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Background: Chronobiological studies with anticancer drugs have shown that their effectiveness and/or toxicity is significantly influenced by the time of their administration in the circadian cycle. Previous studies also have shown that the myelotoxicity of interferons is similarly influenced.Purpose: This study was undertaken to evaluate the antitumor activity of interferons as a function of their administration to animals at defined points in the circadian cycle with equal light and dark periods.Methods: A murine tumor model was employed. Following adaptation to alternating cycles of 12 hours of light and 12 hours of dark for a period of 2-3 weeks, C57BL/6 mice were inoculated with B16 melanoma cells intraperitoneally at different hours after light onset. Exactly 24 hours after inoculation, each group received intraperitoneal injections of either recombinant human interferon alpha (rHuIFN-alpha A/D), recombinant murine IFN-gamma (rMuIFN-gamma), or interferon-carrier solution as control (once a day for 5 days) and were monitored for the length of their survival.Results: The antitumor activity (calculated as percent increased life span) of both rHuIFN-alpha A/D and rMuIFN-gamma varied with the points at which they were administered in the circadian cycle. However, the points showing minimum and maximum activity for rHuIFN-alpha A/D (12-16 and 0-4 hours after light onset, respectively) did not correspond with the points for the rMuIFN-gamma (0-8 and 16 hours after light onset, respectively). To generate maximum antitumor activity, approximately fivefold higher amounts of rHuIFN-alpha A/D were required at 12 than at 4 hours after light onset (dose range, 3333-90,000 IU/d) (P < .0001). Similarly, for rMuIFN-gamma at least 8.5-fold greater amounts were required at 8 than at 16 hours after light onset (dose range, 667-6000 IU/d) (P < .01).Conclusions: In the murine tumor model, administration of rHuIFN-alpha A/D at 4 hours after light onset and rMuIFN-gamma at 16 hours after light onset may produce maximum antitumor activity. [ABSTRACT FROM AUTHOR]

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones. [ABSTRACT FROM AUTHOR]

Vaccination using inactivated B16 melanoma cells that have been treated in vitro for > 2 weeks with interferon-alpha (IFN-alpha) (B16alpha cells) has been shown to elicit a protective host antitumor immunity. In these studies, vaccination with B16alpha cells has been shown to provide protection against primary B16 tumor challenge, established B16 tumors, and metastatic B16 tumors. Specific immune cells and factors that might mediate this tumor immunity have now been evaluated. Macrophage depletion studies suggest that macrophage function is required for expression of tumor immunity either for processing of antigen or for cytokine production but that macrophage function is not involved in direct cytotoxicity against the B16 challenge tumor. CD8(+) T cell depletion studies show that cytotoxic T cell function is required for expression of tumor immunity. Syngeneic knockout mouse experiments offer further insights into the immune cells and factors that mediate the development and expression of tumor immunity. First, interleukin-12 (IL-12) knockout mouse experiments identify IL-12 as an important cytokine in mediating the development of tumor immunity. Second, specific knockout mouse experiments show that tumor immunity requires the function of CD4(+) T cells, CD8(+) T cells, and natural killer (NK) cells. Third, specific knockout mouse experiments show that tumor immunity does not require the function of B cells. The results suggest that vaccination with inactivated B16alpha cells induces an active, cell-mediated immunity to B16 melanoma cells. The tumor vaccination protocol with B16alpha cell vaccinations establishes a potent tumor immunity against B16 melanoma tumors in mice and may serve as a model for induction of tumor immunity against primary or secondary melanoma tumors in humans.

Orally administered interferons (IFN-alpha, IFN-beta, and IFN-gamma) have been shown to exert a number of systemic effects. Orally administered IFNs exert dose-dependent suppressive effects on the peripheral white blood cell (WBC) count. The suppression of the peripheral WBC count is mediated by a suppression of the function of the bone marrow, as measured in an in vitro bone marrow colony-forming assay. The peripheral WBC and bone marrow suppressive effects of orally administered IFNs are at least as potent as those occurring with parenterally administered IFNs. However, the mechanism by which orally administered IFNs exert these peripheral WBC suppressive and bone marrow suppressive effects differs significantly from that of parenterally administered IFNs: orally administered IFN is not detectable in the serum, the effect of orally administered IFN is not blocked by circulating antibody, the effect of orally administered IFN can be adoptively transferred by injection with peripheral white blood cells from donor mice, and the effect of orally administered IFN develops more slowly than that of parenterally administered interferon. Orally administered IFN-alpha employed alone and in synergistic combination with intraperitoneally administered IFN-gamma can exert an antitumor effect. Finally, orally administered interleukin-2 can exert a suppressive effect on both the peripheral white blood cell count and on the bone marrow. These observations suggest that the oral route may be an effective and novel mechanism for the efficacious administration of IFNs and other lymphokines/cytokines.

B16 melanoma cells exposed to >2 weeks of in vitro interferon-alpha (IFN-alpha) treatment (B16alpha cells) were UV inactivated and used for vaccination. This vaccination was efficacious against challenge with parental B16 cells in the absence of adjuvant therapy. Vaccinations based on parental cells and B16 cells exposed to short-term in vitro IFN-alpha treatment were not effective. The efficacy of B16alpha vaccination was evaluated using three B16 tumor models. Using intraperitoneal (i.p.) tumor challenge given after vaccination, vaccination efficacy depended on the concentration of IFN-alpha to which B16alpha cells were exposed, the number of inactivated B16alpha cells inoculated, the number of inoculations administered, and the amount of tumor burden. A significant fraction (30%) of vaccinated mice surviving initial challenge had durable immunity against a second parental tumor challenge. This immunity increased to 92% with administration of a single booster vaccination. Using metastatic tumor challenge given after vaccination, vaccination reduced lung metastases by approximately 67%. Using vaccination begun 3 days after subcutaneous (s.c.) tumor challenge, regression of established tumor occurred when vaccination was given i.p. (39%) or contralaterally s.c. (53%). Taken together, the results suggest that vaccination with inactivated B16alpha cells may serve as a model for induction of host tumor immunity against primary or secondary tumors.

