Interaction of IFN α/β with host cells essential to the early inhibition of friend erythroleukemia visceral metastases in mice

We have previously shown that an intact immune system was essential to the increase in survival time of IFN-α/β-treated mice injected i.v. with an IFN-α/β-resistant line of Friend erythroleukemia cells (FLC) highly metastatic to the liver and spleen. Here, we have investigated the early interactions of IFN α/β with host cells prior to the development of the immune response. IFN α/β treatment resulted in 50- to 100-fold inhibition of FLC multiplication in the liver and spleen of normal DBA/2 mice shortly after tumor inoculation, as evaluated by colony formation in agarose. IFN treatment was far less effective in inhibiting the multiplication of FLC in the livers of NK-cell-deficient DBA/2 beige mice, or in immunocompetent DBA/2 mice treated with antibody to asialo GMI, or silica, or in mice subjected to sub-lethal irradiation. Injection of antibody to CD4 or CD5 did not affect the early inhibitory action of IFN α/β on FLC multiplication but did decrease survival time. Light- and electron-microscope examination of the livers of IFN-treated, FLC-injected mice confirmed the early inhibition of FLC multiplication in the liver and spleen. Our results indicate that IFN α/β inhibits the development of FLC visceral metastases by acting first on host cells, such as NK cells and macrophages, and then continues to act in consort with the developing immune response. © 1994 Wiley-Liss, Inc.     

Sensitized T lymphocytes render DBA/2 beige mice responsive to IFN α/β therapy of friend erythroleukemia visceral metastases

Interferon α/β (IFN α/β) is highly effective in inhibiting the development of Friend erythroleukemia cell (FLC) visceral metastases in DBA/2 mice injected intravenously (i.v.) with FLC, but does not protect FLC-injected DBA/2 beige (bg/bg) mice. Use of IFN α/β-resistant FLC indicated that IFN was acting through host mechanisms in DBA/2 mice and thus pointed to a defect in some host mechanism in bg/bg mice essential for IFN's anti-metastatic action. We undertook experiments to restore in bg/bg mice the marked anti-FLC meta-static effect of IFN a/p observed in DBA/2 and +/bg mice. Adoptive transfer of spleen cells from normal syngeneic mice to IFN-treated bg/bg mice was ineffective, but the transfer of splenic T lymphocytes from FLC-immunized DBA/2 or +/bg mice markedly increased the survival time of FLC-injected bg/bg mice provided that these mice were also treated with IFN a/p. Neither treatment alone resulted in an increase in survival time. As few as 1 × 10⁷ immune spleen cells were effective in IFN-treated FLC-injected bg/bg mice. The T-cell immune response to FLC of bg/bg mice was diminished compared with that of +/bg mice. Likewise, only combination therapy of immune spleen cells and IFN α/β resulted in an increased survival time of ESb-lymphoma-injected bg/bg mice. Our results indicate the essential participation both of T-cell-mediated immune mechanisms and of IFN α/βin the inhibition of FLC visceral metastases.     

Importance of interferon alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend erythroleukemia cells in the liver

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. Our results indicate that interferon alpha (IFN-alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver. (a) After i.v. inoculation of FLC there was an inverse correlation between the presence of IFN-alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC-injected adult C57B1/6 mice had circulating IFN-alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC-injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice. (b) FLC did multiply in the livers of newborn C57B1/6 mice and in the livers of irradiated adult C57B1/6 mice, and IFN-alpha was not detected in their sera. In contrast, after i.v. inoculation of FLC, IFN-alpha was detected in the sera of 3-week-old and athymic nu/nu adult C57B1/6 mice while FLC failed to multiply in the liver. (c) FLC also induced IFN-alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver. We suggest that, depending on the site of tumor implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumor cells, and that IFN-alpha is particularly important when FLC are injected i.v.     

Synergistic anti-tumor effects of combined IL-1/IFN-alpha/beta therapy in mice injected with metastatic Friend erythroleukemia cells

Peritumoral injection of relatively low doses of either mouse interferon (IFN)-alpha/beta (10,000-20,000 units/injection) or of recombinant human interleukin-1 (IL-1) beta (125-250 ng/injection) in mice transplanted s.c. with Friend erythroleukemia cells (FLC) resulted in some inhibition of primary tumor growth, inhibition of liver and splenic metastases and increased survival time. A synergistic anti-tumor effect was observed in mice injected with both IL-1 and IFN-alpha/beta. Highly purified mouse IFN-beta also exerted a synergistic anti-tumor effect when combined with IL-1-beta in mice injected with FLC. The anti-tumor action of IL-1/IFN was markedly reduced in mice treated with antibodies to CD4 antigens. Antibodies to asialo-GM1 also diminished the anti-tumor effect by the combined cytokine treatment. The combined IL-1/IFN therapy was effective in NK-deficient bg/bg mice, although the extent of the anti-tumor response in these mice was less than that observed in bg/+mice.     

