocyte line (P<0.05) (Figure 2b,c). The activity was dsRNA-dependent and interferon-inducible in all cell lines. The specific activity of PKR (autophosphorylation activity/g of protein) was also higher in melanomas than in melanocytes (Figure 2c), implying that the increased activity in the former could not be explained simply by higher levels of protein in the cancer cells.

In nontransformed breast epithelial cells, we found that the activity of PKR is further decreased compared to breast cancer cells by the presence of a PKR inhibitor, p58 (Kim et al., 2000). We wished to determine if PKR-inhibitory activity was also present in the nontransformed melanocytes, thus explaining their lower PKR specific activity as compared to the melanomas. We therefore performed mixing reactions in which a fixed amount of melanoma cell lysate was mixed with increasing amounts of melanocyte lysate. If these nontransformed cells contained a PKR inhibitor of PKR, we should have seen a decrease in kinase activity as increasing amounts of melanocyte lysate was mixed with the melanoma lysates. Instead, PKR activity increased as increasing amounts of melanocyte cellular protein were added to either of the melanoma cell lines (Figure 3), arguing against any PKR inhibitory activity in the nontransformed melanocytes.

PKR immunoreactivity is higher in lymph nodes metastases than in primary melanomas

We next wished to see if there was any clinical correlation for our cell culture data. Immunohistochemical analysis of primary melanomas (n=6) demonstrated weak but detectable levels of PKR in these tumors as well as in pre-neoplastic melanocytic nevi (Figure 4). Because of the difficulty of identifying individual normal melanocytes within the epidermis of these specimens, we were not able to determine the level of PKR expression in these cells. We observed an intense staining within keratinocytes, as reported previously (Haines et al., 1993b). The highest degree of overexpression in our clinical samples was found in lymph node metastases (n=3), which displayed intense cytoplasmic immunoreactivity (Figure 4c). Of note, both melanoma cell lines used in the present study were derived from nodal metastases and not primary tumors, perhaps explaining their high level of PKR expression and activity.

PKR expression is associated with normal and neoplastic proliferation in human colonic epithelium

Since PKR is upregulated in two very different tumor types, breast cancer and melanoma, we extended our analysis to colon cancer. This malignancy has a high incidence and several advantages for immunohistochemical analysis. First, since phenotypically normal colonic mucosa is found adjacent to cancers, an inherent internal control exists (often visible in the same low-power microscopic field). Second, since colon cancers are thought to arise through a pathway involving well-defined pre-malignant and early malignant lesions, we might be able to assess the specific point during the process of malignant transformation at which PKR is upregulated. Third, the normal colonic epithelium is itself divided into two cell compartments that are well defined both geographically as well as on the basis of the state of cellular proliferation and differentiation, i.e., the crypts of Lieberkuhn and the villi, respectively. The former contain immature proliferating stem cells and their early descendants, while intestinal cells on the villi are post-mitotic and terminally differentiated.

Our immunohistochemical analysis of colonic adenocarcinomas showed a clear and sustained overexpression of PKR at the earliest stages of colonic neoplasia (Figure 5). In normal mucosa, minimal immunoreactivity was noted except at the base of the crypts (Figure 5a). A significant increase in PKR protein was seen in tubular adenomas, an early premalignant lesion (Figure 5b). PKR overexpression remained consistently elevated with increasing stages of neoplastic progression, namely carcinoma in situ, primary invasive cancer, and lymph node metastasis (Figure 5c-e). Correspondingly, PKR expression and activity were detected in two independent colon cancer cell lines, CaCo-2 and HT-29. The kinase activity of the colon carcinoma lines was dsRNA-dependent, and their PKR specific activity was similar to that of the Mel 5 melanoma cell line (Figure 5f). No nontransformed colonic epithelial cell line was available as a control for the colon cancer cell lines. In summation, these data suggest that increased PKR expression and activity is associated with cellular proliferation, transformation, and neoplastic progression in both melanocytes and human colonic epithelium.

Discussion

In response to viral infection, upregulation of PKR has been shown to induce translational arrest and subsequent apoptosis (Barber, 2001). Because of these phenomena, PKR has historically been considered a candidate tumor suppressor. Yet, PKR clearly does not fit the classic description of a tumor suppressor gene. Unlike knockouts of p53 or the retinoblastoma gene product (RB), germline deletion of the PKR gene did not lead to late tumor formation in transgenic mice (Abraham et al., 1999; Yang et al., 1995). Furthermore, mutational inactivation of PKR has not to date been reported in any human tumors. At present, the role of PKR (if any) in neoplasia and/or its prevention is unclear.

