Enhanced expression of heat shock protein and mRNA synthesis by type I interferon in human HL-60 leukemic cells

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.     

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.     

Establishment of the antiviral state in alpha, beta-interferon-resistant Friend cells treated with gamma-interferon. Induction of 67-kilodalton protein kinase activity in absence of detectable 2-5A synthetase

Treatment with murine gamma-interferon (IFN) preparations of variant sublines of Friend leukemia cells resistant to the alpha, beta IFN-induced antiviral state (Affabris, E., Jemma, C., and Rossi, G.B. (1982) Virology 120, 441-452; Affabris, E., Romeo, G., Belardelli, F., Jemma, C., Mechti, N., Gresser, I., and Rossi, G. B. (1983) Virology 125, 508-512) results in the establishment of a bona fide antiviral state. In fact, gamma IFN preparations are able to induce a dose-dependent reduction of endogenous virus release and of vesicular stomatitis or encephalomyocarditis viruses yields (up to 1.5 log). Under these experimental conditions, no inducible 2-5A synthetase activity is detectable in cell extracts. The 67-kDa protein kinase, uninducible by treatment with alpha, beta IFN (up to 13,000 units/ml), is instead induced upon treatment with gamma IFN at a similar rate of activity as in wild-type Friend leukemia cells, both when assayed in solution and after immobilization on poly(rI) X poly(rC)-agarose.     

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.     

Ethanol induces 2',5'-oligoadenylate synthetase and antiviral activities through interferon-beta production.

We demonstrate here that ethanol, in contrast to heat shock (Chousterman, S., Chelbi-Alix, M.K., and Thang, M.N. (1987) J. Biol. Chem. 262, 4806-4811), induces interferon (IFN) synthesis and its related activities in Madin-Darby bovine kidney (MDBK) cells. The induced IFN is secreted maximally at 6 h, whereas the induction of 2',5'-oligoadenylate synthetase mRNA peaks between 9 and 12 h and its activity at 15 h. The appearance of both 2',5'-oligoadenylate synthetase activity and the antiviral state upon ethanol treatment is prevented by anti-bovine recombinant IFN-beta antibodies. Bovine diarrhea virus infection-free MDBK cells cultured in medium supplemented with serum substitute also gave similar results, thus indicating that IFN synthesis induced by ethanol is not mediated by the activation of bovine diarrhea virus. Together, these results show that: 1) ethanol induces the 2',5'-oligoadenylate synthetase and antiviral activities through IFN-beta production; and 2) the IFN produced does not act directly from inside the cells, but has to be first secreted to bind to its receptor. In MDBK cells, ethanol induces the synthesis of the 70-kDa protein, which precedes the expression of 2',5'-oligoadenylate synthetase; moreover, the transient nature of the synthesis of the hsp 70 in these cells is similar after both heat shock and ethanol treatment.     

Alteration of heat sensitivity by treatment with nonpermissive temperature or cycloheximide in temperature-sensitive CHO mutant tsH1 cell

1. 1.|The temperature-sensitive mutant CHO-tsH1 and wild type (CHO-SC) cells became thermal resistant when cells were treated for either 2 h at 39.5°C before heating at 43°C or 2 h with 10 μg/ml cycloheximide (CHM) before and during heating at 43°C.

2. 2.|There was a 2000-fold increase in survival after 2.5 h at 43°C by preincubation at 39.5°C in both cell types. There was also a 200- or 700-fold increase in survival after 2.5 h at 43°C by treatment with CHM in tsH1 or SC cell type respectively.

3. 3.|In contrast to the effects at 43°C, at 41.8°C these protective effects were not evident in tsH1 cells. In wild type, however, there was an 800- or 1800-fold increase in survival after 8 h at 41.8°C by preincubation at the temperature of 39.5°C or treatment with CHM, respectively.

4. 4.|Therefore, these results suggest that killing of tsH1 at low temperature hyperthermia (41.8°C) is probably due to denaturation of thermolabile leucyl-tRNA synthetase.