Administration of interferons (IFN) via the intranasal route recently has been shown to exert an antitumor activity against a variety of tumors in mice, including B16 melanoma inoculated intravenously. This study confirms the antitumor activity of orally administered IFN-alpha against B16 melanoma challenge using another route of tumor inoculation, the intraperitoneal route. It further demonstrates that orally administered IFN-alpha can synergistically interact with intraperitoneally administered IFN-gamma but not with intraperitoneally administered IFN-alpha. The results support the interpretation that the oral route may provide an effective alternative or supplement to current methods of IFN administration for the control of malignancies.

Previously we demonstrated that IFN-alpha augments mda-6/WAF1 and inhibits cyclin-dependent kinases in a p53-independent fashion in B 16 murine melanoma cells. On the other hand, IFN-gamma activates p53 expression without affecting the mda-6/WAF1 system. Combination of the two IFNs is additive. B16 cells acquire IFN-alpha resistant but IFN-gamma sensitive phenotype after long term IFN-alpha treatment (B16alpha cells). Here we demonstrate the absence of mda-6/WAF1-associated repression of cyclin-dependent kinases, but the existence of p53-dependent c-myc inhibition in IFN-gamma-treated B16alpha cells. Clearly, selective desensitization of IFN-alpha related growth regulation does not influence the IFN-gamma associated pathway. Our results further support the coexistence of distinct growth regulatory mechanisms in B16 cells that can be activated by different IFN-types independently of each other.

Mice inoculated with B16 melanoma cells exposed to long-term in vitro IFN-alpha treatment (> or = 14 days, B16 alpha cells) but not short-term in vitro IFN-alpha treatment (24 h) exhibited an enhanced survival time. Enhanced survival time also occurred when inactivated B16 alpha cells were inoculated at the same time as live B16 cells. Further, an even greater improvement in survival time was observed when the inactivated B16 alpha cells were inoculated before live B16 cell challenge. No enhancement in survival time was observed when mice were inoculated with inactivated, untreated B16 cells. Enhancement of survival time by B16 alpha cells was unrelated to retrovirus surface antigen expression. Long-lasting protective immunity to B16 cells was observed in mice that survived B16 alpha cell, but not normal B16 cell, challenge and subsequent IFN treatment. It is evident that inoculation with inactivated B16 alpha cells, but not with inactivated untreated B16 cells, was able to prolong significantly the survival time of mice either simultaneously or subsequently challenged with live B16 cells. Additionally, survival of B16 alpha-inoculated but not B16-inoculated mice was accompanied by a durable immunity. Inoculation of inactivated B16 alpha cells may serve as a model for the induction of host immunity to a parental primary or secondary tumor.

Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-alpha become resistant to the in vitro antiproliferative effects of IFN-alpha. However, IFN-alpha-treated mice inoculated with these in vitro IFN-treated cells (B16 alpha res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-alpha-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16 alpha res cells to the in vivo antitumor effects of IFN-alpha was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16 alpha res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16 alpha res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-alpha against B16 alpha res cells. Macrophage killing was unlikely to be mediated by TNF-alpha or IL-1 as B16 and B16 alpha res cells were equally sensitive to TNF-alpha and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16 alpha res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.

A number of different laboratories reported on studies with orally administered interferons and cytokines. Their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. As difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. They also raise the possibility that these effects may have biological relevance for the treatment of human disease. Moreover, they may indicate that the nasal/oral region is a window on the environment. It is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. It is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. Nevertheless, the overall data presented give one the impression of an area that should be pursued.

Resistance to the in vitro antiproliferative effects of INF-alpha rapidly develops in mouse B16 melanoma cells that are maintained in vitro in IFN-alpha (B16 alpha res cells). B16 alpha res cells, however, are significantly more sensitive to the antitumor effects of IFN-alpha when they are injected into mice. This enhanced sensitivity appears to be due, at least in part, to activated macrophages. To investigate enhanced macrophage sensitivity of B16 alpha res cells, macrophage-mediated cytotoxicity assays have been performed using both B16 and B16 alpha res cell targets. Thioglycollate-elicited peritoneal macrophages activated in vitro with IFN-gamma exhibited dose-dependent cytotoxicity against both B16 and B16 alpha res cells, but significantly higher levels of cytotoxicity occurred with B16 alpha res targets. Kinetics experiment results showed that the cytolytic effects against B16 alpha res cells occurred at a very much faster rate than the cytolytic effects against B16 cells (50% cytotoxicity with 2 h of incubation versus 40% cytotoxicity by 24 h, respectively). Finally, peritoneal macrophages from B16-inoculated mice also were significantly more cytotoxic against B16 alpha res cells than against B16 cells. Macrophages from B16 alpha res-inoculated mice were significantly more cytotoxic against B16 alpha res cells than were macrophages from B16-inoculated mice. Taken together, these observations provide in vitro evidence to support the suggestion that peritoneal macrophages are important mediators of the enhanced host-mediated antitumor effects against B16 alpha res cells.

Interferons alpha, beta, and gamma have been shown to exert systemic effects following their oral administration to mice. It was of importance to determine whether oral administration of another biologic response modifier, interleukin-2 (IL-2), could also exert systemic effects in mice. Two systemic effects, peripheral WBC suppression and bone marrow suppression, were evaluated. Oral administration of IL-2 was found to suppress the peripheral WBC count in a dose-dependent manner. Oral administration of IL-2 was also found to suppress the bone marrow proliferative activity. The levels of suppression of both peripheral WBC and myelopoietic progenitor cell numbers observed with orally administered IL-2 were comparable to those seen with subcutaneously administered IL-2. The results demonstrate that orally administered IL-2 can exert systemic effects. Further, the results raise the possibility that oral administration of IL-2 may have therapeutic potential.

