Abstract
Human polynucleotide phosphorylase (hPNPaseold-35) is a type I IFN-inducible 3′,5′ exoribonuclease that mediates mRNA degradation. In melanoma cells, slow and sustained overexpression of hPNPaseold-35 induces G1 cell cycle arrest ultimately culminating in apoptosis, whereas rapid overexpression of hPNPaseold-35 directly promotes apoptosis without cell cycle changes. These observations imply that inhibition of cell cycle progression and induction of apoptosis by hPNPaseold-35 involve multiple intracellular targets and signaling pathways. We now provide evidence that the apoptosis-inducing activity of hPNPaseold-35 is mediated by activation of double-stranded RNA–dependent protein kinase (PKR). Activation of PKR by hPNPaseold-35 precedes phosphorylation of eukaryotic initiation factor-2α and induction of growth arrest and DNA damage-inducible gene 153 (GADD153) that culminates in the shutdown of protein synthesis and apoptosis. Activation of PKR by hPNPaseold-35 also instigates down-regulation of the antiapoptotic protein Bcl-xL. A dominant-negative inhibitor of PKR, as well as GADD153 antisense or bcl-xL overexpression, effectively inhibits apoptosis induction by hPNPaseold-35. These studies elucidate a novel pathway by which an evolutionary conserved RNA-metabolizing enzyme, hPNPaseold-35, regulates cell growth and viability. [Cancer Res 2007;67(17):7948–53]
- human polynucleotide phosphorylase
- hPNPaseold-35
- melanoma
- PKR
- apoptosis
Introduction
Human polynucleotide phosphorylase (hPNPaseold-35) was cloned as a novel gene overexpressed during terminal differentiation of human melanoma cells as well as senescence of progeroid fibroblasts ( 1). It is a 3′,5′ exoribonuclease involved in mRNA degradation and is conserved through evolution from prokaryotes to higher mammals ( 2, 3). hPNPaseold-35 is a type I IFN (IFN-α/β)-inducible early response gene ( 1, 4). Overexpression of hPNPaseold-35 by adenovirus (Ad.hPNPaseold-35) transduction induces a senescent phenotype in primary melanocytes as evidenced by senescence-associated β-galactosidase staining, cell cycle arrest in the G1 phase, and morphologic and gene expression changes ( 5, 6). In cancer cells, a low multiplicity of Ad.hPNPaseold-35 infection causes an initial cell cycle arrest followed by apoptosis ( 5). In contrast, upon infection at a high multiplicity, resulting in sustained elevated levels of hPNPaseold-35 over a short time, no cell cycle arrest is evident with cells rapidly entering into apoptosis ( 5).
As an exoribonuclease, hPNPaseold-35 has the unique property of specifically targeting c-myc mRNA degradation ( 5, 6). Myc is an essential regulator of the G1-S cell cycle checkpoint ( 7), and degradation of c-myc mRNA by hPNPaseold-35 causes cell cycle arrest in G1 phase ( 5, 6). Type I IFNs down-regulate c-myc posttranscriptionally causing G1 arrest ( 8, 9), and this effect is mediated by induction of hPNPaseold-35 ( 9). Accordingly, small interfering RNA (siRNA) inhibition of hPNPaseold-35 significantly protects cells from IFN-β–induced growth inhibition, authenticating that hPNPaseold-35 plays a fundamental role in IFN response ( 9). Because hPNPaseold-35 can induce apoptosis without G1 arrest ( 5), there must be additional cellular pathways modulated by hPNPaseold-35. To elucidate these pathways, we presently focused on an IFN-inducible molecule known to promote apoptosis.
Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an IFN-inducible kinase that plays a decisive role in mediating antiviral and antitumor responses ( 10). PKR is a 551-amino acid protein containing two dsRNA-binding domains in the NH2 terminus and a serine/threonine kinase domain in the COOH terminus. Interactions with dsRNA or other activators modify PKR conformation, resulting in homodimerization, autophosphorylation, and activation. Once activated, PKR phosphorylates several substrate targets, the most well characterized being eukaryotic initiation factor-2α (eIF-2α) on Ser51, which can lead to inhibition of protein synthesis, growth suppression, and apoptosis ( 10). Although phosphorylation of eIF-2α extinguishes global protein synthesis, certain stress response mRNAs, such as growth arrest and DNA damage-inducible gene 153 (GADD153), acquire a selective translational advantage causing growth inhibition and apoptosis ( 11). Overexpression of PKR exerts a strong growth-suppressive and toxic effect on the host cell through translation suppression. In contrast, expression of a catalytically inactive PKR variant, which functions as a dominant-negative inhibitor of PKR, induces malignant transformation of NIH 3T3 cells and tumorigenicity in nude mice, suggesting that PKR may function as a suppressor of cell proliferation and tumorigenesis ( 10, 12).
