Antiproliferative Action Of Interferon Research Articles

CS4-6 Antiproliferative activity of interferon activated monocytes against high concentrations of human tumor cells

CS4-6 Antiproliferative activity of interferon activated monocytes against high concentrations of human tumor cells

616. Enhancement of Antitumor Activity of Interferons by RNAi-Mediated Silencing of SOCS Genes in Tumor Cells

Cytokine-based cancer immunotherapy, which has already been used in clinical practice, is a promising strategy in cancer therapy. However, it has been reported that some tumor cells are not sensitive to cytokine-mediated cell death. Furthermore, tumor cells can acquire resistance to cytokines during their progression, leading to the increase in the administration dose of cytokines. However, this increase would be indispensably associated with severe side effects such as leukopenia and neuropsychiatric symptoms. Suppressor of cytokine signaling (SOCS) proteins are a family of negative regulators of cytokine signaling by inhibiting Jak activation. Of the family members, SOCS-1 and SOCS-3 are the most potent inhibitors of cytokine-induced signals. Recent studies have indicated that high expression of the SOCS proteins is found in several advanced cancer cells. Furthermore, an inverse correlation between the expressionlevel of the SOCS proteins in tumor cells and their sensitivity to cytokines was reported. These pieces of evidence suggest that the silencing of SOCS-1 or -3 in tumor cells by RNA interference can enhance the susceptibility of tumor cells to cytokines. In this study, we selected murine interferon (IFN)-|[beta]| and -|[gamma]| as antitumor cytokines, and examined their effects on the proliferation of cultured tumor cells: B16|[ndash]|BL6 melanoma cells, and CT-26 and colon 26 colon carcinoma cells. A quantitative RT-PCR analysis revealed that CT-26 cells and colon 26 cells had higher mRNA expression of SOCS-1 and -3 than B16-BL6 cells or C2C12 myoblasts, the latter used as a model of normal cells. Proliferation of the tumor cells was inhibited by the addition of IFN-|[beta]| or IFN-|[gamma]| to culture media in an IFN concentration-dependent manner. We found that B16-BL6 cells were more sensitive to IFNs than CT-26 or colon 26 cells. Then, to suppress the expression of SOCS-1 or -3, we constructed small interfering RNA (siRNA)-expressing plasmid DNA targeting one of these genes (psi-SOCS1 and psi-SOCS3). Transfection of psi-SOCS1 or psi-SOCS3 significantly reduced the mRNA expression of the corresponding target gene to 50|[ndash]|60 % of the controls. This reduction in the mRNA level of SOCS-1 or SOCS-3 significantly increased the susceptibility of CT-26 and colon 26 cells to the anti-proliferative activity of IFN-|[beta]| and IFN-|[gamma]|. On the other hand, the reduction in the mRNA levels hardly affected the susceptibility of B16-BL6 cells that showed lower mRNA expression of the SOCS genes than the others. These results indicate that the silencing of SOCS-1 and -3 expression in tumor cells can improve the anti-tumor effect of IFNs under conditions where the expression of these genes are increased.

Expressed Gene Clusters Associated with Cellular Sensitivity and Resistance Towards Anti-viral and Anti-proliferative Actions of Interferon

Interferons (IFN) are multi-functional proteins that induce a large number of genes which mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Bioinformatics analysis of the 3'-untranslated regions of IFN-stimulated genes (ISGs) showed that the AU-rich elements (ARE) exist in approximately 10% of the mRNA induced by IFN. The human epithelial cell lines, WISH and 293, and the human B cell lines, Daudi and RPMI 1788, were assessed for their response to type-I IFN. Due to their differential response to the anti-viral and anti-proliferative action of IFN-alpha, they were used as cellular models for genome wide ARE-gene expression. The anti-viral and anti-proliferative actions of IFN-alpha were substantially more potent against WISH and Daudi cells than 293 and RPMI 1788 cells, respectively. These results correlated with the Stat1-driven gene expression as assessed by monitoring the expression of Stat1-mediated IFN-inducible 6-16 mRNA. Interferons were able to induce a significant proportion of common and distinct ARE-genes, but the patterns of expression were different and dependent on the type of the cell, type of IFN, and status of the cellular sensitivity to IFN. Clustering algorithms generated two informative expressed gene clusters that were selectively associated with cellular sensitivity and resistance to the anti-viral and anti-proliferative action of IFN. Use of rationally designed microarray experiments in IFN biology yielded informative clusters that may provide candidate genes for diagnostic or for evaluation of therapeutic possibilities.