Heat-induced total body hyperthermia has been shown to synergistically enhance the in vivo antitumor effects of MuIFN-gamma directed against B16 melanoma in mice. This observation was made with an 8-hour exposure to hyperthermia that was administered following MuIFN-gamma treatment. It was not known whether this was the most efficacious treatment protocol. Various treatment protocols exploring MuIFN-gamma treatment at different relative times of exposure to hyperthermia and involving different durations of hyperthermia were evaluated. In a 5-day course of therapy, B16 tumor-bearing mice were injected with MuIFN-gamma or mock interferon and incubated in a dry incubator (resulting in a 2 degrees C rise in body temperature). The antitumor efficacy of MuIFN-gamma administered before, in the middle of, or following 8 hours of hyperthermia was evaluated. The antitumor effects of MuIFN-gamma were synergistically enhanced when the MuIFN-gamma was administered before 8 hours of hyperthermia (3.9-fold greater increased life span than with MuIFN-gamma treatment alone). However, when MuIFN-gamma was administered in the middle or following hyperthermia, the life spans were essentially the same as for MuIFN-gamma treatment alone, indicating that the effect of the hyperthermia exposures according to these treatment protocols were less than additive. Administration of MuIFN-gamma before hyperthermia exposures of 2 hours, 5 hours, and 8 hours showed antagonistic, additive, and synergistic interactions of the MuIFN-gamma treatment and hyperthermia exposure, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon alpha (IFN alpha) and interferon beta (IFN beta) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2',5'-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16 alpha res cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16 alpha res-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16 alpha res cell tumors did not seem to be caused by the slower growth rate of the B16 alpha res cells, since experiments performed with a tenfold higher B16 alpha res cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16 alpha res cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.

In this study, we tried to define in vitro interactions of two antitumor agents that have different sites and different mechanisms of action. Vinblastine (VLB) in combination with human recombinant interferon-alpha A/D (rHuIFN-alpha A/D) and in combination with murine recombinant interferon-gamma (rMuIFN-gamma) was studied. The effect of the combination was determined with cell growth kinetics assay on B-16 melanoma and the interaction defined by means of Spector's formula. Both the combination of rHuIFN-alpha A/D with VLB and the combination of rMuIFN-gamma with VLB synergistically inhibited cell growth in vitro. There was a positive biochemical modulation between the two drugs, but it is still unknown whether it occurred at the level of uptake into the cell, metabolism within the cell or egress from the cell.

Background: Chronobiological studies with anticancer drugs have shown that their effectiveness and/or toxicity is significantly influenced by the time of their administration in the circadian cycle. Previous studies also have shown that the myelotoxicity of interferons is similarly influenced., Purpose: This study was undertaken to evaluate the antitumor activity of interferons as a function of their administration to animals at defined points in the circadian cycle with equal light and dark periods., Methods: A murine tumor model was employed. Following adaptation to alternating cycles of 12 hours of light and 12 hours of dark for a period of 2-3 weeks, C57BL/6 mice were inoculated with B16 melanoma cells intraperitoneally at different hours after light onset. Exactly 24 hours after inoculation, each group received intraperitoneal injections of either recombinant human interferon alpha (rHuIFN-alpha A/D), recombinant murine IFN-gamma (rMuIFN-gamma), or interferon-carrier solution as control (once a day for 5 days) and were monitored for the length of their survival., Results: The antitumor activity (calculated as percent increased life span) of both rHuIFN-alpha A/D and rMuIFN-gamma varied with the points at which they were administered in the circadian cycle. However, the points showing minimum and maximum activity for rHuIFN-alpha A/D (12-16 and 0-4 hours after light onset, respectively) did not correspond with the points for the rMuIFN-gamma (0-8 and 16 hours after light onset, respectively). To generate maximum antitumor activity, approximately fivefold higher amounts of rHuIFN-alpha A/D were required at 12 than at 4 hours after light onset (dose range, 3333-90,000 IU/d) (P < .0001). Similarly, for rMuIFN-gamma at least 8.5-fold greater amounts were required at 8 than at 16 hours after light onset (dose range, 667-6000 IU/d) (P < .01)., Conclusions: In the murine tumor model, administration of rHuIFN-alpha A/D at 4 hours after light onset and rMuIFN-gamma at 16 hours after light onset may produce maximum antitumor activity.

The accepted routes of interferon (IFN) administration in clinical applications are intramuscular, subcutaneous, intraperitoneal, intratumor, and intravenous. Recently, oral administration of interferons has been shown to cause a suppression of peripheral white blood cell (WBC) counts. Moreover, orally administered interferons mediate their peripheral WBC suppression via a different mechanism than that of intraperitoneally administered interferons. This study extends the previous studies to show that the peripheral WBC suppression induced by oral interferon treatment reflects an actual bone marrow suppression. The bone marrow-suppressive effects of orally and subcutaneously administered recombinant human IFN-alpha A/D (rHuIFN-alpha A/D) have been partially characterized in kinetics studies and compared with the peripheral WBC-suppressive effects of orally and subcutaneously administered rHuIFN-alpha A/D. Oral and subcutaneous administrations of rHuIFN-alpha A/D cause a significant suppression of peripheral WBC counts with 1 day of rHuIFN-alpha A/D administration. This suppression reaches its maximum level with 3 days of rHuIFN-alpha A/D administration and plateaus over a 12-day treatment time. Similarly, oral and subcutaneous administrations of rHuIFN-alpha A/D cause a significant suppression of bone marrow function with 1 day of rHuIFN-alpha A/D administration. This suppression reaches its maximum level with 3 days of rHuIFN-alpha A/D administration and plateaus over a 12-day treatment time. Thus, the WBC-suppressive and bone marrow-suppressive effects of rHuIFN-alpha A/D administered either orally or subcutaneously parallel each other. The peripheral WBC-suppressive activities of orally and subcutaneously administered rHuIFN-alpha A/D diminish at the same rate, after cessation of rHuIFN-alpha A/D treatment. Peripheral WBC suppression is lost by 5 days after cessation of rHuIFN-alpha A/D treatment. The mechanisms by which orally and subcutaneously administered interferons exert their bone marrow-suppressive effects differ, however. Bone marrow suppression mediated by subcutaneous administration of murine IFN-alpha/beta (MuIFN-alpha/beta) is blocked by the presence of circulating antibodies to MuIFN-alpha/beta. In contrast, the bone marrow suppression mediated by oral administration of MuIFN-alpha/beta occurs even in the presence of circulating antibodies to MuIFN-alpha/beta. These results continue to support a potential clinical role for oral administration of interferons, particularly for the control of diseases of myelogenous origin.