We now establish that hPNPaseold-35 mediates apoptosis by activation of PKR and phosphorylation of eIF-2α. PKR activation results in induction of GADD153 and down-regulation of the antiapoptotic protein Bcl-xL with subsequent promotion of apoptosis. These findings uncover a previously unidentified pathway by which hPNPaseold-35 regulates cell growth and viability and provide new insights into the mechanism of action of this evolutionary conserved RNA-metabolizing enzyme.
Materials and Methods
Cell lines and cell viability assays. HO-1 human metastatic melanoma cells were cultured as described ( 5). Cell viability was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining ( 5).
Construction of adenovirus and infection protocols. The recombinant replication-incompetent adenovirus expressing hPNPaseold-35 (Ad.hPNPaseold-35) was described ( 1, 5). Cells were infected with a multiplicity of infection (MOI) of 100 plaque-forming units (pfu)/cell of Ad.vec (control replication-incompetent adenovirus) or Ad.hPNPaseold-35 ( 5).
Plasmids and transfection protocols. Wild-type PKR (PKR-WT) and catalytically inactive PKR that lacks six amino acids (361–366) and functions as a dominant-negative inhibitor of PKR (PKR-Δ6) expression plasmids were described ( 12). Bcl-2 and Bcl-xL expression plasmids were kindly provided by Dr. John C. Reed (Burnham Institute for Medical Research, La Jolla, CA). Expression plasmid for GADD153 antisense (GADD153AS) was described ( 13). Control and PKR siRNA were obtained from Santa Cruz Biotechnology. For viability studies, cells were seeded in 6-cm dishes and different plasmids were transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. The next day, cells were trypsinized, replated in a 96-well plate, and infected with different adenoviruses and MTT assays were done ( 5).
Colony formation assays. Cells were seeded in 6-cm dishes and transfected the next day with different plasmids, and colony formation assays were done ( 5).
Cell cycle and apoptosis analysis. Cells were seeded in 10-cm dishes, transfected the next day with different plasmids, and the following day infected with different adenoviruses. Cell cycle and apoptosis were analyzed as described ( 5).
RNA isolation and Northern blot analysis. Total RNA was extracted from cells using Qiagen RNeasy Mini kit (Qiagen) according to the manufacturer's protocol and Northern blotting was done as described ( 5). The cDNA probes used were full-length human GADD153, full-length human Bcl-xL, a 500-bp fragment from hPNPaseold-35, and full-length human glyceraldehyde-3-phosphate dehydrogenase.
Western blot analysis. Western blotting was done as described ( 5). The primary antibodies included the following: anti-PKR (1:200; rabbit polyclonal; Santa Cruz Biotechnology), anti-phospho-PKR (1:500; rabbit polyclonal; Invitrogen), anti-eIF-2α (1:200; rabbit polyclonal), anti-phospho-eIF-2α (1:200; rabbit polyclonal), anti-Bcl-2 and anti-Bcl-xL (1:1,000; rabbit polyclonal; kindly provided by Dr. John C. Reed), anti-GADD153 (1:1,000; mouse monoclonal; Abcam), and EF1α (1:1,000; mouse monoclonal; Upstate Biotechnology).
[35S]methionine incorporation assay. Cells were infected with different adenoviruses and cultured for 4 h in methionine-free medium that was replaced by medium containing [35S]methionine (50 μCi/mL). Cells were harvested after 4 h and lysed, and [35S]methionine incorporation was analyzed by a liquid scintillator.
Statistical analysis. Statistical analysis was done using one-way ANOVA followed by Fisher's protected least significant difference analysis.
Results
Infection of cells with Ad.hPNPaseold-35 results in a comparable amount of hPNPaseOLD-35 protein as produced following IFN treatment ( 5, 6). This observation suggests that the phenotypic changes induced by Ad.hPNPaseold-35 infection occur within physiologic ranges of this protein and are not a result of treatment of cells with supraphysiologic levels of this enzyme. Infection of human melanoma cells (HO-1) with Ad.hPNPaseold-35 at a MOI of 100 pfu/cell, infecting ∼90% of cells ( 5), resulted in a temporal increase in phosphorylated PKR over 3 days without changing total PKR levels ( Fig. 1A, top ). No induction of phosphorylated PKR was evident in control-uninfected cells or following infection with an empty adenovirus (Ad.vec). The phosphorylation of PKR by Ad.hPNPaseold-35 correlated with phosphorylation of eIF-2α without changing total eIF-2α levels ( Fig. 1A, bottom). Ad.hPNPaseold-35 infection also delayed the temporal induction of GADD153 mRNA, which followed PKR and eIF-2α phosphorylation ( Fig. 1B). The level of GADD153 mRNA induction correlated with a progressive increase in hPNPaseold-35 mRNA levels following Ad.hPNPaseold-35 infection. Ad.hPNPaseold-35 also inhibited global protein translation as validated by incorporation of [35S]methionine during new protein synthesis ( Fig. 1C). [35S]methionine incorporation was reduced by ∼75% following Ad.hPNPaseold-35 infection when compared with control or Ad.vec infection.