5. 5.|The denaturation of this enzyme may not be protected by inhibition of protein synthesis by preincubation at the nonpermissive temperature of 39.5°C or by CHM.

2',5'-Oligoadenylate synthetase expression is induced in response to heat shock

Hyperthermia (45 degrees C) induced the synthesis of a characteristic heat-shock protein of 70,000 daltons (70 hsp) in Madin-Darby bovine kidney (MDBK) cells. In addition, subsequent to heat shock, there was a substantial increase in the 2',5'-oligoadenylate synthetase (2',5'-A synthetase) activity in both MDBK and human WISH cells. However, in contrast to 70 hsp synthesis, which reached its maximum 3 h after cell transfer from 45 to 37 degrees C, increase in 2',5'-A synthetase expression, conspicuous after 6 h, attained its maximum only 18 h after transfer. Another interesting observation is that, during recovery at 37 degrees C, the cells released into the medium heat-shock-induced factor(s) (HSIF) capable of inducing an increase in 2',5'-A synthetase activity in fresh MDBK cells. HSIF behaves as a polypeptide with a molecular weight of more than 5,000; it is relatively heat stable and sensitive to acidic treatment. HISF seems different from interferon (IFN) since: 1) no detectable antiviral state developed after infection in cells treated with HSIF; 2) antibovine IFN antibodies did not abolish the inducing capacity of HSIF; 3) IFN had an additive effect on the inducing capacity of HSIF, and 4) HSIF released from bovine cells induced a net enhancement of 2',5'-A synthetase activity in human WISH cells. The first three of these observations applied also to heat-shocked MDBK cells.     

Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.     

Effect of interferon on the production of HBsAg and induction of an antiviral state in human hepatoma cell line PLC/PRF/5

The effects of human alpha and beta interferons (IFN) on the production of HBsAG by PLC/PRF/5 cells, an HBsAg-producing human hepatoma cell line, were studied in the exponential and stationary phases of cell growth. When exponential phase cells were treated with 100 or 1,000 U of IFN per ml for 48 hr. the amount of HBsAg in the culture medium decreased. The number of cells and the synthesis of DNA and proteins were also reduced by the IFN treatment. These results suggested that IFN did not affect the production of HBsAg specifically in exponential phase cells. When cells in the stationary phase were similarly treated with IFN, HbsAg production was not inhibited nor did the number of cells decrease. To examine the antiviral state induced by IFN in PLC/PRF/5, induction of 2'5'-oligo (A) synthetase and susceptibility to two kinds of viruses were examined. The 2'5'-oligo (A) synthetase activity was increased in an IFN-dose dependent manner. Susceptibility to vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) was decreased by treatment with 10 and 100 U of IFN per ml for 20 hr. It was concluded that IFN-alpha and IFN-beta induce 2'5'-oligo (A) synthetase and the antiviral state, but do not inhibit HBsAg production by PLC/PRF/5 cells.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

Potentiation of thermal injury in mouse cells by interferon

Mouse cells, when exposed to high temperature (43 degrees), shut off overall protein synthesis and continue to synthesize "heat shock proteins". Such heat shocked cells, upon reincubation at 37 degrees C, recover and proliferate. However, when mouse cells are pretreated with mouse interferon (IFN) and then exposed to 43 degrees, more than 99% of the cell population fail to recover. Synthesis of the major heat shock protein is unaffected in cells treated with IFN. Experiments designed to assess the role of intracellular glutathione (GSH) during cells' recovery from hyperthermia indicated that there is an irreversible depletion of glutathione when IFN treated cells are heat shocked. Neither depletion of GSH, nor potentiation of thermal injury was observed in a IFN-resistant line of mouse cells.     

Enhancement of cell killing by induction of apoptosis after treatment with mild hyperthermia at 42 degrees C and cisplatin.

