Suppression of UV mutagenicity by human interferon

Effects of human interferon (HuIFN)-alpha on UV mutagenicity were examined in a human cell strain, RSa, and xeroderma pigmentosum (XP)-derived fibroblasts (XP1KY). The frequency of ouabain-resistance mutation in UV-irradiated RSa cells was unusually high (Suzuki et al., 1985), but that in cells pretreated with HuIFN-alpha before irradiation was reduced. 6-Thioguanine-resistance mutation was also depressed in XP1KY cells treated with HuIFN-alpha before irradiation. However, the depression of UV mutagenicity by HuIFN-alpha was lessened by treatment with cycloheximide immediately after UV irradiation. The relationship between HuIFN-depressed UV mutagenicity and HuIFN-affected DNA-repair and repair-related functions is discussed.     

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

An interferon analogue, [Ala 30,32,33]HuIFN-alpha 2, acting as a HuIFN-alpha 2 antagonist on bovine cells

We have studied the biological and receptor binding properties of a human alpha 2-interferon (HuIFN-alpha 2) analogue, [Ala30,32,33] HuIFN-alpha 2, which is shown in the accompanying paper (1) to be biologically inactive on homologous cells. Here we demonstrate that this analogue is also devoid of biological activity on bovine MDBK cells. However, whereas the analogue did not inhibit the binding of radiolabeled HuIFN-alpha 2 to WISH cells, it did compete for binding to receptors on the bovine cells. This behavior suggested that [Ala30,32,33] HuIFN-alpha 2 could act as an antagonist of HuIFN-alpha 2 on bovine cells and indeed coaddition of the analogue and native HuIFN-alpha 2 to MDBK cells competitively inhibited both the antiviral and antiproliferative activity of HuIFN-alpha 2.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

Degenerative change in tumor cells caused by human fibroblasts and its enhancement by human interferons

The target cells (KB, HeLa, FL, human hepatoma and murine L929) were cocultured with human embryonic fibroblasts in the Petri dish. The degenerative changes of the target cells except L929 cells by the human fibroblasts were found. Human leukocyte interferon (HuIFN-alpha) and human fibroblast interferon (HuIFN-beta) enhanced these changes, but mouse IFN (MuIFN-alpha, beta) did not. The other human fibroblasts also caused the degenerative changes of the target cell, and HuIFN-alpha enhanced these changes. It was concluded that human fibroblasts play a certain role in the suppression of the human tumor cell.     

Specific binding of human alpha interferon to a high affinity cell surface binding site on bovine kidney cells

Virus-induced human alpha interferon (HuIFN-alpha) derived from Namalwa cells and purified to a specific activity of 2 X 10(8) units/mg of protein was radiolabeled with 125I-labeled Bolton and Hunter reagent to a specific activity of 4-12 microCi/micrograms of protein. The binding of this 125I-IFN to bovine kidney cells was examined at 4 degrees C. Scatchard analysis of the binding data indicate the presence of 650 binding sites/cell and binding of the ligand with an apparent Kd of 6 X 10(-11) M. Trypsin or acid treatment of cells to which 125I-IFN was bound resulted in the release of greater than or equal to 77% of the radioactivity, indicating a majority of radiolabeled material was bound to the cell surface. Antibodies against human leukocyte IFN but not antibodies against human fibroblast IFN inhibited the binding of radiolabeled IFN to the cells. The binding of 125I-IFN was not inhibited by a 75-fold molar excess of mouse IFN but was inhibited 30% by a 200-fold molar excess of human beta (fibroblast) IFN. These data are compatible with the Lower biological activities of these IFNs on bovine kidney cells. Several Escherichia coli derived HuIFN-alpha s inhibited the binding of the radiolabeled IFN to the same extent as native HuIFN-alpha s, but four fragments of HuIFN-alpha 1, an E. coli-derived 86 amino acid NH2-terminal fragment as well as 3 different synthetic carboxy-terminal fragments of 140, 56, or 46 amino acids did not inhibit binding.     

Enhancement of cell growth by human interferons

The effects of human interferons (HuIFN) on the human osteosarcoma cells were examined. HuIFN-alpha, -beta and -gamma enhanced dose-dependently the cell growth. There was no difference in the degree of the enhancement of the cell growth among HuIFN-alpha, -beta and -gamma. The higher the cell density was, the lower the degree of the enhancement of the cell growth. When HuIFN-gamma was neutralized with anti-HuIFN-gamma, the enhancement of cell growth was not found.     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.     