hPNPaseold-35 activates a global translation inhibition pathway. A, HO-1 cells were either uninfected (control) or infected with the indicated adenovirus at a MOI of 100 pfu/cell and the expression of the indicated proteins was analyzed by Western blotting. Ad.vec-infected cells were harvested at day 3 after infection. B, HO-1 cells were treated as in (A) and expression of the indicated mRNAs was analyzed by Northern blotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, HO-1 cells were treated as in (A), and on day 3 after infection, [35S]methionine incorporation assay was done as described in Materials and Methods. D, HO-1 cells were infected as in (A) and, the next day, either mock transfected (control) or transfected with control or PKR siRNA. The expression of the indicated proteins was analyzed by Western blotting 2 days after transfection.
To confirm a functional relationship between Ad.hPNPaseold-35-induced PKR activation and eIF-2α phosphorylation and GADD153 induction, HO-1 cells were infected with either Ad.vec or Ad.hPNPaseold-35 and transfected with control siRNA or PKR siRNA and Western blot analysis was done ( Fig. 1D). Transfection of PKR siRNA, but not control siRNA or untransfected control, resulted in >90% reduction in PKR protein levels ( Fig. 1D, row 2) with inhibition of Ad.hPNPaseold-35-induced phosphorylation of PKR and eIF-2α ( Fig. 1D, rows 1 and 3, respectively) and prevention of GADD153 induction ( Fig. 1D, row 5). However, no change in total eIF-2α or EF1α housekeeping protein levels was observed under any treatment condition ( Fig. 1D, rows 4 and 7, respectively). Western blot analysis confirmed hPNPaseOLD-35 protein synthesis on Ad.hPNPaseold-35 infection ( Fig. 1D, row 6).
To scrutinize the role of PKR activation in controlling hPNPaseold-35 function, HO-1 cells were transfected with either PKR-WT or PKR-Δ6 expression plasmid and then infected with either Ad.vec or Ad.hPNPaseold-35 at 100 pfu/cell and cell viability was assessed by standard MTT assay over 6 days ( Fig. 2A ). Infection with Ad.hPNPaseold-35 profoundly reduced cell viability over control-uninfected or Ad.vec-infected cells. Overexpression of PKR-WT or PKR-Δ6 alone did not significantly affect cell viability. However, PKR-Δ6 afforded significant protection from growth inhibition by Ad.hPNPaseold-35. This ability of PKR-Δ6 to confer protection from Ad.hPNPaseold-35 was also replicated in colony formation assays ( Fig. 2B). As a corollary, apoptosis induction by Ad.hPNPaseold-35, analyzed by propidium iodide staining and flow cytometry, was significantly inhibited by PKR-Δ6 ( Fig. 2C).
Inhibition of PKR protects from Ad.hPNPaseold-35-mediated growth inhibition and apoptosis induction. A, HO-1 cells were either untreated or transfected with the indicated plasmids. The next day, the cells were infected as in Fig. 1A. Cell viability was analyzed by MTT assays on days 3 and 6 after infection. B, HO-1 cells were treated as in (A) and colony formation assays were done as described in Materials and Methods. C, HO-1 cells were treated as in (A) and apoptosis was analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SD.
As a consequence of PKR activation, GADD153 is induced and Bcl-2 and Bcl-xL proteins are down-regulated, resulting in apoptosis. Ad.hPNPaseold-35 infection did not change the level of Bcl-2 protein when compared with Ad.vec infection ( Fig. 3A, row 1 ). However, Bcl-xL protein levels were significantly down-regulated by Ad.hPNPaseold-35 infection ( Fig. 3A, row 2), suggesting specificity, at least in HO-1 cells, of hPNPaseold-35 in regulating the levels of defined antiapoptotic proteins. The down-regulation of Bcl-xL protein levels was inhibited by PKR siRNA or by a GADD153AS expression construct ( Fig. 3A, row 2). The efficacy of GADD153AS to inhibit GADD153 expression was confirmed by Western blotting ( Fig. 3A, row 3). GADD153 is known to down-regulate transcription of Bcl-2 mRNA. To determine whether down-regulation of Bcl-xL protein by Ad.hPNPaseold-35 occurs at the transcriptional level, Bcl-xL mRNA expression was analyzed by Northern blotting following Ad.hPNPaseold-35 infection. Ad.hPNPaseold-35 did not alter Bcl-xL mRNA levels, which remained unchanged in the presence of PKR siRNA or GADD153AS ( Fig. 3B). These findings indicate that, although PKR activation and GADD153 induction are required for Ad.hPNPaseold-35-mediated down-regulation of Bcl-xL, the regulatory mechanism is different from the known mode of regulation of Bcl-2.