For example, two recent studies in human breast cancer cells have led to conflicting conclusions. Savinova et al. (1999) showed elevated protein levels in breast carcinoma cells when compared to normal breast epithelial cells. However, despite the high levels of PKR, its autophosphorylation and eIF2 kinase activity were severely attenuated. In a previous report from our group, we found increases in both PKR protein level and kinase activity in the malignant vs benign breast cells (Kim et al., 2000). These differences cannot be explained simply by differences in methodology. Savinova et al. (1999) performed immunoprecipitation of PKR followed by assay of its kinase activity, whereas in our report (Kim et al., 2000), we did this in the reverse order. However, we repeated the experiments according to Savinova's protocol (data not shown) and found no change in our original results. In either case, both reports clearly demonstrate higher PKR protein levels in transformed vs nontransformed breast cells.

In general, immunohistochemical analyses of PKR in human tumor specimens give further support to this hypothesis (Delhem et al., 2001; Haines et al., 1993b, 1996; Singh et al., 1995; Terada et al., 2000a,b). Our results demonstrate an increase in expression at defined steps of neoplastic progression in two common human cancers. In melanomas, PKR overexpression is associated with lymphogenous metastasis. Both pre-neoplastic melanocytic nevi as well as primary melanomas showed weak immunoreactivity for the protein. This dramatically increased when lymph node metastases were examined (Figure 4). The data in cell culture correlated with this observation. Normal melanocytes had minimal levels of PKR while both lines of melanoma cells were high expressors. Of note, both melanoma cell lines used in the present studies were derived from melanoma lymph node metastases, and this may explain their high levels of PKR protein and activity (Carey et al., 1976). Unlike our previous results in nontransformed and malignant human mammary cells (Kim et al., 2000), we did not note any PKR inhibitory activity in the normal melanocytes. No attenuation of PKR autophosphorylation activity was noted with addition of increasing amounts of normal melanocyte (NHEM) protein to a fixed amount of protein from each melanoma, and in fact, the activity increased with addition of more NHEM protein (Figure 3), suggesting that the higher activity correlates with increasing amount of PKR in each reaction.

The effect of interferon treatment was as expected; PKR expression increased in both the normal and melanoma cells (Figure 2) although we did not notice any effects on eIF2 phosphorylation and cell growth (data not shown). This phenomenon has interesting, if somewhat confounding, implications not only on the mechanism of the therapeutic effect of interferon in melanoma (Kirkwood et al., 1996; Pehamberger et al., 1998), but also more generally on the role of PKR in neoplasia. The possibilities (not all mutually exclusive) are (1) that interferon has a clinical therapeutic effect that is independent of PKR; (2) PKR plays an inhibitory role in clinical melanoma progression that is not phenotypically manifest in cultured cells; (3) PKR activates (or inhibits) pathways that are independent of its kinase activity; or (4) PKR overexpression and activity is a reaction to transformation and not a proximal cause. Recent evidence gives some credence to the fourth possibility. The melanoma differentiation-associated gene (mda7) has tumor suppressive properties and induces apoptosis in a variety of cancer cell lines. Pataer et al. (2002) have demonstrated that adenoviral transfer and upregulation of mda-7 in lung cancer cells results in increased PKR levels and activity, the latter measured by elevated levels of phosphorylated PKR and eIF2 as well as a significant decrease in overall protein synthesis (Pataer et al., 2002). Both the lung cancer cells and normal mouse embryo fibroblasts underwent apoptosis when treated with adenoviral mda-7, a phenomenon that did not occur when PKR-null fibroblasts were treated. This suggested that increased PKR activity was the responsible effector of mda-7's tumor suppressor property. This is the strongest evidence to date that PKR may play an in vivo role in the response to neoplastic transformation and/or progression.

