Diverse roles of STING-dependent signaling on the development of cancer.

Stimulator of interferon genes (STING) is a cellular sensor that controls cytosolic DNA-activated innate immune signaling. We have previously demonstrated that STING-deficient mice are resistant to carcinogen-induced skin cancer, similar to myeloid differentiation primary response gene 88 (MyD88) deficient mice, since the production of STING-dependent DNA-damage-induced proinflammatory cytokines, that likely require MyD88 signaling to exert their growth-promoting activity, are prevented. In contrast, MyD88-deficient mice are sensitive to colitis-associated cancer (CAC), since selected cytokines generated following DNA-damage also activate repair pathways, which can help prevent tumor development. Here, we demonstrate that STING signaling facilitates wound repair processes and that analogous to MyD88-deficient mice, STING-deficient mice (SKO) are prone to CAC induced by DNA-damaging agents. SKO mice harboring tumors exhibited low levels of tumor-suppressive interleukin-22 binding protein (IL-22BP) compared to normal mice, a cytokine considered critical for preventing colon-related cancer. Our data indicate that STING constitutes a critical component of the host early response to intestinal damage and is essential for invigorating tissue repair pathways that may help prevent tumorigenesis.

Potent systemic therapy of multiple myeloma utilizing oncolytic vesicular stomatitis virus coding for interferon-ß.

Multiple myeloma (MM) is an incurable malignancy of plasma secreting B cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV)-expressing interferon-ß (IFNß) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to destroy cancer cells specifically. We demonstrate here that a single, intravenous dose of VSV coding for IFNß (VSV-IFNß) specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune-competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNß expression further delayed myeloma progression and significantly enhanced survival compared with VSV-expressing human IFNß. Evaluation of VSV-IFNß oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma-specific oncolytic activity, but revealed variable susceptibility to VSV-IFNß oncolysis. The results indicate that VSV-IFNß is a potent, safe oncolytic agent that can be systemically administered to target and destroy disseminated myeloma effectively in immune-competent mice. IFNß expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNß to be a promising vector for further development as a potential therapy for the treatment of MM.

Curative one-shot systemic virotherapy in murine myeloma.

Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here, we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity and facilitate noninvasive monitoring of its intratumoral spread. Using high-resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers that expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 h after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Antimyeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.

IRF7: activation, regulation, modification and function.

Interferon regulatory factor 7 (IRF7) was originally identified in the context of Epstein-Barr virus (EBV) infection, and has since emerged as the crucial regulator of type I interferons (IFNs) against pathogenic infections, which activate IRF7 by triggering signaling cascades from pathogen recognition receptors (PRRs) that recognize pathogenic nucleic acids. Moreover, IRF7 is a multifunctional transcription factor, underscored by the fact that it is associated with EBV latency, in which IRF7 is induced as well as activated by the EBV principal oncoprotein latent membrane protein-1 (LMP1). Aberrant production of type I IFNs is associated with many types of diseases such as cancers and autoimmune disorders. Thus, tight regulation of IRF7 expression and activity is imperative in dictating appropriate type I IFN production for normal IFN-mediated physiological functions. Posttranslational modifications have important roles in regulation of IRF7 activity, exemplified by phosphorylation, which is indicative of its activation. Furthermore, mounting evidence has shed light on the importance of regulatory ubiquitination in activation of IRF7. Albeit these exciting findings have been made in the past decade since its discovery, many questions related to IRF7 remain to be addressed.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells.

The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing. We previously showed that these proteins co-localise in the nucleus of interphase cells. In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system. Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers. In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing. Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa). The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72.

Induction of a TRAIL-mediated suicide program by interferon alpha in primary effusion lymphoma.