Free interferon-alpha/beta receptors in the circulation of patients with adenocarcinoma

BACKGROUND: Many viral and neoplastic diseases are resistant to interferon-alpha/beta (IFN-alpha/beta) therapy or develop resistance during the course of IFN treatment. In patients with viral diseases, the authors identified four IFN inhibitors, of which the most important, most likely is a free IFN receptor of type 1 appearing in the circulation that captures and neutralizes IFN-alpha/beta. METHODS: Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-alpha/beta type 1 was studied. The patients were ages 35-75 years. The diagnoses were 24 cases of colon carcinoma, 7 cases of prostate carcinoma, 16 cases of breast carcinoma, 8 cases of ovarian carcinoma, 9 cases of uterine carcinoma, 5 cases of lung carcinoma, 3 cases of astrocytoma, 4 cases of transitional cell carcinoma of the bladder, 1 case of osteosarcoma, 3 cases of multiple myeloma, 4 cases of Hodgkin disease, 2 cases of non-Hodgkin lymphoma, 3 cases of myelodysplastic syndrome, and 2 disseminated tumors of unknown origin. RESULTS: All patients were found to have increased free IFN receptor-alpha/beta type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma. CONCLUSIONS: High IFN inhibitory activity in patients with cancer may be a significant factor in their increased susceptibility to progressive disease, infectious complications, and resistance to IFN therapy. Ongoing studies are being performed with the objective of overcoming this inhibitory activity.     

Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor

Platelet-derived type beta transforming growth factor (TGF beta) is a potent inhibitor of DNA synthesis in primary monolayer cultures of adult rat hepatocytes. TGF beta induced a 50% inhibition of epidermal growth factor (EGF)-mediated DNA synthesis at approximately 5 X 10(12) M. This inhibition did not appear to be due to a delay in the peak of DNA synthesis or a toxic action, nor could it be overcome by increasing concentrations of the mitogens EGF, insulin, or glucagon. Inhibition was observed either when TGF beta and EGF were continuously present together in the culture medium or when TGF beta was added to the hepatocyte cultures after removal of the EGF stimulant. This observation together with a lack of an inhibitory effect of TGF beta on the binding of 125I-labeled EGF to hepatocytes in culture, suggests that the inhibitory action of TGF beta was not caused by a direct competition with EGF at the cell surface. TGF beta could not inhibit DNA synthesis once it had begun; however, the inhibitory action of TGF beta could be partially overcome by increasing amounts of conditioned medium produced by normal hepatocytes. Specific saturable receptors for TGF beta were found on the normal rat hepatocytes, but specific binding could not be detected on hepatocytes from regenerating liver. TGF beta is thus a potent inhibitor of EGF-induced DNA synthesis in adult rat hepatocytes. Its significance for growth control in vivo has yet to be assessed.     

Interleukin-1 beta induces tumor necrosis and early morphologic and metabolic changes in transplantable mouse tumors. Similarities with the anti-tumor effects of tumor necrosis factor alpha or beta

Peri-tumoral injection of recombinant human interleukin-1 beta in mice transplanted s.c. with Friend erythroleukemia cells (FLC) resulted in marked inhibition of tumor growth and increased survival. However, in vitro treatment of FLC (745 or 3Cl-8) with IL-1 beta barely inhibited cell multiplication. IL-1 beta, injected into established solid tumors, induced marked morphologic changes. Vascular congestion and focal extravasation of erythrocytes were observed as early as 6 hr after injection with IL-1 beta of FLC and L1210 tumors and HeJ16 fibrosarcomas. Focal areas of disaggregation of tumor cells and tumor necrosis were observed 6 and 24 hr after IL-1 injection. These morphologic changes were similar to those observed in FLC tumors or HeJ16 fibrosarcomas treated with TNF-alpha or beta. These cytokines determined morphological changes in tumor blood vessels of FLC tumors within 1 hr of injection. Freshly dissected FLC tumors and their tissue extracts were studied by Nuclear Magnetic Resonance (NMR) spectroscopy, shortly after peri-tumoral injection of IL-1 beta or TNF-beta. After 6 hr, both cytokines induced a 3-fold reduction in the levels of two catabolites, glycerophosphorylcholine and glycerophosphorylethanolamine, an accumulation of sn-glycerol 3-phosphate and a more than 10-fold increase in the choline/phosphorylcholine ratio. These results are similar to those reported for TNF-alpha, and can be interpreted on the basis of an activation of glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2) and partial inhibition of choline kinase (EC 2.7.1.32). IL-1 beta and TNF-beta (like TNF-alpha) also induced alkaline shifts (0.10-0.25 units) in the average intratumoral pH value. We suggest that alterations of tumor blood vessels may be the primary events in solid tumors treated with IL-1 beta or TNF. Such alterations lead to early changes in tumor metabolism and subsequent tumor cell degeneration.     