Cloned human interferons alpha: differential affinities for polyinosinic acid and relationship between molecular structure and species specificity

The HuIFN-alpha A and HuIFN-alpha D interferons, produced by two independent recombinant bacterial clones, have different affinities for polyinosinic acid (poly I). The monomeric form HuIFN-alpha A (FMM), but not the HuIFN-alpha D, binds to poly (I)-agarose and is protected by poly (I) from thermal inactivation. Other subtypes of HuIFN-alpha A including the monomer SMM and oligomers have no affinity for this polynucleotide. In addition, these interferons show different target cell preferences in agreement with our previous suggestion (23) that the polynucleotide binding domain may be responsible for species specificity. Two significant observations are 1) the fractions of HuIFN-alpha D and HuIFN-alpha A unbound on poly (I)-agarose show higher antiviral inducing activity on heterologous (MDBK) than on homologous (WISH) cells, whereas they induce about the same activity of 2'5' oligoadenylate synthetase in these two cell lines. These fractions are also active on L929 cells. 2) The bound fraction of HuIFN-alpha A induces almost the same antiviral and 2'5' oligoadenylate synthetase activities in MDBK and in WISH cells but neither activity in L929 cells.     

Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.     

Effect of interferon on the inhibition of malignant cell growth by human peripheral leukocytes. III. Effect of systemic administration

Human leukocyte interferon preparation (HuIFN-alpha LE) was given to the patients with cancer or with chronic hepatitis. Spontaneous tumor cell growth inhibition by human peripheral lymphocytes (STGI) and NK activity were enhanced by the systemic administration of HuIFN-alpha LE, although there were differences in the kinetics between the two activities after one time administration or by the repeated administration. This suggests that IFN acts indirectly on the tumor cells by the medium of normal lymphocytes or NK cells, and that tumor cell growth inhibition is different from NK activity.     

Effects of hyperthermia on the in vitro antiproliferative activities of HuIFN-alpha, HuIFN-beta, and rHuIFN-gamma employed separately and in combination

The relative enhancing effects of hyperthermia on the three types of interferon were evaluated in cloning studies for three human cell lines: G-361 malignant melanoma cells, WISH ammion cells, and AGS stomach adenocarcinoma cells. Hyperthermia enhanced the antiproliferative activity of rHuIFN-gamma against each of the three cell lines and the levels of enhancement by hyperthermia were seen to increase with increasing concentrations of rHuIFN-gamma. The maximum observed levels of enhancement of rHuIFN-gamma activity by hyperthermia varied from cell line to cell line. However, when the relative sensitivities of the cell lines to rHuIFN-gamma were taken into account, the levels of enhancement of rHuIFN-gamma antiproliferative activity by hyperthermia were seen to be similar for each of the cell lines, indicating that hyperthermia consistently enhanced rHuIFN-gamma antiproliferative activity. Hyperthermia did not consistently enhance the antiproliferative activities of HuIFN-alpha and HuIFN-beta. Further studies indicated that hyperthermia enhanced by approximately 6-fold the antiproliferative effects of combinations of rHuIFN-gamma with HuIFN-alpha and HuIFN-beta. The results support the possibility that a combination treatment protocol of hyperthermia and interferon administration (particularly HuIFN-gamma or combinations of HuIFN-gamma with HuIFN-alpha or HuIFN-beta) may provide an enhanced antitumor effect in man.     

Detection of the deletion of interferon-alpha and beta genes in lymphoblastoid cells by PCR]

The HuIFN-alpha and beta genes were examined by the PCR method in the 11 human lymphoblastoid cell lines. The results showed that the homozygous deletion of HuIFN-alpha and beta genes was detected in 5 of 11 cell lines and in 5 of 11 cell lines, respectively. The deletions of both the HuIFN-alpha and beta genes were observed in 4 of 11 cell lines. One T cell leukemia cell line deleted only HuIFN-alpha gene, while the other T cell leukemia line deleted only HuIFN-beta gene. This suggests that the deletions of HuIFN-alpha and beta genes may be related the development of leukemia or lymphoma.     

Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhance of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells.     

Interferon receptor interaction. Internalization of interferon alpha 2 and modulation of its receptor on human cells

Studies reported earlier [ Joshi et al. (1982) J. Biol. Chem. 257, 13884-13887] have indicated that human interferon-alpha 2 (HuIFN-alpha 2) binds to a specific macromolecular receptor on human cells as identified by cross-linking with bifunctional cross-linking reagents and analysis by polyacrylamide gel electrophoresis. We have carried out experiments to investigate the fate of the interferon-receptor complex on the cell surface under conditions which lead to cellular response. As analyzed by cross-linking and gel electrophoresis, the interferon-receptor complex, formed on incubation with 125I-IFN-alpha 2 at 4 degrees C, persisted at the cell surface for several hours at 4 degrees C; however, if the cells were switched to 37 degrees C, there was a rapid decline in the complex, apparently due to a loss of the interferon receptors from the cell surface. This was associated with an internalization of the 125I-interferon as indicated by the fact that, on incubation at 37 degrees C, an appreciable fraction of the cell-associated interferon (approximately equal to 50%) became resistant to trypsin digestion, or dissociation on incubation in growth medium or low-pH buffer. A large fraction of the trypsin-resistant (internalized) 125I-labeled material migrated as intact interferon in polyacrylamide gels, and it was immunoprecipitated by anti-(HuIFN-alpha)antibodies but not by anti-(HuIFN-beta)antibodies. The bulk of the internalized 125I-interferon was recovered in a particulate fraction and, on cross-linking with disuccinimidyl suberate, a 150000-Mr complex could be detected. The results suggest that interferon may be internalized as a complex with the receptor, which may account for the loss of the interferon-receptors on the cell surface. This modulation of the IFN-alpha/beta receptors was induced by HuIFN-alpha and HuIFN-beta but not by HuIFN-gamma. The recovery of the IFN-alpha/beta receptors, lost upon incubation with HuIFN-alpha, took several hours and required protein synthesis. The significance of the results is discussed.     