There is a marked, reproducible circadian variation in the toxicity of a number of antineoplastic drugs. A recent study has employed a murine model to show that recombinant human interferon-alpha A/D (rHuIFN-alpha A/D) exhibited a differential potency in its peripheral white blood cell (WBC)-suppressive and bone marrow-suppressive activities according to the time in the circadian cycle at which it was administered. It was of interest to determine whether another biological response modifier such as IFN-gamma would also exhibit a differential potency during the circadian cycle. A mouse model was used to study peripheral WBC suppression, a toxicity associated with IFN-gamma therapy. Recombinant murine (rMu)IFN-gamma was employed to induce peripheral WBC suppression and was evaluated for its ability to induce peripheral WBC suppression as a function of the time of rMuIFN-gamma administration. Mice were maintained on cycles of 12 h of light and 12 h of darkness. The rMuIFN-gamma was administered at various hours after light onset (HALO). The rMuIFN-gamma-induced peripheral WBC-suppressive effect varied in its intensity in a cyclical manner. Administration of rMuIFN-gamma at 4 HALO caused the greatest suppressive effect, whereas administration of rMuIFN-gamma at 14 HALO caused the least suppressive effect. Mice treated at 14 HALO were found to be about 20-fold less sensitive to the peripheral WBC-suppressive effects of rMuIFN-gamma than mice treated at 4 HALO. This differential sensitivity to the peripheral WBC-suppressive effects of rMuIFN-gamma was examined at six different times in the circadian cycle and was found to be a general effect, occurring throughout the circadian cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-genetic resistance to the antiproliferative effects of interferon-alpha (IFN-alpha) develops in murine and human melanoma cells within 2-4 days of exposure of the cells to IFN-alpha. Simultaneous treatment of murine B16 melanoma cells with MuIFN-gamma and MuIFN-alpha prevents the development of resistance. In this study, the ability of MuIFN-gamma pretreatment to prevent the development of resistance was assessed for varying concentrations of MuIFN-gamma and for varying lengths of time of pretreatment. Pretreatment of the cells for 48 h with MuIFN-gamma using concentrations as low as 5 U/ml prevents the subsequent development of resistance when the cells are cloned in the presence of MuIFN-alpha. Higher concentrations of MuIFN-gamma are more effective in preventing the development of resistance. In addition, short MuIFN-gamma pretreatment times, such as 2-4 h, appeared to be most effective in preventing the development of resistance. In order to determine the mechanism for this biological effect, various second messenger perturbing chemical agents and several other biological agents were screened for ability to prevent the development of resistance. Neither interleukin-2 (IL-2), epidermal growth factor (EGF), nor any of the chemical agents examined could prevent the development of resistance, nor did they alter the ability of MuIFN-gamma to prevent the development of resistance. Tumor necrosis factor (TNF), however, was able to substitute for MuIFN-gamma in preventing the development of resistance, using concentrations of 125 ng/ml and higher.(ABSTRACT TRUNCATED AT 250 WORDS)

A number of antitumor drugs have been shown to vary in their toxicity and in their antitumor potency according to the time in the circadian cycle at which they are administered. It was of interest to determine whether other agents, such as a biological response modifier, would also exhibit differential potency during the circadian cycle. Interferons (IFNs) are biological response modifiers which have antitumor and antiviral activity and which also have toxic side effects. A mouse model was used to study one of these toxic side effects, peripheral white blood cell (WBC) suppression. Interferon-induced peripheral WBC suppression was evaluated as a function of the time of recombinant human (rh) IFN-alpha A/D administration. Mice were maintained on cycles of 12 hours of light and 12 hours of darkness. The rhIFN-alpha A/D was administered at various hours after light onset (HALO). The rhIFN-alpha A/D-induced peripheral WBC suppressive effect varied in its intensity in a cyclical manner. Administration of rhIFN-alpha A/D at 0 HALO caused the greatest suppressive effect, while administration of rhIFN-alpha A/D at 8 HALO caused the least suppressive effect. Mice treated at 8 HALO were found to be about 10-fold less sensitive to the peripheral WBC suppressive effects of rhIFN-alpha A/D than mice treated at 0 HALO. This differential sensitivity to the peripheral WBC suppressive effects of rhIFN-alpha A/D was examined for 6 different times in the circadian cycle and was found to be a general effect, occurring throughout the circadian cycle. Using a granulocyte/macrophage colony-forming unit (GM-CFU) assay, bone marrow function was also shown to be differentially affected by treatment with rhIFN-alpha A/D at 0 HALO and 8 HALO in a manner parallel to that seen with peripheral WBC. Thus, rhIFN-alpha A/D exerts a differential effect on peripheral WBC counts and on bone marrow function according to the time in the circadian cycle at which it is administered to the mouse. Such temporal variation in the myelosuppressive activity of interferons could be important in designing future clinical trials with these antiviral and antitumor agents. Administration of interferons at empirically determined times in the circadian cycle could be used to reduce the myelotoxic side effects of interferons in humans.

WISH and Hep-2 cells were incubated in an environment with atmospheric oxygen (20% O2, approximately 140 mmHg partial pressure), and under hypoxic conditions (2% O2, approximately 14 mmHg). The oxygen tension greatly affected the metabolism of the cells and their response to interferon-alpha (IFN-alpha) and IFN-gamma. Under hypoxic conditions, the cytopathogenicity of vesicular stomatitis virus (VSV) was reduced by about 50%, and the antiviral effects of the interferons (IFNs) were increased, both in terms of VSV-induced cytopathic effect (CPE), and yields of infectious virus. Local hypoxia is a nonspecific host defense against virus infection. The present results suggest that one of the mechanisms is by potentiation of the effects of the IFN produced at the sites of virus replication.

