Biomedical subjects
Publications and source records attributed to T Kuwata.
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Characterization was performed of a UV-resistant variant strain. UVr-10, derived from a human clonal cell line, RSb, with high sensitivity not only to the lethal effect of 254-nm far-ultraviolet (UV) irradiation but also to the effects of 4-nitroquinoline 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to the cell proliferation inhibition (CPI) effect of human leukocyte interferon (HuIFN-alpha) preparations. Colony-formation assays confirmed the increased resistance of UVr-10 cells to both UV and 4NQO, but no increased resistance to MNNG. The marked recovery from the inhibition of the total cellular DNA synthesis of UVr-10 cells, estimated by [methyl-3H]thymidine ([3H]dThd) uptake into the cellular DNA materials, was seen during 6 h after UV irradiation or 4NQO treatment even under the conditions without the recovery uptake into those of the parent RSb cells, but not during 6 h after MNNG treatment. Comparative studies on the activity of DNA repair synthesis between UVr-10 and RSb cells, by measuring the extent of UV-, 4NQO- or MNNG-induced unscheduled DNA synthesis (UDS) and DNA repair replication, revealed an increased activity of UVr-10 cells to UV and 4NQO but no significant increase of the activity to MNNG. These results suggest that increased DNA repair activities of a UVr-10 cell line may account for its becoming resistant to the lethal effect of UV and 4NQO. Concerning the CPI effect of HuIFN-alpha, UVr-10 cells showed increased resistance. Further, the DNA synthesis activity of UVr-10 cells was not so inhibited by HuIFN-alpha exposure as that of RSb cells. However, HuIFN-alpha-exposed UVr-10 cells showed more enhanced levels of activity of pppA(2'p5'A)n synthetase (2-5A synthetase) than the exposed RSb, thus suggesting that HuIFN-alpha could exert enough intracellular effect even in UVr-10 cells. The implication of the increased resistance of UVr-10 cells to the effects of UV, 4NQO and HuIFN-alpha, but not to those of MNNG, is discussed.
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We have studied neutralization of the antiviral activity of human fibroblast (IFN-beta) and immune interferon (IFN-gamma) by incubation with glycolipids (including the gangliosides GM1, GM2 and GM3, as well as various other glycolipids) and with the sialoglycoprotein, glycophorin. When 100 units/ml of IFN-beta were preincubated with 30-80 microM of the gangliosides, all antiviral activity was abolished. Similarly, 120 microM of glycophorin completely reversed the antiviral activity of 100 units/ml of IFN-beta. Glycolipids containing more than two sugars also showed moderate inhibitory effects. GM2 at a concentration of 200 microM almost completely inhibited the antiviral activity of 100 units/ml of IFN-gamma, but GM1, GM3 and glycophorin had only a moderate inhibitory effect. These results suggest that the terminal N-acetylneuraminic acid (NANA) residues of gangliosides and of glycophorin play an important role in the inhibition of IFN-beta, and that they may be similarly involved in the inhibition of IFN-gamma.
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.
Natural killing (NK) activities were measured in peripheral blood lymphocytes (PBL) from 14 patients with Werner's syndrome (WS), and the results were compared with those of 187 normal individuals at different ages. The NK activities of healthy donors were demonstrated a characteristic and invariable manner in accordance with the age and the sex, whereas the activities of WS patients were considerably reduced irrespective of the age and the sex. When normal PBL were preincubated with WS sera containing anti-lymphocyte antibodies (ALA) plus rabbit complement, the NK activities of the surviving PBL were markedly reduced when compared with those of PBL treated with other heterologous ALA of rabbit origin in the same manner. PBL from WS patients treated with purified human leucocyte interferon augmented the NK activity even beyond the range of controls. These results suggested that NK activities in WS patients were reduced in a manner similar to that in normal old individuals. The ALA reactive to NK cells in WS patients may participate in the reduction of NK activities. The augmentative effect of interferon on NK activities in WS patients may also suggest the possible blocking of potent NK activities by an unknown mechanism rather than an intrinsic defect of their NK cells.