The pkr kinase promoter binds both Sp1 and Sp3, but only Sp3 functions as part of the interferon-inducible complex with ISGF-3 proteins

The protein kinase regulated by RNA (PKR) is an important mediator of the antiviral and antiproliferative actions of interferon (IFN). The promoter of the PKR gene contains a novel 15-bp element designated KCS that is required for both basal and IFN-inducible transcription, with KCS function dependent upon both position and orientation relative to the ISRE element. Novel inducible protein complexes (iKIBP1, iKIBP2) that require both the KCS and the ISRE element sequences for their formation have been identified and characterized. Transcription factors Sp1 and Sp3 were found to be KCS-binding proteins by electrophoretic mobility shift analyses (EMSA) and Sepharose bead-KCS oligonucleotide pull-down assays. However, only Sp3 but not Sp1 was a constituent of the inducible iKIBP complexes. EMSA also identified STAT1, STAT2, and IRF-9 as components of the iKIBP complexes, indicating that ISGF-3 participates in iKIBP complex formation. Proteins bound at the KCS element in the absence of ISRE were able to recruit both STAT1 and STAT2 to the KCS element; recruitment was dependent upon IFN-α treatment. Chromatin immunoprecipitation assays revealed that the binding of Sp3, similar to STAT1 and STAT2, at the PKR promoter in vivo was IFN-dependent, but that Sp1 binding was not dependent upon IFN treatment. These results, taken together, strongly suggest a role for Sp1 in basal and Sp3 in inducible transcription of PKR and that a potential function of the KCS element is to facilitate the recruitment of ISGF-3 complex components to the PKR promoter to stimulate transcription.

Functional Characterization of pkr Gene Products Expressed in Cells from Mice with a Targeted Deletion of the N terminus or C terminus Domain of PKR

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays an important role in messenger (m) RNA translation by phosphorylating the alpha subunit of eukaryotic initiation factor 2. Through this capacity PKR is thought to be a mediator of the antiviral and antiproliferative actions of interferon. In addition to translational function, PKR has been implicated in many signaling pathways to gene transcription by modulating the activities of a number of transcription factors, including NF-kappa B and STATs. However, experiments with two different PKR knockout (PKR(-/-)) mouse models have failed to verify many of the biological functions attributed to PKR. In addition, results with cells from the two PKR(-/-) mice have been contradictory and confusing. Here, we show that the first PKR(-/-) mouse with deletion of exons 2 and 3, corresponding to the N terminus domain of PKR (N-PKR(-/-)), expresses a truncated protein, resulting from the translation of the exon-skipped mouse PKR (ES-mPKR) mRNA. The ES-mPKR protein is defective in dsRNA binding but remains catalytically active both in vitro and in vivo. Furthermore, we show that the second PKR(-/-) mouse with a targeted deletion of exon 12, which corresponds to the C terminus of the molecule (C-PKR(-/-)), expresses a truncated mPKR produced by alternative splicing of exon 12. Although the spliced form of mPKR (SF-mPKR) is catalytically inactive, it retains the dsRNA-binding properties of the wild type mPKR. Reverse transcription-PCRs demonstrate that SF-mPKR mRNA is expressed in several normal mouse tissues, and appears to be under developmental control during embryogenesis. Our data demonstrate that both PKR(-/-) models are incomplete knockouts, and expression of the PKR variants may account, at least in part, for the significant signaling differences between cells from the two PKR(-/-) mice.

The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR).

One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR). PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein-protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and inter action with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that, although dispensable for PACT's high-affinity interaction with PKR, the third motif is essential for PKR activation. In addition, domain 3 and eukaryotic initiation factor 2alpha both interact with PKR through the same region within PKR, which we have mapped to lie between amino acid residues 318 and 551.

Dominant Negative Function by an Alternatively Spliced Form of the Interferon-inducible Protein Kinase PKR

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.

Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor.

The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the lambda repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the lambda repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58IPK. We demonstrated, utilizing both the yeast and lambda systems, that P58IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization. In contrast, a nonfunctional form of P58IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers. These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors. Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor. On the basis of these results we propose a model for the regulation of PKR dimerization.

Interaction of the Human Protein Kinase PKR with the Mouse PKR Homolog Occurs via the N-Terminal Region of PKR and Does Not Inactivate Autophosphorylation Activity of Mouse PKR

The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes,HIS3 andlacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of theHIS3 andlacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathioneS-transferase (GST) fusions inEscherichia coliwere purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1–184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.