Effect of protein kinase C inhibitors on the antiviral activity of human alpha interferon in herpes simplex virus-infected human neuroblastoma cells.

Pretreatment of human neuroblastoma cells with an inhibitor of protein kinase C (PKC), staurosporine or H-7, prior to the addition of human alpha interferon (HuIFN-alpha), recombinant HuIFN-alpha, or recombinant HuIFN-beta blocked the inhibitory effect of these IFNs on the release of infectious herpes simplex virus type 1 from treated cells. In addition, staurosporine blocked the inhibitory effect of HuIFNs on the expressions of herpes simplex type 1 glycoproteins B, C, and D in treated neuroblastoma cells. Furthermore, addition of HuIFNs resulted in an increased expression of PKC in treated neuroblastoma cells. These results suggest that inhibitors of PKC block the expression of HuIFN-induced genes in treated human neuroblastoma cells. Thus, the activation of PKC is an important step in the HuIFN-treated cells of neuronal origin.     

Suppression of N-myc over-expression in human neuroblastic cells by human gamma-interferon

In the previous study, it was shown that the treatment of human neuroblastoma cells with human interferon-gamma (HuIFN-gamma) induced the morphological changes. However, the treatment with human interferon-alpha (HuIFN-alpha) or -beta (HuIFN-beta) did not induce them. In the present study, the effect of HuIFNs on the overexpression of N-myc of the human neuroblastoma cells (GOTO strain) is examined. The treatment of GOTO cells with rHuIFN-gamma inhibits the overexpression of N-myc, and its degree is dependent on the duration of the treatment. However, HuIFN-alpha and HuIFN-beta did not inhibit the overexpression of N-myc. This suggests that the oncogene N-myc may have relation to the morphological differentiation of human neuroblastoma cells because only the HuIFN-gamma, which induces the morphological differentiation, inhibits the overexpression of N-myc.     

Inhibitory effect of interferons and tumor necrosis factor on syncytium formation by bovine leukemia virus

The treatments of the bovine spleen cells BESp with recombinant human leukocyte interferon (HuIFN-alpha A) and human fibroblast interferon (HuIFN-beta) inhibit the syncytium formation by bovine leukemia virus (BLV) and the virus yield, depending on the IFN doses. But HuIFN-gamma did not inhibit the syncytium formation. The tumor-necrosis factor (TNF) also inhibits the syncytium formation and the virus yield, depending on the TNF doses. Furthermore, the combination of the HuIFN-alpha A with the TNF inhibits the syncytium formation more strongly than the HuIFN-alpha A alone or the TNF alone.     

Efficient repair of 8-oxo-7,8-dihydrodeoxyguanosine in human and hamster xeroderma pigmentosum D cells

The repair of the endogenous lesion 8-oxo-7,8-dihydrodeoxyguanosine (8-oxodG) was investigated in the nucleotide excision repair mutant xeroderma pigmentosum D (XPD), using human normal or transformed XPD fibroblasts and the Chinese hamster XPD cell line UV5. In vivo repair of 8-oxodG induced by hydrogen peroxide treatment and analyzed by high-performance liquid chromatography/electrochemical detection was normal in the XPD mutant fibroblasts XP15PV and GM434, as compared to normal human fibroblasts GM970, GM5757, and GM6114. Similar results were obtained with the human SV40-transformed XPD mutant cell line GM8207 in comparison to the control cell line GM637. Repair of 8-oxodG was even slightly (2-3-fold) but reproducibly increased in Chinese hamster XPD mutant UV5 cells, as compared to parental AA8 cells. This unexpected effect was reversed by transfection in UV5 cells of a wild-type XPD cDNA and confirmed in in vitro experiments in which a plasmid substrate containing a single 8-oxoG was repaired by UV5 cell extracts. The data show that repair of 8-oxodG is normal in XPD cells, thus indicating that the neurological complications of XPD patients may not be linked to in vivo accumulation of this lesion.