Gammaherpes viruses are often detected in lymphomas arising in immunocompromised patients. We have found that Azidothymidine (AZT) alone induces apoptosis in Epstein Barr Virus (EBV) positive Burkitt's lymphoma (BL) cells but requires interferon alpha (IFN-alpha) to induce apoptosis in Human Herpes Virus Type 8 (HHV-8) positive Primary Effusion Lymphomas (PEL). Our analysis of a series of AIDS lymphomas revealed that IFN-alpha selectively induced very high levels of the Death Receptor (DR) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HHV-8 positive PEL lines and primary tumor cells whereas little or no induction was observed in primary EBV+ AIDS lymphomas and EBV-Burkitt's lines. AZT and IFN-alpha mediated apoptosis in PEL was blocked by stable overexpression of dominant negative Fas Associated Death Domain (FADD), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins (DR-4 and DR-5) and thymidine. Trimeric TRAIL (in place of IFN-alpha) similarly synergized with AZT to induce apoptosis in HHV-8 positive PEL cells. This is the first demonstration that IFN-alpha induces functional TRAIL in a malignancy that can be exploited to effect a suicide program. This novel antiviral approach to Primary Effusion lymphomas is targeted and may represent a highly effective and relatively non-toxic therapy.

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

Host defense, viruses and apoptosis.

To thwart viral infection, the host has developed a formidable and integrated defense network that comprises our innate and adaptive immune response. In recent years, it has become clear that in an attempt to prevent viral replication, viral dissemination or persistent viral infection of the cell, many of these protective measures actually involve the induction of programmed cell death, or apoptosis. An initial response to viral infection primarily involves the innate arm of immunity and the killing of infected cells with cytotoxic lymphocytes such as natural killer (NK) cells through mechanisms that include the employment of perforin and granzymes. Once the virus has invaded the cell, however, a second host defense-mediated response is also triggered which involves the induction of a family of cytokines known as the interferons (IFNs). The IFNs, which are essential for initiating and coordinating a successful antiviral response, function by stimulating the adaptive arm of immunity involving cytotoxic T cells (CTLs), and by inducing a number of intracellular genes that directly prevent virus replication/cytolysis or that facilitate apoptosis. The IFN-induced gene family is now known to comprise the death ligand TRAIL, the dsRNA-dependent protein kinase (PKR), interferon regulatory factors (IRFs) and the promyelocytic leukemia gene (PML), all of which have been reported to be mediators of cell death. That DNA array analyses indicate that numerous cellular genes, many as yet uncharacterized, may similarly be induced by IFN, further emphasizes the likely importance that these cytokines have in the modulation of apoptosis. This likelihood is additionally underlined by the elaborate strategies developed by viruses to inhibit IFN-antiviral function and the mechanisms of cell death.

HHV-8 encoded vIRF-1 represses the interferon antiviral response by blocking IRF-3 recruitment of the CBP/p300 coactivators.

Human herpes virus 8 (HHV-8) has developed unique mechanisms for altering cellular proliferative and apoptotic control pathways by incorporating viral homologs to several cellular regulatory genes into its genome. One of the important pirated genes encoded by the ORF K9 reading frame is a viral homolog of the interferon regulatory factors (IRF), a family of cellular transcription proteins that regulates expression of genes involved in pathogen response, immune modulation and cell proliferation. vIRF-1 has been shown to downregulate the interferon- and IRF-mediated transcriptional activation of ISG and murine IFNA4 gene promoters. In this study we demonstrate that vIRF-1 efficiently inhibited virus-induced expression of endogenous interferon B, CC chemokine RANTES and CXC chemokine IP-10 genes. Co-expression analysis revealed that vIRF-1 selectively blocked IRF-3 but not IRF-7-mediated transactivation. vIRF-1 was able to bind to both IRF-3 and IRF-7 in vivo as detected by coimmunoprecipitation analysis, but did not affect IRF-3 dimerization, nuclear translocation and DNA binding activity. Rather, vIRF-1 interacted with the CBP/p300 coactivators and efficiently inhibited the formation of transcriptionally competent IRF-3-CBP/p300 complexes. These results illustrate that vIRF-1 is able to block the early stages of the IFN response to virus infection by interfering with the activation of IRF-3 responsive, immediate early IFN genes.

Oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, Ras, or myc function and involves the induction of apoptosis.

We have recently shown that vesicular stomatitis virus (VSV) exhibits potent oncolytic activity both in vitro and in vivo (S. Balachandran and G. N. Barber, IUBMB Life 50:135-138, 2000). In this study, we further demonstrated, in vivo, the efficacy of VSV antitumor action by showing that tumors that are defective in p53 function or transformed with myc or activated ras are also susceptible to viral cytolysis. The mechanism of viral oncolytic activity involved the induction of multiple caspase-dependent apoptotic pathways was effective in the absence of any significant cytotoxic T-lymphocyte response, and occurred despite normal PKR activity and eIF2alpha phosphorylation. In addition, VSV caused significant inhibition of tumor growth when administered intravenously in immunocompetent hosts. Our data indicate that VSV shows significant promise as an effective oncolytic agent against a wide variety of malignant diseases that harbor a diversity of genetic defects.

