Antiproliferative Effects Of Interferon Research Articles

Interferon-α enhances the susceptibility of renal cell carcinoma to rapamycin by suppressing mTOR activity.

The aim of the present study was to investigate the antiproliferative effects of interferon (IFN)-α and rapamycin (RPM) on renal cell carcinoma (RCC) cells and examine the synergistic growth suppression conferred by IFN-α and RPM. The effects of IFN-α and/or RPM on RCC cells were determined using a WST-1 assay and the synergy of IFN-α and RPM against three RCC cell lines was analyzed with isobolographic analysis. The expression of mammalian target of rapamycin (mTOR) was downregulated by RNAi, and the expression and phosphorylation of proteins in the mTOR pathway following treatment with IFN-α and/or RPM was examined by western blot analysis. The observations indicated that IFN-α significantly increased the susceptibility of RCC cells to RPM and the synergistic effect of IFN-α and RPM against RCC cells was confirmed in all three RCC cell lines. The mTOR pathway was shown to be associated with the synergistic effect of IFN-α and RPM against RCC. IFN-α and RPM alone decreased the phosphorylation of mTOR, p70 S6 kinase, S6 and 4E binding protein 1, and IFN-α significantly enhanced the RPM-induced suppression of the mTOR pathway. However, in RCC cells with low mTOR activity, the synergy of IFN-α and RPM was eliminated. Therefore, the results of the present study indicate that the mTOR pathway plays an important role in the synergistic effect of IFN-α and RPM against RCC cells. Thus, mTOR may serve as an effective therapeutic target in the treatment of advanced RCC.

Interferon (IFNγ) induces cell cycle arrest in RCC cell lines

We have recently demonstrated that the anti-proliferative effect of interferon (IFNγ) in RCC is due to an increased production of nitric oxide (NO) which in turn inhibits the induction of arginase (ARG2). The growth of cultured murine RCC cell lines was inhibited in 85% in IFNγ treated when compared to un-treated cells. In contrast only 33% of these cells were inhibited by IFNγ. These results indicate that even though IFNγ show a more potent anti-proliferative effect, still there is a certain degree of treatment- resistance (15%) that is higher when cells are treated with IFNγ. This study was undertaken to study the mechanisms by which IFNγ inhibits RCC growth and to provide new insights in understanding how this cytokine alone or in combination with other therapies could benefit RCC clinical outcome. Cell survival, cell cycle arrest-apoptosis and cell signaling targets were identified by MTT assay, flow cytometry and immunoblotting respectively in RCC cell lines CL-2 (low-ARG/low-iNOS), CL-19 (high-ARG/low iNOS) and Renca (med-ARG/zero-iNOS). The results showed that 1-100 U/ml IFNγ suppressed proliferation of both CL-2 and CL-19 in a dose- and time-dependent manner. IFNγ inhibited the growth of CL 2 and CL 19 tumor cells with IC50-values of 10.2 ± 1.5 U/ml and 13.5 ± 1.0 U/ml, respectively. There was no inhibition of growth in the Renca cell line. The results on cell cycle progression have shown that the anti-proliferative effect of IFNγ is associated with a G2/M cell cycle arrest of CL-2 and CL-19 cells. The event of cell cycle arrest was associated with a marked decline in protein levels of G2/M regulatory proteins cyclin B1, cyclin A and cyclin-dependent kinase 1 (Cdk1/cdc2), whereas an up-regulation in CDC25c was observed. Elimination of IFNγ resulted in increased tumor growth that was contained with the sustained addiction of the cytokine. No apoptosis was observed. Our study demonstrated that IFNγ suppress the growth of murine RCC cells by inducing cell-cycle arrest at G2/GM through the down-regulation of cyclin B1 and cyclin C. These findings suggest that IFNγ acts as an effective anti-proliferative agent by modulating cell growth regulators in renal carcinoma cells and can be used as a election treatment to inhibit tumor proliferation in patients scheduled for surgery.