Ohtsubo, T., Igawa, H., Saito, T., Matsumoto, H., Park, H. J., Song, C. W., Kano, E. and Saito, H. Enhancement of Cell Killing by Induction of Apoptosis after Treatment with Mild Hyperthermia at 42 degrees C and Cisplatin. Radiat. Res. 156, 103-109 (2001).We examined the interactive effects of cisplatin (1.0 microg/ml) combined with hyperthermia on cell killing and on the induction of apoptosis in IMC-3 human maxillary carcinoma cells. The cytotoxic effects of hyperthermia on IMC-3 cells at 44 degrees C were greater than at 42 degrees C, as has been reported for many other cells. The induction of apoptosis, DNA fragmentation and poly(ADP-ribose) polymerase cleavage were greater after hyperthermia at 44 degrees C for 30 min compared with treatment at 42 degrees C for 105 min, even though both of these heat doses were isoeffective in reducing cell survival to 50%. Treatment with cisplatin at 37 degrees C for up to 120 min did not result in cytotoxicity or the induction of apoptosis. The enhancement ratio for treatment with cisplatin at 42 degrees C was greater than that at 44 degrees C. More apoptosis was induced after the treatment with cisplatin at 42 degrees C compared to treatment with cisplatin at 44 degrees C. Taking these findings together, the combination of cisplatin and hyperthermia at 42 degrees C appeared to be more effective than cisplatin with hyperthermia at 44 degrees C for the induction of apoptosis in IMC-3 cells.     

The Testicular Antiviral Defense System: Localization, Expression, and Regulation of 2′5′ Oligoadenylate Synthetase, Double-Stranded RNA-activated Protein Kinase, and Mx Proteins in the Rat Seminiferous Tubule

Although the involvement of viruses in alterations of testicular function and in sexually transmitted diseases is well known, paradoxically, the testicular antiviral defense system has virtually not been studied. The well known antiviral activity of interferons (IFNs) occurs via the action of several IFN-induced proteins, among which the 2′5′ oligoadenylate synthetase (2′5′ A synthetase), the double-stranded RNA-activated protein kinase (PKR), and the Mx proteins are the best known. To explore the antiviral capacity of the testis and to study the testicular action of IFNs, we looked for the presence and regulation of these three proteins in isolated seminiferous tubule cells, cultured in the presence or in the absence of IFN α, IFN γ, or Sendai virus. In all conditions tested, the meiotic pachytene spermatocytes and the post-meiotic early spermatids lacked 2′5′ A synthetase, PKR, and Mx mRNAs and proteins. In contrast, Sertoli cells constitutively expressed these mRNAs and proteins, and their levels were greatly increased after IFN α or Sendai virus exposure. While peritubular cells were also able to markedly express 2′5′ A synthetase, PKR, and Mx mRNA and proteins after IFN α or viral exposure, only PKR was constitutively present in these cells. Interestingly, IFN γ had no effect on peritubular cells' 2′5′ A synthetase and Mx production but it enhanced Mx proteins in Sertoli cells. In conclusion, this study reveals that the seminiferous tubules are particularly well equipped to react to a virus attack. The fact that the two key tubular elements of the blood–testis barrier, namely, Sertoli and peritubular cells, were found to assume this protection allows the extension of the concept of blood–testis barrier to the testicular antiviral defense.     

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.     

A hamster temperature-sensitive alanyl-tRNA synthetase mutant causes degradation of cell-cycle related proteins and apoptosis

We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as ts ET12. It has a single nucleotide mutation, converting the 321st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: ts BN250 and ts BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5 degrees C. ts ET12 cells, however, did not undergo apoptosis until 48 h after a temperature-shift to 39.5 degrees C, while mutated alanyl-tRNA synthetase of ts ET12 cells was lost within 4 h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in ts ET12 cells at 39.5 degrees C, as shown in ts BN250. In contrast, the mutated aminoacyl-tRNA synthetases of ts BN250 and ts BN269 were stable at 39.5 degrees C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5 degrees C. No release of EMAPII occurred in ts ET12 cells at 39.5 degrees C, consistent with the delay of apoptosis in these cells.