Ad.hPNPaseold-35 down-regulates Bcl-xL. A, HO-1 cells were infected with the indicated adenovirus at a MOI of 100 pfu/cell and, the next day, transfected with empty pcDNA3.1 vector, control siRNA, PKR siRNA, or GADD153AS expression construct. The cells were harvested on day 3 after infection, and the expression of the indicated proteins was analyzed by Western blotting. B, HO-1 cells were treated as in (A). The cells were harvested on day 3 after infection, and the expression of the indicated mRNAs was analyzed by Northern blotting.
Further confirmation of an involvement of GADD153 up-regulation and Bcl-xL down-regulation in Ad.hPNPaseold-35-mediated growth inhibition and apoptosis induction was provided with a GADD153AS construct and Bcl-2 and Bcl-xL expression constructs ( Fig. 4A–C ). It should be noted that the experimental results presented in Figs. 2 and 4 were done simultaneously at least three or more times to maintain the same controls for all experiments. To provide better clarity for presentation, these results are separated and presented in two figures, although some of the control data points in these two figures are the same. Both GADD153AS and Bcl-xL, but not Bcl-2, afforded significant protection from Ad.hPNPaseold-35-mediated growth inhibition as confirmed by cell viability (MTT) and colony formation assays ( Fig. 4A and B). Apoptosis assays also showed that GADD153AS and Bcl-xL provided significant protection from Ad.hPNPaseold-35-induced apoptosis ( Fig. 4C). Complete protection from hPNPaseold-35-mediated growth suppression, viability alterations, and apoptosis induction was not evident using these independent inhibitory approaches, suggesting that blocking multiple downstream pathways activated by PKR may be mandatory for complete protection.
Inhibition of GADD153 and overexpression of Bcl-xL protect from Ad.hPNPaseold-35-mediated growth inhibition and apoptosis induction and a model of hPNPaseold-35 action. A, HO-1 cells were either untreated or transfected with the indicated plasmids. The next day, the cells were infected as in Fig. 1A. Cell viability was analyzed by MTT assay on days 3 and 6 after infection. B, HO-1 cells were treated as in (A) and colony formation assays were done as described in Materials and Methods. C, HO-1 cells were treated as in (A) and apoptosis was analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SD. D, schematic representation of the molecular pathways by which hPNPaseold-35 induces apoptosis. The generation of dsRNA by hPNPaseold-35 is hypothetical and remains to be determined.
Discussion
hPNPaseold-35 potentially inhibits cell growth and viability through multiple mechanisms. A slow, sustained increase in hPNPaseold-35 levels in melanoma cells, resulting from infection with Ad.hPNPaseold-35 at low MOI, causes initial cell cycle arrest followed by apoptosis, which is similar to what occurs following IFN-β treatment in HO-1 cells ( 5, 6). In contrast, rapid, high-level hPNPaseold-35 overexpression, still within the range of maximal induction by IFN-β resulting from infection with high MOI of Ad.hPNPaseold-35, promotes frank apoptosis without cell cycle arrest ( 5). These observations suggest that inhibition of cell cycle progression and induction of apoptosis by hPNPaseold-35 might involve multiple and potentially distinct intracellular targets and signaling pathways. Cell cycle arrest by hPNPaseold-35 involves specific degradation of c-myc mRNA ( 5, 6), and in the present studies, we verify that activation of PKR might be an important pathway for apoptosis induction.