In colon cancers, PKR overexpression was associated with a much earlier step in carcinogenesis, i.e., from phenotypically normal mucosa to adenoma (the earliest pre-cancerous colonic lesion) (Kinzler and Vogelstein, 1996) (Figure 5). We also detected PKR kinase activity in two well-characterized lines of colon cancer cells (Figure 5f), suggesting that PKR is functional in these tumors. The upregulation of expression at such an early stage in neoplasia might imply an important role for PKR in colonic epithelial transformation, again, either as a pro-neoplastic agent or as a response to the earliest changes of cancer formation. One of the first genetic alterations in the colonic adenoma-carcinoma sequence is mutation of the APC gene (Adenomatous Polyposis Coli) that is thought to activate a number of oncogenic proteins, such as c-myc (Sieber et al., 2000). Since PKR has been shown to play a tumor suppressive role in response to activation of this oncogene (Raveh et al., 1996; Shang et al., 1998), one could hypothesize its upregulation on this basis.

Materials and methods

Cell culture

Melanoma cell lines SK-MEL-5 and SK-MEL-24 were obtained from ATCC (Carey et al., 1976) and grown in Eagle's minimal essential medium with nonessential amino acids and 1 mM sodium pyruvate supplemented with 10% fetal bovine serum. Normal human epidermal melanocytes were purchased from BioWhittaker, Inc., Walkersville, MD, USA and grown in their melanocyte medium. Treatment with interferon-2b (Sigma, St. Louis, MO, USA) was done for 18 h by adding the reagent to a final concentration of 500 U/ml in each plate. Cells were harvested for lysis when they were approximately 80-90% confluent. Lysis protocol has been previously described (Kim et al., 2000).

Immunoblotting

Cell lysates (25-50 g protein) were run on SDS/polyacrylamide gels and transferred to PVDF Immobilon membranes using the Biorad^Ò semi-dry blotting apparatus. After probing with the relevant antibody, antigens were detected using the ECL kit from ICN^Ò. Polyclonal antibody to purified human PKR was raised in rabbits (Green and Mathews, 1992). Monoclonal antibody to human eIF2 was a generous gift from Dr MJ Clemens. Polyclonal antibody to phospho-serine⁵¹ eIF2 peptide was purchased from Research Genetics, Inc. Antibodies to eIF2B subunits were kindly provided by Dr Chris Proud (Oldfield et al., 1994). Monoclonal antibody to -actin was obtained from ICN^Ò. Signal intensity was quantified by densitometry.

PKR autophosphorylation assay

PKR activation and autophosphorylation activity was measured in kinase reactions by the protocol described previously (Kim et al., 2000). Reactions contained cell lysates prepared by non-detergent lysis (25-50 g protein/reaction), 15 mM HEPES-KOH (pH 7.4), 5 mM MgCl₂, 1 mM DTT, 10 mg/ml each of leupeptin, pepstatin and aprotinin, 10 mM PMSF, 100 mM ATP, and 0.5 Ci/ml [-³²P]ATP, in the presence or absence of 20 ng/ml of dsRNA. After incubation for 20 min at 30°C, PKR was immunoprecipitated for 1 h on ice using rabbit polyclonal antibody at a dilution of 1 : 500 (Green and Mathews, 1992). Immune complexes were collected by addition of 50 l of a 10% slurry (v/v: 0.043 g in 1.5 ml) of protein A-sepharose in NET²⁺ buffer containing 2 mg/ml of bovine serum albumin (BSA). The mixture was agitated for 30 min at 4°C, and the beads washed several times with 1 ml NET²⁺ buffer. Finally, the beads were boiled in Laemmli sample buffer (Laemmli, 1970) for 15 min, centrifuged at low speed, and the supernatant analysed by SDS/polyacrylamide gel electrophoresis and autoradiography. Signal intensity was quantified by densitometry.

Immunohistochemical analysis

Cells were grown to near confluence and harvested by gentle scraping from the plates. After resuspension in phosphate buffered saline (PBS) the cells were pelleted, fixed for 5 h in phosphate buffered formalin, and then embedded overnight in paraffin. Blocks were cut into 3.5-m-thick sections. For human tissue analysis, similar sections were cut from archived tumor samples. After deparaffinization, the samples were rehydrated and then boiled in citrate buffer (Antigen Retrieval Citra, BioGenex Laboratories, Inc., San Ramon, CA, USA) for 10 min. The cooled sections were washed in PBS and then blocking buffer (PBS, 1% BSA, 0.2% skimmed milk, 0.3% Triton X-100). Primary antibody was added at a dilution of 1 : 500 (Green and Mathews, 1992). Normal rabbit serum was used as a control. Antigens were detected using biotinylated secondary antibody (BioGenex MultiLink), followed by alkaline phosphatase-conjugated streptavidin, and then Fast Red as the final chromagen (BioGenex). Slides were lightly counterstained with hematoxylin. Previously performed blocking controls demonstrated the specificity of the primary antibody (Kim et al., 2000).