Human interferon beta (IFN-beta) stimulated the synthesis of prostaglandin E (PGE) and prostaglandin F2 alpha in IFN-sensitive RSa and GM258 cell lines, but not in IFN-resistant HEC-1 cells. IFN-beta at a concentration of 1000 units/ml elicited 2- to 3-fold increases in PGE production in these cell lines. In the presence of exogenous arachidonic acid (1 microgram/ml), IFN-pretreated cells produced 5-fold more PGE compared to the cell cultures in the absence of arachidonic acid. Prednisolone, an inhibitor of phospholipase A2, at a concentration of 2 micrograms/ml inhibited the enhanced synthesis of PGE by IFN-pretreated cells. Indomethacin (4 micrograms/ml), a potent fatty acid cyclooxygenase inhibitor, also inhibited the increased synthesis of PGE. IFN stimulated the release of [14C]arachidonic acid from phospholipids but did not stimulate the activity of fatty acid cyclooxygenase. These data suggest that IFN stimulates prostaglandin synthesis by promoting the release of arachidonic acid from phospholipids. Since cycloheximide and actinomycin D inhibited the stimulation of PGE synthesis, the stimulation of prostaglandin synthesis by IFN seemed to be due to de novo enzyme synthesis which catalyzes the release of fatty acid. Addition of exogenous PGE suppressed the growth of RSa and GM258 cells. Prednisolone and iodomethacin partially inhibited anti-cell growth activity of IFN, suggesting a possibility that IFN-inhibited cell growth was partly mediated by prostaglandin.
Cholera toxin inhibits human natural cell-mediated cytotoxicity in a dose- and time-dependent manner. Pretreatment of lymphocytes with 10 ng/ml of cholera toxin for 2 h almost completely inhibited cytolysis. Interferon augmented human natural cell-mediated cytolysis, but when lymphocytes were pretreated with cholera toxin before interferon treatment, no enhancement of cytolysis occurred. Cholera toxin could inhibit the enhancement of cytolysis by interferon even when lymphocytes were treated with cholera toxin after 2 h interferon treatment. Cholera toxin subunit B which binds cell surface ganglioside galactosyl-N-acetylgalactosaminyl - [N-acetylneuraminyl] - galactosylglucosylceramide (GM1) without activating adenyl cyclase had no effect either on natural cytolysis or on the enhancement of natural cytolysis by interferon, suggesting that mere binding of cholera toxin to the cellular receptor was not enough to inhibit natural cell-mediated cytolysis. Cyclic adenosine 3',5'-monophosphate (cAMP) levels increased in cholera toxin-treated lymphocytes and the time course of cAMP accumulation was similar to that of cytotoxicity inhibition. Exogenous dibutyryl-cAMP (db-cAMP) and theophylline inhibited cytolysis, while exogenous dibutyryl cyclic guanosine 3',5'-phosphate (db-cGMP) enhanced cytolysis slightly, suggesting that the process of inhibition of human natural cell-mediated cytolysis was at least partly modulated by intracellular cyclic nucleotides.
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A human endometrial cancer cell line, HEC-1, was found to be resistant to the antiviral and anticellular action of interferon. However, HEC-1 cells were susceptible to the cytotoxicity of natural killer (NK) cells, and interferon enhanced such NK activity. When HEC-1 cells were treated with interferon, sensitivity of HEC cells to the cytotoxicity of NK cells was not suppressed.