Analyzing the mechanisms of interferon-induced apoptosis using CrmA and hepatitis C virus NS5A.

The dsRNA-dependent protein kinase, PKR, is a key component of interferon (IFN)-mediated anti-viral action and is frequently inhibited by many viruses following infection of the cell. Recently, we have demonstrated that IFN and PKR can sensitize cells to apoptosis predominantly through the FADD/caspase-8 pathway (S. Balachandran, P. C. Roberts, T. Kipperman, K. N. Bhalla, R. W. Compans, D. R. Archer, and G. N. Barber. (2000b) J. Virol. 74, 1513-1523). Given these findings, it is thus plausible that rather than specifically target IFN-inducible genes such as PKR, viruses could also subvert the mechanisms of IFN action, in part, at locations that could block the apoptotic cascade. To explore this possibility, we analyzed whether the poxvirus caspase-8 inhibitor, CrmA, was able to inhibit IFN or PKR/dsRNA-mediated apoptosis. Our findings indicated that CrmA could indeed inhibit apoptosis induced by both viral infection and dsRNA without blocking PKR activity or inhibiting IFN signaling. In contrast HCV-encoded NS5A, a putative inhibitor of PKR, did not appear to inhibit cell death mediated by a number of apoptotic stimuli, including IFN, TRAIL, and etoposide. Our data imply that viral-encoded inhibitors of apoptosis, such as CrmA, can block the innate arms of the immune response, including IFN-mediated apoptosis, and therefore potentially constitute an alternative family of inhibitors of IFN action in the cell.

The interferons and cell death: guardians of the cell or accomplices of apoptosis?

The interferons (IFNs) play an integral role in cellular host defense against virus infection and conceivably tumorigenesis. Despite over 50 years of research, however, the molecular mechanisms underlining IFN action remain to be fully elucidated, in part because of the large number of genes, with an uncharacterized function that appears to be induced by these cytokines. Although the majority of in vitro studies indicate that IFNs antiviral properties involve inhibiting viral replication while maintaining the integrity of the cell, numerous reports have now implicated that a number of IFN-induced genes, IFN transcriptional regulatory factors and IFN signaling molecules can also mediate apoptosis. Here, we review some of what is known about IFN's ability to invoke programmed cell death as part of an intricate arsenal intended to prevent viral infection and malignant disease.

Vesicular stomatitis virus (VSV) therapy of tumors.

Vesicular stomatitis virus (VSV) is an essentially nonpathogenic negative-stranded RNA virus, the replication of which is extremely sensitive to the antiviral effects of interferon (IFN). We demonstrate here that VSV selectively induces the cytolysis of numerous transformed human cell lines in vitro, with all the morphological characteristics of apoptotic cell death. Importantly, VSV can also potently inhibit the growth of p53-null C6 glioblastoma tumors in vivo without infecting and replicating in normal tissue. With our previous findings demonstrating that primary cells containing the double-stranded RNA-activated protein kinase PKR and a functional IFN system are not permissive to VSV replication, these results suggest that signaling by IFN may be defective in many malignancies. Thus VSV might be useful in novel therapeutic strategies for targeting neoplastic disease.

Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.

Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Here it is shown that the HCV envelope protein E2 contains a sequence identical with phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2alpha, a target of PKR. E2 inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth. This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon.

PKR, apoptosis and cancer.

The double-stranded (ds) RNA-regulated serine/threonine protein kinase, PKR, is an interferon-inducible enzyme of widespread occurrence in mammalian cells. PKR is activated by dsRNA via a mechanism involving autophosphorylation. Once activated, the enzyme phosphorylates the alpha-subunit of protein synthesis initiation factor eIF2, thereby inhibiting translation. Accumulating data suggest that PKR has additional substrates, and that the kinase may also regulate gene transcription and signal transduction pathways. Although PKR plays an important role in mediating the antiviral effects of interferons, PKR is also implicated in regulating cell proliferation in uninfected cells and may have a tumor suppressor function under normal conditions. Studies of human malignancies and tumor cell lines suggest that, in general, patients bearing tumors with a higher PKR content have a more favorable prognosis. However, in human breast carcinoma cells, dysregulation of PKR may be associated with the establishment or maintenance of the transformed state.

Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling.

The dsRNA-dependent protein kinase (PKR) is considered to play a key role in interferon-mediated host defense against viral infection and conceivably malignant transformation. To investigate further the mechanisms of PKR-induced growth inhibition, we have developed tetracycline-inducible murine cell lines that express wild-type PKR or a catalytically inactive PKR variant, PKRdelta6. Following induction, the growth of the wild-type PKR-expressing cells was similar to that of cells transfected with vector alone, while cells expressing PKRdelta6 became malignantly transformed. Significantly, treatment with dsRNA caused the wild-type PKR-overexpressing cells to undergo programed cell death while, conversely, cells expressing PKRdelta6 were completely resistant. Our studies demonstrated that activation of PKR induces the expression of members of the tumor necrosis factor receptor (TNFR) family, including Fas (CD95/Apo-1) and pro-apopotic Bax. In contrast, transcripts representing Fas, TNFR-1, FADD (Fas-associated death domain), FLICE, Bad and Bax were ablated in cells expressing PKRdelta6. The involvement of the death receptors in PKR-induced apoptosis was underscored by demonstrating that murine fibroblasts lacking FADD were almost completely resistant to dsRNA-mediated cell death. Thus, PKR, a key cellular target for viral repression, is a receptor/inducer for the induction of pro-apoptotic genes by dsRNA and probably functions in interferon-mediated host defense to trigger cell death in response to virus infection and perhaps tumorigenesis.

The murine PKR tumor suppressor gene is rearranged in a lymphocytic leukemia.

The double-stranded RNA-dependent kinase, PKR, is encoded by an interferon inducible gene and is largely responsible for the anti-viral effects of this cytokine. Recent studies have shown that PKR may also play a role in the regulation of normal cellular growth. Although numerous examples of viral strategies for inactivation of PKR exist, there is no evidence of PKR inactivation in tumors. We demonstrate here that the Tik gene, which encodes a dual-specificity kinase, is the murine homolog of PKR, the dsRNA-dependent kinase, and has undergone a rearrangement of one allele in a murine lymphocytic leukemia cell. We have cloned a cDNA that corresponds to a mutated transcript from the rearranged mPKR gene and show that while the mutated polypeptide retains its ability to dimerize and bind dsRNA, it is catalytically inactive. Although this mutated mPKR lacks apparent dominant-negative function, the net effect of reduced PKR activity in these cells may be significant.

Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) is suggested to play an important role in both the antiviral and antiproliferative arms of the interferon response. To gain insights into the molecular mechanisms underlying PKR's growth regulatory properties, we examined the biological and biochemical properties of PKR variants containing either a mutation in catalytic domain II (PKR-M1) or a deletion of RNA binding domain I (PKR-M7) in both reticulocyte translation extracts and in vitro kinase assays with purified reagents and compared these results with those using the same mutants stably expressed in vivo. While wild-type PKR (PKR-WT) efficiently inhibited mRNA translation in a reticulocyte extract, the inactive PKR-M1 had no effect. The PKR-M7 mutant was modestly inhibitory in this assay. The PKR-M1 variant was able to reverse the translational inhibitory effects and increased eukaryotic initiation factor (eIF)-2 alpha phosphorylation levels caused by addition of double-stranded RNA to reticulocyte extract, whereas PKR-M7 could not. Both PKR-M1 and PKR-M7 functioned as transdominant inhibitors of PKR-WT in our in vitro kinase assays. While the inhibition by PKR-M1 required a vast excess of mutant to shut down PKR function, PKR-M7 inhibited PKR-WT at approximately stoichiometric levels. To complement these experiments, we compared growth rates and alpha phosphorylation levels in transformed cell lines overexpressing either PKR-M1 or PKR-M7. Levels of endogenous eIF-2 alpha phosphorylation were significantly more diminished in PKR-M7 overexpressing cells compared with PKR-M1. These paradoxical data will be discussed in terms of the potential molecular mechanisms underlying malignant transformation caused by the PKR variants.

Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation.

Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.