Abstract B60: HPP1-mediated tumor suppression and interferon signaling

Abstract Interferon resistance is common to the majority of cancer cell lines and primary tumors, and confers increased tumorigenicity and evasion of immune surveillance. HPP1, a recently characterized tumor suppressor gene associated with STAT1 signaling, is silenced in the majority of colorectal neoplasms. We have sought to determine if HPP1's tumor suppressive effects are mediated by interferon-associated pathways and whether HPP1 is capable of restoring sensitivity to interferon. We have overexpressed HPP1 in the HCT 116 colorectal cancer cell line and, by microarray analysis, have demonstrated that ectopic expression of HPP1 resulted in a dramatic upregulation of STAT1 as well as a number of associated interferon-inducible genes. We have previously shown that downregulation of STAT1 by siRNA in HPP1 transfectants results in a restoration of growth potential. Given that STAT1 homodimers bind specifically to promoter GAS (gamma activating sites) sites causing transcriptional upregulation of target genes, we transfected luciferase GAS reporter (pLucGAS) and control plasmids into HPP1 and control transfectants. Overexpression of HPP1 was associated with a significant increase in GAS site binding (p=0.007), suggesting that HPP1 mediates tumor suppression via its association with Type II-like (gamma) interferon pathways. To investigate the importance of Type II-like pathways, we treated HCT 116 cells, with and without overexpression of HPP1, with increasing doses of interferon gamma. Induction of activated STAT1 (phosphoSTAT1), was demonstrable in HPP1-overexpressing transfectants with a progressive rise concordant with interferon dose escalation. Interferon gamma was unable to induce any augmentation of activated STAT1 in controls. Despite progressive induction of phosphoSTAT1, no significant alterations in proliferation, apoptosis or cell cycle distribution were observed with interferon gamma treatment of HPP1 transfectants. We conclude that activated STAT1 and Type II-like interferon-associated pathways are important in mediating the tumor suppressive effects of HPP1. The lack of additional anti-proliferative effects of interferon on HPP1 transfectants may be due to saturation of the pathway. However, further investigation of the role of other STAT1-associated mechanisms in HPP1 tumor suppression, such as the Type I-like interferon (alpha) pathway is warranted. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B60.

In vitro antiproliferative effects of interferons in combination with the proteasome inhibitor bortezomib

In vitro antiproliferative effects of interferons in combination with the proteasome inhibitor bortezomib

Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation

Interferons are produced following neural damage as part of the inflammatory response and may thus affect neural stem cell function. We compared the effects of interferon-γ and interferon-β on the proliferation and differentiation of adult murine neural progenitors. Both interferons inhibited neurosphere proliferation due to cell cycle arrest in G1 but only interferon-γ induced neuronal differentiation. Both interferons induced differential phosphorylation of STAT proteins and a modest and late upregulation of the cell cycle regulator p27 but not several other likely cell cycle regulators. Thus in neural progenitor cells, anti-proliferative effects of interferons are not necessarily linked to differentiation.

Does HCV antiviral therapy decrease the risk of hepatocellular carcinoma?

Whether antiviral therapy for hepatitis C virus (HCV) infection can prevent hepatocellular carcinoma (HCC) independent of a sustained virologic response (SVR) has been debated since a randomized trial from Japan in 1995. Incidence of HCC in patients with chronic hepatitis C with SVR is reduced through cirrhosis prevention, but HCC is also prevented in cirrhosis by reduction of necroinflammatory activity and possibly cell turnover. However, HCV patients with cirrhosis who achieve an SVR still have a risk, although a lower one, of HCC, so surveillance must continue. The potential antiproliferative effect of interferon (IFN) with respect to HCC has been discounted by a prospective randomized clinical trial in nonresponders to IFN (Hepatitis C Antiviral Long-term Treatment Against Cirrhosis [HALT-C]), in which IFN did not modify any outcomes. Thus, the best strategy to reduce HCC is to treat HCV-infected patients before they develop cirrhosis and to increase the SVR rate.

Mapping of the Auto-inhibitory Interactions of Protein Kinase R by Nuclear Magnetic Resonance

The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed 15N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2α docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2α docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1 + 2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2α binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.

A newly discovered function for RNase L in regulating translation termination

The antiviral and antiproliferative effects of interferons are mediated in part by the 2'-5' oligoadenylate-RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2'-5' oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.

