Skip to main content
  • AACR Publications
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

  • Register
  • Log in
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Focus on Computer Resources
    • 75th Anniversary
    • Meeting Abstracts
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • OnlineFirst
    • Editors' Picks
    • Citations
    • Author/Keyword
  • News
    • Cancer Discovery News
  • AACR Publications
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in

Search

  • Advanced search
Cancer Research
Cancer Research

Advanced Search

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Focus on Computer Resources
    • 75th Anniversary
    • Meeting Abstracts
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • OnlineFirst
    • Editors' Picks
    • Citations
    • Author/Keyword
  • News
    • Cancer Discovery News
Priority Reports

Activation of Double-Stranded RNA–Dependent Protein Kinase, A New Pathway by Which Human Polynucleotide Phosphorylase (hPNPaseold-35) Induces Apoptosis

Devanand Sarkar, Eun Sook Park, Glen N. Barber and Paul B. Fisher
Devanand Sarkar
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eun Sook Park
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Glen N. Barber
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul B. Fisher
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1158/0008-5472.CAN-07-0872 Published September 2007
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

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.

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

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).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

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.

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

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.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

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

    1. ↵
      Leszczyniecka M, Kang DC, Sarkar D, et al. Identification and cloning of human polynucleotide phosphorylase, hPNPaseold-35, in the context of terminal differentiation and cellular senescence. Proc Natl Acad Sci U S A 2002; 99: 16636–41.
      OpenUrlAbstract/FREE Full Text
    2. ↵
      Sarkar D, Fisher PB. Polynucleotide phosphorylase: an evolutionary conserved gene with an expanding repertoire of functions. Pharmacol Ther 2006; 112: 243–63.
      OpenUrlCrossRefPubMed
    3. ↵
      Leszczyniecka M, DeSalle R, Kang DC, Fisher PB. The origin of polynucleotide phosphorylase domains. Mol Phylogenet Evol 2004; 31: 123–30.
      OpenUrlCrossRefPubMed
    4. ↵
      Leszczyniecka M, Su ZZ, Kang DC, Sarkar D, Fisher PB. Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPaseold-35, a type I interferon inducible early response gene. Gene 2003; 316: 143–56.
      OpenUrlCrossRefPubMed
    5. ↵
      Sarkar D, Leszczyniecka M, Kang DC, et al. Down-regulation of Myc as a potential target for growth arrest induced by human polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells. J Biol Chem 2003; 278: 24542–51.
      OpenUrlAbstract/FREE Full Text
    6. ↵
      Sarkar D, Park ES, Emdad L, Randolph A, Valerie K, Fisher PB. Defining the domains of human polynucleotide phosphorylase (hPNPaseOLD-35) mediating cellular senescence. Mol Cell Biol 2005; 25: 7333–43.
      OpenUrlAbstract/FREE Full Text
    7. ↵
      Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol 2000; 16: 653–99.
      OpenUrlCrossRefPubMed
    8. ↵
      Dani C, Mechti N, Piechaczyk M, Lebleu B, Jeanteur P, Blanchard JM. Increased rate of degradation of c-myc mRNA in interferon-treated Daudi cells. Proc Natl Acad Sci U S A 1985; 82: 4896–9.
      OpenUrlAbstract/FREE Full Text
    9. ↵
      Sarkar D, Park ES, Fisher PB. Defining the mechanism by which IFN-β downregulates c-myc expression in human melanoma cells: pivotal role for human polynucleotide phosphorylase (hPNPaseold-35). Cell Death Differ 2006; 13: 1541–53.
      OpenUrlCrossRefPubMed
    10. ↵
      Barber GN. The dsRNA-dependent protein kinase, PKR and cell death. Cell Death Differ 2005; 12: 563–70.
      OpenUrlCrossRefPubMed
    11. ↵
      Harding HP, Novoa I, Zhang Y, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 2000; 6: 1099–108.
      OpenUrlCrossRefPubMed
    12. ↵
      Barber GN, Jagus R, Meurs EF, Hovanessian AG, Katze MG. Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase. J Biol Chem 1995; 270: 17423–8.
      OpenUrlAbstract/FREE Full Text
    13. ↵
      Sarkar D, Su ZZ, Lebedeva IV, et al. mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci U S A 2002; 99: 10054–9.
      OpenUrlAbstract/FREE Full Text
    14. ↵
      Meurs E, Chong K, Galabru J, et al. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 1990; 62: 379–90.
      OpenUrlCrossRefPubMed
    15. ↵
      Deutscher MP, Li Z. Exoribonucleases and their multiple roles in RNA metabolism. Prog Nucleic Acid Res Mol Biol 2001; 66: 67–105.
      OpenUrlPubMed
    16. ↵
      Zhan Q, Lord KA, Alamo I, Jr., et al. The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol 1994; 14: 2361–71.
      OpenUrlAbstract/FREE Full Text
    17. ↵
      Ron D, Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 1992; 6: 439–53.
      OpenUrlAbstract/FREE Full Text
    18. ↵
      Ubeda M, Wang XZ, Zinszner H, Wu I, Habener JF, Ron D. Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element. Mol Cell Biol 1996; 16: 1479–89.
      OpenUrlAbstract/FREE Full Text
    19. ↵
      McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol 2001; 21: 1249–59.
      OpenUrlAbstract/FREE Full Text
    20. ↵
      Su ZZ, Sarkar D, Emdad L, et al. Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter. Proc Natl Acad Sci U S A 2005; 102: 1059–64.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      Bilsland AE, Merron A, Vassaux G, Keith WN. Modulation of telomerase promoter tumor selectivity in the context of oncolytic adenoviruses. Cancer Res 2007; 67: 1299–307.
      OpenUrlAbstract/FREE Full Text
    22. ↵
      Chan I, Lebedeva IV, Su ZZ, et al. Progression-elevated gene-3 promoter (PEG–Prom) confers cancer cell-selectivity to human polynucleotide phosphorylase (hPNPaseold-35)–mediated growth suppression. J Cell Physiol. In press, 2007.
    View Abstract
    PreviousNext
    Back to top
    Cancer Research: 67 (17)
    September 2007
    Volume 67, Issue 17
    • Table of Contents
    • Table of Contents (PDF)
    • About the Cover
    • Index by Author