Interferons (IFN) have been approved for a number of clinical uses. The accepted routes of administration are intramuscular, subcutaneous, and intravenous. Recently, interferons administered by the oral route have been shown to exert a systemic effect. Oral administrations of IFN-alpha, IFN-beta, and IFN-gamma have been shown to cause a suppression of the peripheral white blood cell (WBC) count in mice. This study investigates the mechanism by which this suppression occurs. The results show that, in contrast to their intraperitoneal administration, oral administration of rHuIFN-alpha A/D or rMuIFN-gamma does not result in the presence of detectable levels of interferons in the blood. In addition, although the presence of circulating specific antibody to interferon blocks the peripheral WBC suppressive effects of intraperitoneally administered MuIFN-beta or rMuIFN-gamma, the presence of those antibodies does not block the peripheral WBC suppressive effects of the orally administered interferons. The peripheral WBC suppressive effect of orally administered rHuIFN-alpha A/D and rMuIFN-gamma can be transferred by injection of blood from oral interferon-treated donor mice to recipient mice. Recipient mice receiving plasma from donor mice showed no peripheral WBC suppression. Recipient mice receiving blood cells from donor mice showed significant peripheral WBC suppression. No effect of orally administered rHuIFN-alpha A/D on the relative percentages of lymphocytes, neutrophils, and monocytes was noted. These results indicate that the mechanism by which orally administered interferons exert their WBC suppressive effect differs from that of intraperitoneally administered interferons. WBC suppression resulting from orally administered interferons may involve cell to cell transfer of the interferons' effects, rather than the systemic distribution of the interferons in the blood. These studies further suggest that there may be a role for oral administration as a new route of interferon administration and provide a glimpse into the mechanism by which the orally administered interferons exert their systemic effects.

Hyperthermia treatment has been shown to enhance the in vitro antiproliferative effects of IFN-alpha, IFN-beta, and IFN-gamma, with IFN-gamma being more strongly enhanced than IFN-alpha. The comparative effects of hyperthermia on the in vivo antitumor activities of IFN-alpha and IFN-gamma were evaluated in the murine system using both subcutaneous and intraperitoneal B16 melanoma tumor model systems. Heat-induced whole body hyperthermia, resulting in a 2 degree C rise in body temperature, was administered by incubating the mice for 8 hours in a dry incubator at 37.1 degrees C. Whole body hyperthermia was found to enhance the antitumor activity of IFN-alpha by approximately 1.0 fold and 1.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented an additive effect of hyperthermia and IFN-alpha. Hyperthermia was found to enhance the antitumor activity of IFN-gamma by approximately 2.9 fold and 2.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented a synergistic effect of hyperthermia and IFN-gamma. The results of this in vivo study confirm and extend the in vitro observation that hyperthermia more strongly enhances the antitumor action of IFN-gamma than IFN-alpha. These results may have clinical importance because they suggest that hyperthermia may be used in combination with IFN-gamma to provide a synergistically enhanced antitumor action.

The ability of interferons (IFN) to exert a systemic effect following their oral administration was evaluated. One systemic effect of parenteral interferon administration has been shown to be a suppression of the number of peripheral white blood cells both in man and in mouse models. Using the mouse model of peripheral white blood cell suppression, the relative systemic effects of orally and subcutaneously administered interferons were determined. Murine IFN-beta, murine IFN-gamma and cross-reactive recombinant human IFN-alpha A/D were examined. The oral administrations of each of the three interferons were found to cause a dose-dependent suppression of the peripheral white blood cell counts. Significant levels of suppression were seen with as little as 5 units/day of murine IFN-beta and with 500 units/day of recombinant human IFN-alpha A/D and murine IFN-gamma. The dose-response curves obtained with orally administered interferons were much more shallow than those obtained with subcutaneously administered interferons. The results demonstrate that oral administration of interferons can provide a significant systemic effect. Further, the results support the possibility that the oral administration of interferons may have therapeutic potential.

Mouse B16 melanoma cells have been shown to rapidly develop resistance to the antiproliferative effects of MuIFN-alpha or MuIFN-beta when exposed to these interferons. In cloning studies, the maximal antiproliferative effects of MuIFN-alpha were seen with 2-4 days treatment. This resistance has been further characterized. The level of resistance which develops in B16 melanoma cells is dependent upon the concentration of MuIFN-alpha to which the cells are exposed. In addition, B16 melanoma cells which are resistant to the antiproliferative effects of MuIFN-alpha have greatly elevated levels of the interferon-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase. Since it has previously been shown that B16 melanoma cells do not develop resistance to the antiproliferative effects of MuIFN-gamma, several experiments studied the influence of MuIFN-gamma on the development of resistance to MuIFN-alpha. Combinations of IFN-gamma and IFN-alpha have previously been shown to result in a synergistic enhancement of the antiproliferative effects. Kinetic studies show that the response of the cells to the MuIFN-gamma antiproliferative effect appears to be dominant over the development of resistance since no resistance develops in response to combination treatment. Not only is MuIFN-gamma able to prevent development of resistance when it is present continuously, but also when it is used for the sequential treatment of the cells before their exposure to MuIFN-alpha. A 2-day pretreatment with MuIFN-gamma is sufficient to prevent the development of resistance during later exposure of the cells to MuIFN-alpha alone for up to 6 additional days.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative effects of treatment with an anticonvulsant, phenytoin, on the production of interferons were determined for both the murine and human systems. Phenytoin treatment was found to have differential effects on the in vitro production of Type I and Type II interferons. Phenytoin had either no effect (HuIFN-alpha) or an enhancing effect (MuIFN-alpha/beta) on the in vitro production of Type I interferons. In contrast, phenytoin pretreatment had an inhibitory effect on the in vitro production of Type II interferons (IFN-gamma) for both the murine and human systems. Phenytoin appeared to exert its inhibitory effect directly on the IFN-gamma-producing cell and was active even when added as late as 6 h after IFN-gamma induction. This inhibition was not related to a toxic effect of the phenytoin and occurred at phenytoin concentrations which were pharmacologically relevant (10-20 micrograms/ml). The effects of phenytoin on the in vivo production of MuIFN-gamma were also examined. In parallel to the in vitro observations, phenytoin treatment of mice significantly reduced the in vivo induction of MuIFN-gamma. The results raise the possibility that phenytoin therapy in humans may significantly affect the production of HuIFN-gamma.

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.

The interaction of the interferons (IFNs) that cooperate to potentiate the antiviral action of IFN was studied. Serial dilutions of MuIFN-gamma and MuIFN-alpha/beta were employed separately and in combination to block virus replication in one-step, virus yield reduction experiments. To calculate the potentiation of IFN activity, protection levels obtained for each combination of MuIFN-gamma and MuIFN-alpha/beta were compared with those obtained for the separate IFNs. Potentiation levels increased with increasing concentrations of each of the IFNs in a dose-dependent manner, suggesting that potentiation of IFN's antiviral activity was the result of the mutually synergistic interaction of the IFNs. Three challenge viruses were employed: Mengo virus (positive-strand RNA virus), vesicular stomatitis virus (negative-strand RNA virus), and vaccinia virus (DNA virus). Identical results were observed with the three different viruses, suggesting that mutual synergism was a basic feature of the potentiation of IFN's antiviral activity by combined preparations of MuIFN-gamma and MuIFN-alpha/beta.

