Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired.

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.     

The ppp(A2'p)nA and protein kinase systems in wild-type and interferon-resistant Daudi cells

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2'p)nA(n = 2 to less than or equal to 4) in these effects has been investigated. No (less than 1 nM) ppp(A2'p)nA or (A2'p)nA or alternative products of the ppp(A2'p)nA synthetase [e.g. NAD (2'pA)2] were detected in interferon-treated cells. In addition no evidence was obtained for the occurrence of ppp(A2'p)nA-mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon. A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2'p)nA and interferon and double-stranded RNA (dsRNA)-mediated protein kinase systems. The resistant line was not receptor-negative as it responded to interferon by the production of elevated levels of the ppp(A2'p)nA synthetase similar to those observed in extracts from wild-type cells. There was no detectable difference between the lines in the levels of the (2'-5')phosphodiesterase responsible for the degradation of ppp(A2'p)nA. There was, however, about a twofold increase in the ppp(A2'p)nA-dependent endoribonuclease activity in response to interferon with extracts from the wild-type but not the resistant cells. In addition, although the dsRNA-dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild-type but not the resistant cells.     

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.     

Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.     

Interferon- and sarcolectin-dependent cellular regulatory interactions

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.     

Protein synthesis in extracts from interferon-treated Mengo virus infected cells

We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [³⁵S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.     

Early plasma membrane depolarization by alpha interferon: biologic correlation with antiproliferative signal

Daudi lymphoma cells, of a line sensitive to growth inhibition by alpha interferon, showed dose-dependent plasma membrane depolarization within 10 min after exposure to natural or recombinant alpha interferons (10 to 1000 IU/ml). This biophysical change was detected flow cytometrically by measuring the intensity of fluorescent emission from cells stained with dye indicators of membrane potential. Subclones of Daudi lymphoma cells, resistant to growth inhibition by alpha interferon, showed no membrane depolarization. Parallel results were obtained in initial tests of an isologous pair of T cell and B cell lines which differ in sensitivity to growth inhibition. Thus, decreased membrane potential may herald an interferon signal for antiproliferative action.     

2',5'-Oligoadenylate synthetase activity in lymphocytes from normal mouse.

The activity of 2',5'-oligoadenylate synthetase, an enzyme recently discovered in interferon-treated cells, was found in lymphocytes from normal mouse spleen that had received neither exogenous interferon nor its inducers. The oligoadenylate synthesized by lymphocyte cell extracts inhibited protein synthesis in rabbit reticulocyte lysates. The oligomers were composed mainly of trimer and were resistant to digestion by T2 ribonuclease. The level of the enzyme in lymphocytes was about 20 to 30% of that in L929 cells treated with interferon. The activity of the enzyme was further enhanced in lymphocytes in vitro by addition of interferon. The 2',5'-oligoadenylate synthetase was distributed among several lymphoid tissues, but was not detected in cell extracts from brain or liver. The enzyme may play an important role in the regulation of the immune system.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Independent regulation of ppp(A2'p)nA-dependent RNase in NIH 3T3, clone 1 cells by growth arrest and interferon treatment

The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.     

Cellular RNA is not degraded in interferon-treated HeLa cells after poliovirus infection

A drastic inhibition of protein synthesis occurs in HeLa cells treated with human lymphoblastoid interferon and infected with poliovirus. At the time when this inhibition has been established no degradation of ³²P-labelled ribosomal RNA can be detected. Isolation of the mRNAs from poliovirus-infected cells plus or minus interferon treatment, followed by translation in a reticulocyte lysate indicates that cellular mRNAs remain active. These results suggest that gross degradation of cellular RNA does not occur in interferon-treated poliovirus-infected HeLa cells and that a non-specific nuclease induced by 2′–5′ A is not responsible for the inhibition of protein synthesis observed.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway

The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein synthesis, a blockade in ribosome component biosynthesis, and G1 cell cycle arrest. G1 arrest may occur by inhibiting the protein synthesis of critical factors required for cell cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT activity are relatively resistant. Our previous data suggest that low AKT activity induces resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-Myc proteins. In support of this notion, the current study demonstrates that the human cyclin D1 mRNA 5' untranslated region contains an internal ribosome entry site (IRES) and that both this IRES and the c-myc IRES are negatively regulated by AKT activity. Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as specific inhibitors of these pathways reduce IRES-mediated translation and protein levels under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation initiation may permit cell cycle progression upon mTOR inactivation in cells in which AKT kinase activity is relatively low.     

Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.     

Inhibition of cell division by interferons: Changes in the transport and intracellular metabolism of thymidine in human lymphoblastoid (Daudi) cells

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon-treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.