Place des thérapeutiques médicales dans la prise en charge des tumeurs desmoïdes de l'enfant

Aggressive fibromatoses (desmoids tumors) are a rare nonmetastasizing fibrous tumor characterized by a local invasive capacity, a high recurrence rate after excision. Surgery remains the standard treatment. Wide excision with negative margins seems to decrease the chance of recurrence but must be not mutilating. Adjuvant high dose radiotherapy may have a good result but must be employed in selected patients. Drug therapy, essentially with continuous low dose adjuvant chemotherapy using methotrexate and vinblastine may be a reasonable choice, with low toxicity. Non-cytotoxic drug therapy represented by antiestrogens and non-steroidal anti-inflammatory drugs may have a beneficial effect. Antiproliferative effects of interferon and other drugs such as retinoïc acid have shown an encouraging response rates. The exactly role of this therapies remains controversial. Cellular, molecular and genetic investigations may be helpful to understand the mechanism of desmoid tumor proliferation. The aim of this study is to discuss the place of different medical therapies in the treatment of desmoid tumors, with review of literature.

Therapy-Induced Antibodies Against the Antiviral and Antiproliferative Effects of Interferons in Patients with Chronic Hepatitis C Virus Infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.

Genetically targeted cancer therapy: tumor destruction by PKR activation.

The is a double-stranded RNA-activated protein kinase (PKR) has been largely investigated for its key role in viral host defense. Although best characterized by its function in mediating the antiviral and antiproliferative effects of interferon (IFN), PKR is also implicated in transcriptional regulation, cell differentiation, signal transduction, and tumor suppression. However, recent findings identifying PKR as an important effector of apoptosis have led to an increased interest in PKR modulation as an antitumor strategy. PKR can either be up-regulated through direct induction by the transcription factor E2F-1, or it can be activated through direct protein-protein interactions with the melanoma differentiation-associated gene-7 (MDA7, IL-24). Additionally, the intracellular formation of double-stranded RNA by transfection with antisense RNA complementary to tumor-specific RNA sequences can induce PKR activation and apoptosis selective to these tumor cells. The growing application of viral vector-based gene therapies and oncolytic, replicating viruses that must elude viral defense in order to be effective, has also drawn attention to PKR. Oncolytic viruses, like the attenuated herpes simplex virus R3616, the vesicular stomatitis virus, or reovirus, specifically replicate in tumor cells only because the viral host defense in the permissive cells is suppressed. In this article we review the role of PKR as an effector of apoptosis and a target for tumor treatment strategies and discuss the potential of PKR-modifying agents to treat patients with cancer. Targeted gene therapy against cancer can be approached by activation of PKR with the down-regulation of protein synthesis and induction of apoptosis, or by suppression of PKR with the propagation of oncolytic virus. Since the PKR pathway can be modified by many routes, antitumor therapies combining oncolytic virus, gene therapies, and chemotherapy with PKR modifiers are likely to emerge in the near future as therapeutic options in the treatment of patients with cancer.

Expression of Unphosphorylated Form of Human Double-Stranded RNA-Activated Protein Kinase in Escherichia coli

Interferon (IFN)-inducible, double-stranded (dsRNA)-activated protein kinase (PKR) is a key mediator of the antiviral and antiproliferative effects of IFN. PKR is present within cells in a latent state. In response to binding dsRNA, the enzyme becomes activated, causing autophosphorylation and an increase in specific kinase activity. In order to study PKR and its inhibitors, a large amount of the enzyme in its latent, unphosphorylated state is required. When PKR is fused to glutathione S-transferase (GST-PKR) and the fusion protein is expressed in Escherichia coli, the PKR obtained is fully activated by autophosphorylation. Therefore, we have developed an expression plasmid in which both GST-PKR and bacteriophage λ protein phosphatase (λ-PPase) genes were placed downstream of a T7 promoter. After induction of expression, unphosphorylated GST-PKR was obtained in good yield, and purified to near homogeneity. The purified enzyme has dsRNA-dependent activation and phosphorylates the translation initiation factor eIF2α. Using the recombinant protein, we analyzed the inhibition mechanisms of two viral inhibitors, vaccinia virus K3L protein and adenovirus virus-associated RNA I (VAI RNA). K3L inhibited both autophosphorylation of PKR and phosphorylation of eIF2α, whereas VAI RNA inhibited only autophosphorylation. The separation of autophosphorylation and catalytic activity shows that the recombinant PKR is useful in analyzing the functions of PKR, its inhibitors, and its regulatory molecules. The coexpression system of protein kinase with λ-PPase described here will be applicable to obtaining unphosphorylated and unactivated forms of other protein kinases.

Control of cell cycle progression in human mesothelioma cells treated with gamma interferon.