For the determination of the conditions for the most effective cytolysis of human melanoma cells, leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta), and immune interferon (IFN-gamma) were compared for their abilities to kill cultured human melanoma cells in the presence and absence of peripheral blood mononuclear leukocytes (PBL). A microassay was employed in which the viability of melanoma target cells was determined after various times of incubation with interferons alone or with PBL. On 7 human melanoma cell lines (from 6 different patients), IFN-gamma had significantly greater direct anticellular effect than IFN-alpha or IFN-beta. When PBL were added, all target cells were killed after 48 hours with IFN-gamma, but they were not killed with IFN-alpha or IFN-beta. When IFN-gamma was added to either IFN-alpha or IFN-beta, a potentiation of the anticellular effect was observed both with and without PBL. The actions of this "natural" IFN-gamma could be reproduced with recombinant IFN-gamma and could be neutralized by an antibody to a synthetic peptide encoded by the 5'-end of IFN-gamma complementary DNA. It was concluded that IFN-gamma is significantly more active against these human melanoma cell lines than either IFN-alpha or IFN-beta and, most significantly, that eradication can occur in the presence of IFN-gamma and PBL. Furthermore, synergistic anticellular action can be observed when IFN-gamma is added to IFN-alpha or IFN-beta. These findings point to the need for preclinical trials to evaluate eradication in vivo.

Previously an inhibitor of interferon action had been isolated from mitogen stimulated mouse spleen cells. This inhibitor was associated with the IFN-gamma molecule and might possibly represent an altered IFN-gamma molecule which can no longer induce an effective antiviral state. This report further investigates the biological properties of this inhibitor. Inhibitor activity is independent of the virus used in the interferon assay. Inhibitor activity has also been found to be species specific. Mouse inhibitor does not inhibit the antiviral activity of either human IFN-gamma or human IFN-beta. However, inhibitor production is not limited to the mouse system. Inhibitor is also present in preparations of human IFN-gamma. The inhibitor does not appear to directly compete with IFN-gamma for a specific cell surface receptor since inhibitor activity is independent of interferon concentration. The presence of inhibitor allows a unique, albeit reduced level of antiviral protection to develop. Increasing concentrations of interferon do not increase the level of antiviral protection allowed by the inhibitor. This inhibitor of interferon action may represent a natural mechanism whereby IFN-gamma-induced effects are regulated in vivo.

In this study, murine interferons (IFNs) were employed separately and in combination at subeffective and effective antitumor concentrations in a mouse B-16 melanoma system. Murine IFN-gamma (MuIFN-gamma) was demonstrated to be approximately 20 times more potent than MuIFN-alpha and MuIFN-beta in this system. Potentiation was observed with combinations of both subeffective and effective concentrations of MuIFN-gamma with MuIFN-alpha or MuIFN-beta. The level of potentiation was observed to increase from undetectable to two-fold to fourfold with three increasing IFN concentrations. Thirty percent of the mice treated for 14 days with 332 U/day MuIFN-gamma plus 7,500 U/day MuIFN-alpha survived apparently tumor-free for 100 days. Forty percent of the mice treated for 14 days with 332 U/day MuIFN-gamma plus 7,500 U/day MuIFN-beta survived apparently tumor-free for 100 days. All control mice died by day 41. The results are consistent with the suggestion that combination IFN therapy may have some use in the control of at least some tumors in humans.

We have previously established that type I interferon (IFN), a mixture of alpha- and beta-IFN, augments the antitumor activity of alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, against B16 melanoma. The objective of the present investigation was to extend these earlier observations to metastatic Lewis lung carcinoma and to determine specifically which component(s) of type I IFN potentiates the antitumor activity of DFMO. Furthermore, we wanted to determine whether type II (gamma) IFN can also potentiate the antitumor activity of DFMO. Treatment of animals bearing Lewis lung carcinoma with DFMO, 2% in drinking water (3 g/kg/day), or IFN-alpha/beta (1000 units/mouse) given subcutaneously on alternate days for a total of ten doses alone resulted in 42 and 5% inhibition of tumor growth, respectively. A combination of DFMO and interferon brought about complete elimination of tumors in 12 of the 18 animals, and 94% inhibition of tumor growth in the remainder. DFMO or type I IFN administered alone caused 94 and 26% inhibition of metastasis, respectively. Combination treatment with these two agents resulted in complete elimination of visible metastases. Treatment of mice bearing B16 melanoma with DFMO resulted in 81% inhibition of tumor growth compared to controls. The administration of interferons alone resulted in tumor growth inhibition of 15, 3, 1, and 43% for type I, alpha-, beta-, and gamma-interferons, respectively. Treatment of animals with combination of DFMO and various interferons resulted in inhibition of 94, 93, 86, and 94% of B16 tumor growth for type I, alpha-, beta-, and gamma-interferons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Human lymphocytes were treated with human alpha (IFN-alpha), beta (IFN-beta), or recombinant gamma (IFN-gamma) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-gamma was approximately 50 times more active per unit of antiviral activity than either IFN-alpha or IFN-beta. Moreover, the levels of natural killing by lymphocytes treated with combinations of IFN-alpha and IFN-beta were additive, whereas combinations of recombinant IFN-gamma and IFN-alpha or recombinant IFN-gamma and IFN-beta were synergistic. The development of natural killing in lymphocytes treated with recombinant IFN-gamma did not occur more rapidly but reached higher levels (62%) than that observed with lymphocytes treated with IFN-alpha or IFN-beta (15%). The results suggest the importance of IFN-gamma and mixtures of IFN-gamma with IFN-alpha or IFN-beta in the enhancement of natural killing activity against virus infections and neoplasia.