    Sign up for alerts

    View this article with LENS

    Open full page PDF
    Article Alerts
    Sign In to Email Alerts with your Email Address
    Email Article

    Thank you for sharing this Cancer Research article.

    NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

    Enter multiple addresses on separate lines or separate them with commas.
    Activation of Double-Stranded RNA–Dependent Protein Kinase, A New Pathway by Which Human Polynucleotide Phosphorylase (hPNPaseold-35) Induces Apoptosis
    (Your Name) has forwarded a page to you from Cancer Research
    (Your Name) thought you would be interested in this article in Cancer Research.
    Citation Tools
    Activation of Double-Stranded RNA–Dependent Protein Kinase, A New Pathway by Which Human Polynucleotide Phosphorylase (hPNPaseold-35) Induces Apoptosis
    Devanand Sarkar, Eun Sook Park, Glen N. Barber and Paul B. Fisher
    Cancer Res September 1 2007 (67) (17) 7948-7953; DOI: 10.1158/0008-5472.CAN-07-0872

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
    Share
    Activation of Double-Stranded RNA–Dependent Protein Kinase, A New Pathway by Which Human Polynucleotide Phosphorylase (hPNPaseold-35) Induces Apoptosis
    Devanand Sarkar, Eun Sook Park, Glen N. Barber and Paul B. Fisher
    Cancer Res September 1 2007 (67) (17) 7948-7953; DOI: 10.1158/0008-5472.CAN-07-0872
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
    • Tweet Widget
    • Facebook Like
    • Google Plus One

    Jump to section

    • Article
      • Abstract
      • Introduction
      • Materials and Methods
      • Results
      • Discussion
      • Acknowledgments
      • Footnotes
      • References
    • Figures & Data
    • Info & Metrics
    • PDF
    Advertisement

    Related Articles

    Cited By...

    More in this TOC Section

    • RAGE/PR3 Interaction Mediates Bone Metastasis
    • MCPyV Small T Ag Initiates Mouse Merkel Cell Carcinoma
    • Colon Cancer Stemness Is Regulated through LUST
    Show more Priority Reports
    • Home
    • Alerts
    • Feedback
    Facebook  Twitter  LinkedIn  YouTube  RSS

    Articles

    • Online First
    • Current Issue
    • Past Issues
    • Meeting Abstracts

    Info for

    • Authors
    • Subscribers
    • Advertisers
    • Librarians
    • Reviewers

    About Cancer Research

    • About the Journal
    • Editorial Board
    • Permissions
    • Submit a Manuscript
    AACR logo

    Copyright © 2018 by the American Association for Cancer Research.

    Cancer Research Online ISSN: 1538-7445
    Cancer Research Print ISSN: 0008-5472
    Journal of Cancer Research ISSN: 0099-7013
    American Journal of Cancer ISSN: 0099-7374

    Advertisement