Recombinant human interferon gamma (r-hu-IFNgamma) exerts both antitumoral activity in the early stages of human malignant mesothelioma and a cytostatic effect in human mesothelioma (HM) cell lines in vitro. The antiproliferative effect of interferons (IFNs) reported in a variety of cells has been attributed to several mechanisms. In order to progress in the understanding of HM cell growth modulation by r-hu-IFNgamma, modifications of cell cycle progression and expression of key cell cycle regulator proteins in response to r-hu-IFNgamma were examined. Nine HM cell lines were studied, including one resistant to the antiproliferative effect of r-hu-IFNgamma. Except in the resistant cell line r-hu-IFNgamma produced an arrest in the G1 and G2-M phases of the cell cycle, associated with a reduction in both cyclin A and cyclin dependent kinase inhibitors (CDKIs) expression. Moreover cyclin B1/cdc2 activity was decreased. The present study provides the first evidence of a G2-arrest in r-hu-IFNgamma-treated HM cell lines and indicates that HM cell lines, despite their tumorigenic origin still support cell cycle control. The cell cycle arrest induced by r-hu-IFNgamma seems to depend on cyclin regulation through p21(WAF1/CIP1)- and p27(Kip1)-independent mechanisms and is not directly related to the induced DNA damage.

Defective Jak-STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon-alpha.

Advanced malignant melanoma is an aggressive malignancy with poor prognosis. Current therapeutic strategies have a modest success rate. The most promising treatment consists of a combination of chemotherapy with interferon-alpha, but complete response rates remain less than 15%. Interferon-alpha is also effective in adjuvant therapy for non-advanced melanoma treated surgically. The molecular mechanisms leading to loss of growth restraints and gain of growth-promoting functions during carcinogenesis of malignant melanoma are not understood in detail. Here, we studied 9 human melanoma cell lines with regard to growth inhibition by interferon-alpha and defects in intracellular signal transduction through the Jak-STAT pathway. In 3 cell lines, we found a complete loss of growth restraint by interferon-alpha. In all of them, different components of the Jak-STAT pathway were defective. Since signal transduction through the Jak-STAT pathway is necessary for antiviral and antiproliferative effects of interferons, we conclude that defects in this pathway may be one of the mechanisms that lead to cancer progression through loss of growth-restraining functions. Moreover, our results indicate that a subgroup of melanomas could be completely resistant to interferon-alpha and should therefore not be treated with this cytokine.

The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro.

The tumor suppressor p53 is a multifunctional protein that plays a critical role in modulating cellular responses upon DNA damage or other stresses. These functions of p53 are regulated both by protein-protein interactions and phosphorylation. The double-stranded RNA activated protein kinase PKR is a serine/threonine kinase that modulates protein synthesis through the phosphorylation of translation initiation factor eIF-2alpha. PKR is an interferon (IFN)-inducible protein that is thought to mediate the anti-viral and anti-proliferative effects of IFN via its capacity to inhibit protein synthesis. Here we report that PKR physically associates with p53. The interaction of PKR with p53 is enhanced by IFNs and upon conditions that p53 acquires a wild type conformation. PKR/p53 complex formation in vitro requires the N-terminal regulatory domain of PKR and the last 30 amino acids of the C-terminus of human p53. In addition, p53 may function as a substrate of PKR since phosphorylation of human p53 on serine392 is induced by activated PKR in vitro. These novel findings raise the possibility of a functional interaction between PKR and p53 in vivo, which may account, at least in part, for the ability of each protein to regulate gene expression at both the transcriptional and the translational levels.

Mechanisms of resistance off NSCLC to interferons

Interferons (IFNs) are naturally occurring cytokines which have pleiotropic actions including regulation of cell growth and differentiation, important for control of tumour growth and development. In this study we investigated the association between the presence of IFN genes in NSCLC cell lines, receptor expression and function, and sensitivity to IFNs. Some of the NSCLC lines had partial or complete IFN gene deletion but others contained all genes. However, all NSCLC lines were resistant to the antiproliferative effects of IFN alpha 2 and IFN beta ser. While the lack of sensitivity to IFNs did not appear to be associated with the presence of IFN genes, numbers of receptors or with low binding affinities it did correlate with abnormal regulation of receptor expression. When analyzed by Northern blotting it was notable that IFNA receptor expression on a sensitive cell line, Daudi, was upregulated following IFN exposure however, in the insensitive NSCLC lines IFN mediated upregulation of receptors did not occur. This defect in the regulation of receptor expression in NSCLC lines could contribute to the insensitivity of the antiproliferative effects of IFN's and thus potentiate tumour development or progression.