The effect of different types of interferon (IFN) on macrophage and granulocyte colony formation (CFU-C) in vitro was studied using bone marrow cells from C57BL/6 mice. CFU-C formation was measured at 7 days using a standard methylcellulose culture system with L-cell conditioned medium (LCCM) as a source of colony-stimulating factor activity (CSF), IFN gamma was obtained from staphylococcal enterotoxin A- (SEA) stimulated mouse spleen cells and was partially purified (50-fold) using affinity chromatography. IFN alpha/beta was obtained from Newcastle disease virus-infected mouse L-cells and was partially purified (286-fold) using an AcA 202 column. IFN gamma inhibited CFU-C formation in a dose-dependent manner and was 100 times more inhibitory than IFN alpha/beta. IFN gamma completely suppressed CFU-C formation with as little as 15 units of antiviral activity. In contrast, 1000 to 2000 units of antiviral activity of IFN alpha/beta was required for complete suppression of CFU-C formation. The ability of IFN gamma to inhibit CFU-C formation was completely abolished after pH 2 treatment (24 hr), heat treatment (60 degrees C, 2 hr), or treatment with a specific antiserum against IFN gamma, which indicated that IFN gamma was the mediator of CFU-C inhibition. When IFN gamma and IFN alpha/beta were mixed together and added to bone marrow cells, they had a synergistic effect on the suppression of CFU-C formation. The level of inhibition mediated by both IFN gamma and IFN alpha/beta was totally dependent upon the amount of CSF present in the culture system; increasing the level of CSF could completely overcome the IFN suppressive effect. These results suggest that IFN gamma and IFN alpha/beta may have regulatory roles in hematopoiesis.

Mixed preparations of murine interferon-gamma (MuIFN-gamma) and murine interferon-alpha/beta (MuIFN-alpha/beta) have been shown to induce more than additive levels of antiviral protection, when compared to those induced by these interferons given separately. MuIFN-gamma preparations contain many lymphokines and several of these as well as MuIFN-gamma itself may participate in this potentiation. In the present study, natural as well as three recombinant DNA-derived MuIFN-gamma's, in combination with antibody to MuIFN-gamma have been employed to examine the precise role of MuIFN-gamma. The antiviral effect was examined with a single cycle virus yield reduction assay and the antiproliferative effect with a colony formation inhibition assay. Recombinant DNA-derived MuIFN-gamma was as effective as natural MuIFN-gamma at participating in the potentiation of both the antiviral and antiproliferative activities. Antibody to MuIFN-gamma effectively blocked the potentiation of both the antiviral and the antiproliferative activities of natural and recombinant DNA-derived MuIFN-gamma's. Since the recombinant DNA-derived preparations from E. colie can be assumed not to contain mammalian proteins other than MuIFN-gamma, the data conclusively demonstrate that the potentiation factor in MuIFN-gamma preparations is MuIFN-gamma itself.

We have previously shown that murine bone marrow cells cultured with interleukin 2 (IL-2) produce interferon-alpha/beta (MuIFN-alpha/beta) and that IFN-alpha/beta can suppress in vitro granulocyte-macrophage colony-forming cell formation (GM-CFC). In this study, IL-2 was directly assessed for its ability to inhibit in vitro granulocyte and/or macrophage colony-forming cell formation (GM-CFC/M-CFC). C57BL/6 bone marrow cells were cultured with different colony-stimulating factors (CSF), i.e., partially purified macrophage-CSF (M-CSF) or recombinant granulocyte and macrophage CSF (GM-CSF) in the presence or absence of different IL-2 preparations. Partially purified mouse IL-2 or recombinant human or mouse IL-2 (rHuIL-2 and rMuIL-2) totally inhibit GM-CFC and M-CFC formation at 7 days of culture. The level of inhibition mediated by IL-2 was concentration-dependent, with as little as 1 U/ml giving total inhibition of colony formation. The ability of IL-2 to inhibit colony formation was completely abolished by treatment with antisera to IL-2. MuIFN-alpha/beta and MuIFN-gamma appeared to play no role in IL-2-induced myelo-suppression in that addition of antisera to these IFN failed to block IL-2-induced suppression. Myelo-suppression mediated by IL-2 was independent of the concentration of CSF used in the bone marrow cultures. Suppression was also not dependent upon the initial presence of T cells or natural killer (NK) cells. Bone marrow cells depleted of Thy-1+, Lyt-1+, Lyt-2+, NK-1.1+, Asialo GM1+, or Qa-5+ cells were as susceptible to IL-2 induced suppression as untreated or complement-treated bone marrow cells. These results suggest that IL-2 may play an important role in regulating different aspects of hematopoiesis.

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.

In inbred DBA/2 mice, the antitumor activities of separate and combined preparations of mouse immune interferon and mouse virus-induced interferon on the development of P388 tumors were studied. Immune interferon alone (25 U/day) did not affect tumor development. Virus-induced interferon alone (25,000 U/day) delayed tumor development and increased survival time. The mouse immune interferon preparations significantly enhanced or potentiated the antitumor effects of mouse virus-induced interferon when the interferons were used in combined therapy.

Colony stimulating factors have been shown to antagonize the bone marrow suppressive effects of interferons in vitro. The effect of partially purified murine macrophage colony stimulating factor (CSF-1) was evaluated for its ability to antagonize interferon's bone marrow suppressive effect and was measured for its effect against two other interferon activities. The effect of CSF-1 against interferon's bone marrow suppressive effect was measured in a bone marrow colony growth assay. The effect of CSF-1 against interferon's antiviral activity was measured in single-cycle vesicular stomatitis virus growth experiments. The effect of CSF-1 against interferon's antiproliferative activity was measured in 3-day cell growth kinetics assays using B-16 melanoma cells and J-774 reticulum sarcoma cells of macrophage origin. Each of the three types of interferon (MuIFN-alpha, MuIFN-beta, and MuIFN-gamma) were employed and had common effects, though they differed in the levels of their effective concentrations. Concomitant treatment with CSF-1 and interferon blocked the interferon mediated bone marrow suppression in a dose dependent manner but had no effect on the antiviral or antiproliferative activities of the interferons. Importantly, CSF-1 did not affect interferon's antiproliferative activity against the growth of J-774 reticulum sarcoma cells, even though CSF-1 is a macrophage colony stimulating factor and J-774 cells are of macrophage origin. The results suggest that colony stimulating factors might be employed to specifically protect bone marrow function during interferon therapy while not interfering with interferon's antiviral and antitumor activities.