Ad.hPNPaseold-35 infection phosphorylates and activates PKR without changing total PKR levels. HO-1 cell growth and viability are not significantly affected by overexpression of a PKR-WT expression plasmid. These observations suggest that a triggering event(s) is required to activate PKR and prompt the question of how hPNPaseold-35 activates PKR. It is well established that PKR activation is induced by its binding to dsRNA ( 14). As a RNA degradation enzyme, hPNPaseold-35 can degrade only single-stranded RNA and its activity is stalled when it encounters a secondary hairpin structure in a mRNA molecule ( 15). Hypothetically, an incomplete RNA degradation process caused by hPNPaseold-35 might generate intracellular dsRNA molecules resulting in the activation of PKR. Additionally, PKR activation by hPNPaseold-35 might be a consequence of hPNPaseold-35, generating physiologically relevant small-inhibitory RNAs or microRNAs, a possibility that is currently being investigated. In principle, these RNA intermediates that are generated by hPNPaseold-35 might be involved in activation of PKR, triggering a cascade of events leading to phosphorylation of eIF-2α, induction of GADD153, and inhibition of Bcl-xL culminating in translational inhibition and apoptosis. Because overexpression of PKR-Δ6, GADD153AS, or Bcl-xL only provided partial protection from Ad.hPNPaseold-35-induced apoptosis, it seems that additional mechanism(s) might be involved in this process. One potential mechanism that we unraveled previously is degradation of c-myc mRNA by hPNPaseold-35, leading to cell cycle arrest and apoptosis ( 5). Additional as yet unidentified events might also play a role in this phenomenon. An overall schematic model of the events that may mediate hPNPaseold-35-induced apoptosis is presented in Fig. 4D. Overexpression of PKR-WT together with Ad.hPNPaseold-35 infection did not further increase growth inhibition and apoptosis induction by hPNPaseold-35 ( Fig. 2A and C), but there was a modest increase in inhibition of colony formation ( Fig. 2B). A possibility is that Ad.hPNPaseold-35-induced PKR activation is already saturated so that an exogenous PKR effect might not be observed in short-term assays, whereas it can be detected in long-term colony formation assays.
GADD153, also known as CHOP10 [CAAT/enhancer binding protein (C/EBP) homologous protein], is a stress response gene that can induce growth inhibition and/or apoptosis ( 16). GADD153 is a transcription factor that heterodimerizes with members of the C/EBP family of transcription factors and interferes with C/EBP-mediated transcription ( 17). In addition, GADD153 itself can enhance gene transcription by binding to a sequence-specific DNA element or through interactions with other transcription factors, such as activator protein-1 ( 18). A mechanism by which GADD153 induces apoptosis is by down-regulating the activity of the bcl-2 promoter ( 19). However, we did not observe any change in Bcl-2 protein levels on Ad.hPNPaseold-35 infection, whereas a significant reduction in Bcl-xL levels was observed. The functional significance of Bcl-xL reduction was apparent from cell growth and apoptosis studies, confirming that it is Bcl-xL, not Bcl-2, that protects cells from Ad.hPNPaseold-35 action. The molecular mechanism of this differential effect of GADD153 is undefined and raises several interesting questions. Does GADD153 also regulate the Bcl-xL promoter? This does not seem to be the case as evidenced by a lack of alteration in bcl-xL mRNA level on Ad.hPNPaseold-35 infection ( Fig. 3B), indicating that Ad.hPNPaseold-35-induced signaling events induce a posttranslational modification in Bcl-xL protein. Is there any specific event in melanoma cells that increases stability of Bcl-2 protein? Is Bcl-2 function redundant in melanoma cells so that down-regulation of Bcl-xL alone can activate an apoptotic program? Answering these questions will significantly extend our understanding of the molecular mechanism of apoptosis induction in human melanoma cells.
In summary, we identify a novel pathway in which two IFN-inducible molecules, hPNPaseold-35 and PKR, cross-talk to induce an apoptotic cascade. Because PKR, a dsRNA sensor, functions as an antiviral molecule, current findings highlight the possibility that as an integral component of IFN signaling hPNPaseold-35 might also be involved in antiviral responses. Studies are in progress to address these relevant issues to comprehend the pleiotropic functions of this intriguing enzyme. Moreover, based on the ability of hPNPaseold-35 to strongly inhibit cell growth and reduce viability, by inducing senescence and apoptosis, this novel gene could serve as a possible genetic tool for cancer gene therapy. To achieve this objective, it will be mandatory to target expression of this enzyme uniquely in cancer cells, which can be achieved by using a cancer-specific/selective promoter derived from progression elevated gene-3 or human telomerase ( 20, 21). These possibilities are currently being actively pursued ( 22).
Acknowledgments
Grant support: NIH grant CA035675, Samuel Waxman Cancer Research Foundation, and Chernow Endowment. P.B. Fisher is the Michael and Stella Chernow Urological Cancer Research Scientist and a Samuel Waxman Cancer Research Foundation Investigator.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
- Received March 5, 2007.
- Revision received June 11, 2007.
- Accepted July 12, 2007.
- ©2007 American Association for Cancer Research.
References
Volume 67, Issue 17