Isolation of the Interferon-Inducible RNA-Dependent Protein KinasePkrPromoter and Identification of a Novel DNA Element within the 5′-Flanking Region of Human and MousePkrGenes

The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of thePkrgene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5′-flanking fragments of thePkrgene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed an element (5′GGAAAACGAAACT3′) involved in IFN inducibility that corresponds to the consensus sequence of the IFN-stimulated response element (ISRE). Comparison of the promoter sequence of the humanPkrgene to that of the mouse homolog identified a novel element (5′GGGAAGGCGGAGTCC3′) immediately upstream of the ISRE element which so far is unique to the human and mousePkrgene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of thePkrpromoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the humanPkrpromoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mousePkrgenes. The strict conservation of sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.

Differential regulation of human indoleamine 2,3-dioxygenase gene expression by interferons-gamma and -alpha. Analysis of the regulatory region of the gene and identification of an interferon-gamma-inducible DNA-binding factor.

The induction of indoleamine 2,3-dioxygenase (IDO) activity has been implicated in the antiproliferative action of interferon (IFN)-gamma on tumor cells and the inhibition of intracellular pathogens. Earlier studies have demonstrated that the expression of the IDO gene is induced strongly by IFN-gamma, but very poorly by IFN-alpha despite the presence of a sequence highly homologous to the IFN-alpha-responsive sequence element (interferon-stimulated response element (ISRE)) in its IFN-gamma-responsive control region. In addition, a sequence with a partial homology to the IFN-gamma-responsive sequence (GAS) identified by Lew et al. (Lew, D.J., Decker, T., Strehlow, I., and Darnell, J.E., Jr. (1991) Mol. Cell. Biol. 11, 182-191) in a human gene for a guanylate-binding protein and to the X box sequence found in all major histocompatibility complex class II genes was found. Deletion experiments have indicated that the ISRE homolog (but not the GAS-related or the X box-related sequence) was essential for an inducibility by IFN-gamma. To investigate the lack of inducibility by IFN-alpha despite the presence of an ISRE homolog, the binding of this ISRE homolog to the IFN-alpha-stimulated gene factor 3 (ISGF3) was examined. Gel mobility shift experiments and competition experiments indicated that this ISRE homolog did not form a stable complex with ISGF3. This may account for a poor inducibility by IFN-alpha. This inability to bind ISGF3 appears to be (at least in part) due to minor differences between the nucleotide sequence of the ISRE homolog present in the IDO gene promoter and the ISRE consensus sequence found in IFN-alpha-inducible genes. An IFN-gamma-inducible DNA-binding factor was identified with characteristics different from ISGF3: (i) the IFN-gamma-inducible factor was detected in the nuclear extracts, but not in the cytoplasmic extracts; and (ii) the appearance of this DNA-binding factor required new protein synthesis, which could explain the dependence on new protein synthesis for the induction of IDO by IFN-gamma.

Biochemical characterization of the membrane-associated phosphorylating proteins involved in the anti-proliferative effect of human recombinant interferon-alpha 2a (rIFN-.ALPHA.2a) in daudi cells.

The anti-proliferative effect of interferons (IFNs) was investigated, focusing on the physiological activities of membrane-associated proteins involved in the progress of cell proliferation. In preliminary screening of the inhibitory effect of IFNs against the cell growth of various human hematopoietic tumor cells, Daudi cell was the most sensitive to rIFN-alpha 2a. SDS-PAGE followed by autoradiography detected that treatment of Daudi cells with rIFN-alpha 2a significantly reduced phosphorylation of the membrane-associated Mr 98,000 and 70,000 polypeptides. To determine the physiological significance of these phosphorylating polypeptides in mediation of the IFN effects, they were purified from Daudi cells. It was found that the molecular weight of the purified polypeptides was approximately 330,000 and it (designated 330-kDa protein) was consist of two distinct subunits [alpha-subunit (Mr 98,000) and beta-subunit (Mr 70,000)]. The biochemical properties, such as subunit structure, subunit phosphorylation, requirements for phosphorylating activity and protease sensitivity of the 330-kDa protein were similar to those reported for Na+, K(+)-ATPase. Evidence provided here suggests that the anti-proliferative action of IFNs may, at least in part, be implicated in the IFN-induced physiological impairment of the membrane-associated 330-kDa protein.

Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.

Activation of 2′,5′-Oligoadenylate Synthetase Activity on Induction of HL-60 Leukemia Cell Differentiation

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.

Enhanced Antiproliferative Action of Interferon Targeted by Bispecific Monoclonal Antibodies

It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.