Suppressive effects of human IFN-alpha, IFN-beta and mouse-IFN on syncytium formation in human and mouse transformed cells have been studied using u.v.-irradiated Sendai virus (UV-Sendai virus). After treatment of human RSa cells with 500 units/ml human IFN-alpha for 16 h, the syncytium formation induced by UV-Sendai virus was reduced to less than 10% of controls. The suppressive effect was dependent on the amount of interferon added, and it was blocked by the addition of cycloheximide. Suppression of syncytium formation was also observed on human IFr cells, which are partially resistant to the anticellular effect of interferon but are sensitive to the antiviral effect of interferon. Human IFN-beta also had a suppressive effect on syncytium formation in human transformed cells, and human IFN-alpha, IFN-beta and mouse IFN showed species specificity in their effect on fusion. This anti-cell fusion activity was developed in IFr cells from about 5 h after addition, of IFN-alpha and when the cells were treated with IFN-alpha for 16 h, the resistant state of cell continued for over 20 h after removal of IFN-alpha.
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Interferon induces two double-stranded RNA-dependent enzymatic activities: an oligoisoadenylate synthetase that converts ATP to ppp(A2'p)n5'A, and a protein phosphokinase. We have explored the level and inducibility of these two enzymes in a human cell line (HEC-1) totally insensitive to both the antiviral and the anticellular actions of interferon. The activities of both enzymes are high in untreated cells and only minor changes occur after treatment with interferon, even at high concentrations. Interferon-treated HEC-1 cells do not contain an inhibitor of the oligoisoadenylate synthetase activity. The products of this HEC-1 oligoisoadenylate synthetase consist mainly of dimers, trimers, and tetramers as found in other cell lines after interferon treatment. The synthetase level is unaffected by treating the cells with anti-interferon antiserum, indicating that the results cannot be explained by a spontaneous low production of interferon by these cells. Furthermore, virus multiplication is not inhibited, even after treatment with interferon. These observations suggest that either the two enzymatic activities do not suffice for the establishment of an antiviral state in vivo or that a regulatory control mechanism, lost in these cells and common for both enzymes, is required for the expression of the antiviral action of interferon. This might explain both the constitutivity of the two enzymes and the interferon resistance observed.
Sensitivities of human transformed cell line RSa and its variant cell line IFr to the cytotoxicity of Con A were compared. IFr cells were more resistant than RSa to Con A. Con A-resistant cell lines, Con Ar-1 and Con Ar-3, were isolated from RSa, and they were slightly more sensitive than RSa cells to the cell growth-inhibitory actions of interferons. Agglutinability of RSa, IFr, and Con Ar cells by Con A was compared and found to be almost equal. The combined effects of Con A and interferon upon growth and viability of these cell lines were tested. When RSa and IFr cells were treated simultaneously with Con A and Le-IF, growth of the cells was suppressed more markedly than when treatment was with Con A or Le-IF alone. To clarify the mechanism of this phenomenon, binding of 125I-labeled Con A was examined. Though ther wee some differences, both leukocyte and fibroblast interferon enhanced the binding of Con A to RSa cells and also in Con Ar cells but, in interferon-resistant IFr and HEC cells, enhancement of Con A binding was low or not observed. Therefore, the combined effect of Con A and interferon on the inhibition of cell growth is not considered to be merely due to the enhanced binding of Con A by interferon action. Successive treatment of RSa or Con Ar cells with Con A and interferon did not enhance the antiviral action of interferon at all. On the contrary, simultaneous treatment with Con A and interferon suppressed the antiviral action of interferon, depending on the concentration of Con A used. Thus, the effect of Con A on the antiviral and cell growth-inhibitory actin of interferon seems rather different.
Treatment of HeLa cells with human interferons inhibited 51Cr release from cells induced by ultraviolet-inactivated Sendai virus. The inhibitory effect became apparent about 6 h after interferon treatment and persisted for 24 to 48 h. In the interferon-treated cells, the cytolysis was inhibited within 10 min after adding virus and the inhibitory action was suppressed by the treatment of the cells with cycloheximide. Mock interferon and mouse interferon did not inhibit the cytolysis and antiinterferon serum neutralized the effect of interferon. All these findings indicate that Sendai virus-induced cytolysis is inhibited by interferon per se. However, interferon did not have any influence on Sendai virus hemolysis.
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