An inhibitor of interferon's (IFN) antiviral action was evaluated for its ability to block interferon's anticellular action. Crude and partially purified mouse immune interferon (MuIFN-gamma) preparations with high and low levels of an inhibitor of IFN's antiviral action were prepared. These preparations were examined for their relative antiviral and direct anticellular activities. The inhibitor present in the IFN-gamma preparations was found to be an equally potent inhibitor of both the antiviral and the direct anticellular activities of IFN-gamma. Further, while the inhibitor of IFN-gamma was inherently present in most IFN-gamma preparations, it was not an inherent feature of all IFN-gamma molecules. Inhibitor-free IFN-gamma could be separated from inhibitor-containing IFN-gamma by column chromatography. The results suggest that the inhibitor generally antagonizes interferon action and should be taken into account when evaluating the biological properties of IFN-gamma preparations.

The activity of interferons was tested in ultraviolet light-induced skin tumors in mice. After the tumors were well established, they were injected and measured daily for 19 days. Mouse virus type (IF-alpha + IF-beta) and immune (IF-gamma) interferons were injected intralesionally into three groups of test animals and compared with a fourth group which received mock interferon (control). When used separately, virus type and immune interferons did not affect tumor growth; however, we observed regression in tumor size when the two interferons were used in combination.

Mouse immune (IFN-gamma)2 and virus-type (IFN-alpha/beta) interferons were used separately and in combination in cloning studies with B-16 melanoma cells. IFN-gamma was found to be a more potent mediator of the direct anticellular effect of interferon than was IFN-alpha/beta, as shown not only by a greater sensitivity of B-16 cells to IFN-gamma but also by a steeper slope of the anticellular sensitivity curve of the IFN-gamma. The differences in the slopes of the curves defining their anticellular effect appeared to be inherent in the interferons themselves and not due to an inhibitor of interferon, a stimulator of cell growth, or another factor possessing anticellular activity. The results are consistent with the interpretation that IFN-gamma and IFN-alpha/beta exert their anticellular effects by different mechanisms. The anticellular activity of interferon against B-16 melanoma replication until development was potentiated by mixed preparations of IFN-gamma and IFN-alpha/beta. The potentiation appeared to be an expression of a property of the interferons themselves. Replication units resistant to the potentiated activity of the interferons were not detected. Potentiation levels were dependent on the concentrations of both IFN-gamma and IFN-alpha/beta and continued to increase dramatically as the interferon concentrations increased. Maximum potentiation observed was 214-fold at the highest interferon concentrations used. The data suggested that potentiation is a mutual, synergistic interaction of IFN-gamma and IFN-alpha/beta.

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse IFN gamma preparations purified 30-fold were found to have direct cytolytic activity against a number of tumor and normal cells. Cell killing was determined using a sensitive, rapid and accurate assay which employed very low numbers of cells and very small quantities of interferon. The cytolytic activity of IFN gamma on 11 murine tumor cell lines was investigated. A 20-fold difference was found between the most-sensitive cell type, P-388 lymphoma, versus the most resistant cell type, C127v leukemia. A number of normal mouse cells was also found to have low to intermediate sensitivity to the cytolytic action of IFN gamma. Human IFN gamma was also shown to have cytolytic activity which, like mouse IFN gamma, was relatively species-specific. Direct cytolysis was not found to be a characteristic of IFN-alpha/beta. Different mechanisms of action for the antiviral and cytolytic activities of IFN gamma are indicated because the cytolytic titer of IFN gamma did not parallel its antiviral titer on most cell types and increasing the cell number produced a decrease in the cytolytic titer and an increase in the anti-viral titer. High concentrations of IFN gamma (i.e., 2,900 units/ml) resulted in complete lysis of cells within 24 h, while lower concentrations (i.e., 700 units/ml) resulted in a reversible inhibition of cell growth during this time period. Evidence that the cytolytic substance in the IFN gamma preparation was IFN gamma include the following: (1) both antiviral and anticellular activities copurified through a 30-fold purification; and both activities were (2) relatively species-specific; (3) sensitive to heat; (4) inactivated by low pH and (5) neutralized by antibodies to IFN gamma. Therefore, we propose the possibility that direct cytolysis is yet another of IFN gamma's distinctive antivities.

Fever is frequently an important side effect of interferon (IFN) therapy. Studies have shown that culturing interferon-treated cells at elevated temperature heightens the antiproliferative activity of IFN-alpha and IFN-beta. Since IFN-gamma has also been shown to be a potent antiproliferative agent, the effect of elevated temperature on IFN-gamma activity was compared to its effect on IFN-alpha and IFN-beta. Mouse B-16 melanoma cells were simultaneously cultured under cloning conditions at a range of temperatures (37.3, 38.1, 38.6, and 39.4 degrees C) in the presence of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma. The antiproliferative activities of all three interferons were enhanced by incubation at the elevated temperatures. However, the elevated temperatures had a more dramatic enhancing effect on the antiproliferative activity of MuIFN-gamma (10-fold enhancement) than of either MuIFN-alpha or MuIFN-beta (2.9- and 3.4-fold enhancement, respectively). Next, the enhancing effect of elevated temperature (39.4 degrees C) was examined for a range of interferon concentrations. The degree of the enhancing effect increased with increasing concentrations of MuIFN-gamma but not with increasing concentrations of MuIFN-alpha or MuIFN-beta. Enhancing effects of temperature as high as 14-fold were observed for 100 units of MuIFN-gamma/ml. This dramatic enhancement was observed for both natural and recombinant MuIFN-gamma and was neither a function of greater relative perception of MuIFN-gamma titer at elevated temperature nor a function of greater relative stability of MuIFN-gamma at the elevated temperature. The differential enhancement of MuIFN-gamma activity by elevated temperature appeared to be specific for the antiproliferative activity, since the antiviral activity of MuIFN-gamma was not relatively more enhanced at 39.4 degrees C than were the antiviral activities of MuIFN-alpha and MuIFN-beta. These results suggest that fever may be an important factor in maximizing the antitumor effects of MuIFN-gamma and perhaps of human IFN-gamma. They also raise the possibility that a combination treatment regimen of hyperthermia and interferon therapy, particularly IFN-gamma therapy, may provide a significant antitumor effect.