Identification of interferon-modulated proliferation-related cDNA sequences.

To identify genes mediating the antiproliferative action of interferon (IFN), two cDNA libraries were constructed with mRNA from IFN-treated and untreated human fibrosarcoma (HT1080) cells previously shown to be highly sensitive to the antiproliferative effects of IFN. Differential screening of these two libraries identified cloned sequences whose expression was either induced or repressed with IFN treatment. Rescreening of these sequences with cDNA probes constructed from proliferating or quiescent cells led to the identification of one IFN-induced and three IFN-repressed sequences whose expressions also appeared to be modulated by cell proliferation. Blot-hybridization analysis revealed that RNA levels corresponding to the three repressed genes decreased when HT1080 cells were treated with IFN or when proliferation of normal CUA foreskin fibroblast cells became naturally arrested by contact inhibition. Levels of RNA corresponding to the induced gene increased in HT1080 cells within 24 hr after IFN-treatment but declined below basal levels by 48 hr. Expression of these genes was unaffected or only slightly affected by IFN treatment in variant cells resistant to the antiproliferative effects of IFN. Collectively, these results suggest that the identified cDNAs correspond to genes that are involved in the antiproliferative action of IFN. Moreover, these results also suggest that IFN's antiproliferative action may be exerted through genes that contribute to arresting cell proliferation during contact inhibition.

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events: Studies with vascular smooth muscle cells and endothelial cells

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [ 3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFNα, recombinant human IFNα2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.

Isolation of Daudi cells with reduced sensitivity to interferon. III. Interferon-induced proteins in relation to the phenotype of interferon resistance.

The pattern of both constitutive and interferon-induced proteins was determined by two-dimensional gel electrophoresis in parental and interferon-resistant clones of Daudi cells in relation to the phenotype of interferon resistance. The complement of constitutive proteins present in clones DIF3, DIF8, DIF9 and DIF10 appeared to be identical to that of parental Daudi cells even though these cells were resistant to both the antiviral and anti-proliferative actions of interferon. Treatment of Daudi cells for 20 h with 10(3) reference units/ml of electrophoretically pure human interferon-alpha resulted in the induction of 15 proteins of molecular weights ranging from 15000 to 62000 detected after a 4 h labelling period with L-[35S]methionine. A number of these proteins were also induced in interferon-resistant clones of Daudi cells although some of these proteins appeared later and in smaller amounts than in the interferon-treated parental cells. However, seven proteins with molecular weights ranging from 18000 to 58000 which were induced by interferon in parental Daudi cells were not induced in any of the four interferon-resistant clones, suggesting that the phenotype of interferon resistance of these cells may be related to a reduction or absence of certain interferon-induced protein(s).

Effects of long-term treatment of human carcinoma cells with interferon α

Three tumor cell lines derived from human colon, urinary bladder and ovarian carcinoma were serially passaged in the continuous presence of human interferon α for extended periods of time. Phenotypic changes induced by interferon differed among these three cell lines. Thus interferon enhanced colon tumor cell aggregation but inhibited the aggregation of bladder tumor cells. The antiproliferative activity of interferon was more pronounced in bladder and ovarian cells than in colonic cells. However, the tumorigenicity of parental and cloned colon tumor cells injected i.p. or s.c. was markedly reduced by passage of the cells with interferon. Interferon treatment reduced the tumorigenicity of ovarian tumor cells when these cells were injected i.p. but not when injected s.c. The tumorigenicity of bladder tumor cells was not affected by interferon.

Growth of Wild-Type and Interferon-Sensitive Mengovirus in tk- L Cells

Cells lacking thymidine kinase (tk) have been reported to fail to respond to the action of interferon (IFN). While some laboratories have confirmed this observation, others have failed to do so. We studied the effect of IFN on four freshly isolated tk- lines of mouse L cells infected with mengovirus. In all cases, normal antiviral activity was induced. The antiproliferative activity of IFN was studied using the parental L cell line, a tk- derivative, and a tk- (tk+) subline into which the tk gene of herpes simplex virus was introduced. All three lines had a doubling time of about 20 h. In all cases, 2,000 U/ml of IFN increased this time to about 50 h. In contrast to the above results, an IFN-sensitive mutant of mengovirus (is-1) grew much better in protected tk- cells than in protected normal cells. This phenomenon appears to be dependent on the fact that tk- cells were routinely maintained in medium containing 5-bromodeoxyuridine (BUDR). In the absence of this drug, the virus behaved normally. The implications of this observation are discussed.