Statistical analysis

In order to compare the PKR protein levels and activities between cell lines, signal intensities from densitometry analysis were normalized between different blots. Mean values for each cell line were obtained and were compared statistically using the independent samples t-test.

Acknowledgements

We wish to thank Gene Marquet for valuable technical assistance with the immunohistochemistry. This work was supported by grant AI34552 to MBM from the National Institute of Health.

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Figure 1 (a) PKR expression was assessed in two melanoma cell lines (Mel 5 and Mel 24) as well as in a nontransformed melanocyte line (NHEM) using both immunohistochemistry and Western blotting. As a positive control, we used a known overexpressing breast cancer line (BC). Actin expression was used to control for loading. Both melanomas demonstrated higher levels of expression than the melanocytes. (b) High power view (100´) demonstrating the subcellular localization of PKR protein detectable as Fast Red chromagen in this immunohistochemical analysis. Expression was predominantly cytoplasmic, however, intense nucleolar reactivity was noted as well (arrow)

Figure 2 (a) Western blot showing that PKR expression is inducible by treatment with IFN-2b in both transformed and nontransformed cells. An increase in the phosphorylated form of eIF2 was noted in both melanomas, suggesting an increase in PKR kinase activity. (b) PKR autophosphorylation assay demonstrating that PKR kinase activity is elevated in melanomas (Mel 5 and 24) as compared to melanocytes (NHEM). This is inducible with interferon treatment and is double-stranded RNA dependent. Activity of purified PKR protein is shown at far right as a positive control. (c) PKR protein levels as measured by Western blot and densitometry are slightly higher in melanomas than in nontransformed melanocytes. However, PKR kinase activities (as measured by autophosphorylation assays) are significantly higher in melanomas (assays were performed as in Materials and methods and quantitated by densitometry). As a result, the specific activity (activity/g of PKR) is much lower in the nontransformed cells. Y-axis units are relative and arbitrary, and the data reflect the mean and standard deviation (error bars) from three separate experiments using cell lines not previously treated with interferon. *Denotes that the values for NHEM were significantly lower than that for either melanoma cell line (P<0.05)

Figure 3 The results of a mixing reaction are shown, in which increasing amounts of normal melanocyte lysate was added to a fixed amount of lysate from melanoma cells. This was done for both Mel 5 and Mel 24 tumor lines. Results demonstrate an increase in kinase activity as more melanocyte protein is added, making it unlikely that a PKR inhibitor is present in the melanocytes, as has been reported in benign breast epithelial cells

Figure 4 (a and b) Immunohistochemical analyses of human epidermis demonstrate weak but detectable signal in primary melanomas (solid arrow). Intense immunoreactivity is noted in keratinocytes (hashed arrow). (c) In contrast, melanoma metastatic to lymph nodes demonstrated very high levels of PKR overexpression

Figure 5 Immunohistochemical analysis of samples from human colon cancer specimens. (a) Phenotypically normal colonic mucosa is shown. PKR is detectable as Fast Red and is noted with more intensity in the crypts of Lieberkuhn (arrow). Minimal staining is noted in the villi. (b) Intense cytoplasmic immunoreactivity is observed in tubular adenomas, neoplastic lesions considered the first step in the progression to colon cancer. (c) An area of carcinoma in situ is shown. These lesions are considered malignant but at a step just prior to invasion through the epithelial basement membrane. PKR overexpression is again noted. Both primary invasive cancers (d) and lymph node metastases (e) also demonstrated overexpression. In (d) an adjacent area of phenotypically normal epithelium on the left graphically shows the differential PKR expression levels. (f) PKR autophosphorylation assay showing kinase activity in Caco-2 and HT-29 colon cancer cell lines. The reaction is dsRNA dependent and of similar activity as that in Mel 5 cells. PKR protein levels and corresponding actin levels in the three cell lines are shown in the bottom panel (these are all taken from the same blot)