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

The molecular chaperone hsp40 regulates the activity of P58IPK, the cellular inhibitor of PKR.

The interferon-induced double-stranded RNA-activated protein kinase, PKR, likely contributes to both the antiviral and the antiproliferative effects of interferon. We previously found that influenza virus avoids the translational inhibitory effects of activated PKR by activating a cellular inhibitory protein, termed P58IPK, based on its Mr of 58,000. P58IPK is a member of the tetratricopeptide family of proteins and possesses significant homology to the conserved J region of the DnaJ family of heat shock proteins. We earlier hypothesized that P58IPK was kept in an inactive state with its own inhibitor (termed I-P58IPK) in uninfected cells. We therefore attempted the purification and characterization of I-P58IPK. The following data suggest that we have identified the molecular chaperone, hsp40, as 1-P58IPK. (i) The MonoP-purified I-P58IPK protein reacted with hsp40 antibody. (ii) This preparation demonstrated high specific activity in an in vitro functional assay containing only purified recombinant and native components. (iii) Purified, recombinant hsp40 protein inhibited P58IPK in an identical in vitro assay. (iv) Finally, we demonstrate that hsp40 directly complexes with P58IPK, in vitro, suggesting the inhibition occurs through a direct interaction. Our data, taken together, provide evidence for a novel intersection between the heat shock and interferon pathways, and suggest that influenza virus regulates PKR activity through the recruitment of a cellular stress pathway.

Characterization of a novel IRF-1-deficient mutant cell line.

The transcriptional activation of the major histocompatibility complex (MHC) class I genes by both type I (alpha/beta) and II (gamma) interferons (IFNs) has been extensively studied, and it has been shown that the upregulation of several DNA-binding proteins is critical for this process. In our laboratory, we introduced the mouse H-2Kb gene into the AKR mouse leukaemia cell line K36.16 to effect the generation of tumor-specific immunity. Individual clones were selected and studied. Whereas the MHC class I genes in most of the clones obtained could be stimulated by interferons, one of the clones obtained, clone Kb-S27, failed to be induced, or was at best poorly induced by IFN-alpha/beta and -gamma. Both the exogenous H-2Kb and the endogenous H-2Dk genes behaved in the same manner and were not stimulated by IFNs. The lack of response to IFNs by clone Kb-S27 also resulted in its resistance to the antiproliferative effects of IFNs. This lack of IFN-induction by clone Kb-S27 was not simply due to a change in its surface interferon receptors. Gel-retardation assay and northern blot analysis both demonstrated the lack of induction of the IRF-1 DNA-binding factor in clone Kb-S27. In addition, northern blot analysis showed that the IRF-2 gene expression in clone Kb-S27 was upregulated when compared with the other IFN-inducible clones.

Interferon-Resistant Daudi Cells Are Deficient in Interferon-α–Induced ISGF3α Activation, but Remain Sensitive to the Interferon-α–Induced Increase in ISGF3γ Content

Low levels of the transcription factor ISGF3 alpha were detected in the cytoplasm and nucleus of untreated Daudi cells, which increased markedly following interferon (IFN) treatment. In contrast no ISGF3 alpha was detected in an IFN-resistant clone of Daudi cells, DIF8, and only low levels were detected in these cells after IFN-alpha treatment. High levels of ISGF3 were produced in vitro, however, by the addition of ISGF3 alpha to extracts of IFN-treated DIF8 cells, indicating that IFN is unable to produce substantial amounts of functional ISGF3 alpha in DIF8 cells. A second clone of IFN-resistant Daudi cells, DIF3, also exhibited defective ISGF3 alpha production, which was restored to normal in the subclone DIF3REV5 that had reverted to high IFN sensitivity. Thus, the antiproliferative effect of IFN on Daudi cells and derived clones is closely related to the level of ISGF3 present in the nucleus of these cells. IFN-alpha, however, also enhances the content of ISGF3 gamma in IFN-resistant cells as well as certain proteins of unknown function, raising the possibility that a second pathway of IFN-alpha signal transduction, distinct from the ISGF3 pathway, remains functional in both DIF8 and DIF3 cells.