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

Kinetics of inhibition of in vitro interferon-alpha/beta production after benzo-(a)-pyrene exposure

L-929 cells were treated with benzo-(a)-pyrene (BP), washed, and then interferon(IFN)-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid (poly-I-C). IFN-alpha/beta production by these cells was measured at 6-hour intervals. Up to 24 h after commencement of IFN induction, IFN production was severely inhibited in BP-treated cells as compared to controls. After 24 h, IFN-alpha/beta production in BP-treated cells began to rise and reached levels seen in untreated cells by 48 h. These data indicated that IFN induction was not completely inhibited by BP treatment, but, rather, the peak of IFN production was delayed. Mouse embryo cultures from SENCAR mice showed the same pattern of delayed IFN production when treated with BP. Direct exposure of the cells to BP for at least 15 min prior to removal of the carcinogen was required for IFN induction to be inhibited; however, at least 24 h of further incubation in fresh medium before addition of poly-I-C was required for inhibition of IFN induction by BP.     

Effect of endogenous and exogenous interferons on the differentiation of human monocyte cell line U937

The effect of interferons (IFNs) on the differentiation of hematopoietic cells was examined with the human monocyte cell line U937. The differentiation of U937 was induced by hydroxyvitamin D3 and was evaluated through the study of specific markers. The induction of the U937 differentiation was associated with a production of IFN and with a marked increase in (2'5') oligoadenylate synthetase. Addition of anti-IFN-alpha/beta antibodies inhibited the enhancement of (2'5') oligoadenylate synthetase and reduced the inhibitory effect of hydroxyvitamin D3 on cell growth. Nevertheless, neutralization of endogenous IFN excreted during U937 cell maturation did not modify the expression of the differentiation markers examined. Exogenous natural IFN-alpha, IFN-beta, or recombinant (r) IFN-gamma, when added to the culture medium, did not promote a "global" U937 differentiation. Most of the differentiation markers, except for reduction of nitroblue-tetrazolium, were not induced by IFN-alpha or -beta. However, rIFN-gamma was able to induce the appearance of several monocytic membrane markers at an extent comparable or slightly inferior to that elicited by hydroxyvitamin D3. Different effects on the expression of HLA antigens were obtained with these IFNs: IFN-alpha or -beta enhanced mainly class I HLA antigen expression, whereas rIFN-gamma increased selectively the expression of class II HLA DC1 but not HLA DR antigens. In contrast, phytohemagglutinin-leukocyte conditioned medium elicited a marked and selective enhancement of the expression of HLA-DR antigens. This induction of HLA DC1 antigens by rIFN-gamma was not observed in two other leukemic cell lines (HL60 and HEL). The present study shows that IFN-alpha or -beta may participate in the antiproliferative effect occurring during cellular differentiation, while IFN-gamma may be involved in the induction of the expression of specific monocytic markers involved in cellular immunoregulation.     

Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells.

The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified.     

Interferon-alpha/beta in virus-induced mouse mammary carcinogenesis: effects on the spontaneous process and on the progression of transplanted pre-neoplastic lesions.

Low levels of anti-viral activity, mainly interferon alpha/beta (IFN-alpha/beta), are regularly found in lymphoid tissues of BALB/c mice infected with the C3H strain of mammary tumor virus. At the time of tumor development, significant amounts of anti-viral activity were detected in homogenates of spleen and mammary tumors, but not in blood and normal mammary glands. This activity is pH2-resistant and neutralized by antibody to IFN/alpha-beta. The pathogenetic role of IFN in mammary carcinogenesis was investigated in 2 ways: (a) by treating virus-infected newborn mice with antibody to IFN-alpha/beta, and (b) by giving either the latter antibody or IFN-alpha/beta to virus-free animals transplanted with pre-neoplastic lesions. Mice were treated only for 2 months, starting either 1 week after birth or immediately after tumor transplant. In case (a), treatment with antibody to IFN-alpha/beta shortened the incubation period of mammary carcinomas and decreased the mean survival time. In case (b), anti-IFN antibody did not significantly affect the development of mammary tumors. However, exogenous IFN-alpha/beta markedly reduced both tumor incidence and mortality rate. These results indicate that endogenous IFN-alpha/beta plays a crucial role in the in vivo restriction of the early infectious phase of spontaneous carcinogenesis and that relatively high doses of IFN-alpha/beta may inhibit the progression of pre-neoplastic lesions.     

Inhibition of intravascular mouse melanoma dissemination by recombinant human interferon alpha A/D

The effects of pure recombinant human interferon alpha A/D (IFN alpha A/D) on natural killer (NK) activity and the experimental lung metastasis of B16-F10 melanoma were studied. Treatment of C57BL/6 mice with IFN alpha A/D augmented splenic NK activity and also inhibited the experimental lung metastasis of B16-F10 melanoma in a dose-dependent manner. The augmentation of NK activity and the inhibition of experimental lung metastasis by IFN alpha A/D were completely abolished in anti-asialo GM1-pretreated mice. These results suggested that the effector cells which inhibited melanoma metastasis in the present system were mainly NK cells, and that it was by activating NK cells that IFN alpha A/D had its effect. We next studied the timing of IFN alpha A/D administration for the most effective prevention of melanoma metastasis. The inhibitory effect of IFN alpha A/D was most pronounced when it was given 12 hr before or at the same time as melanoma inoculation. This suggested that melanoma cells were susceptible to NK cells only for a short period of time after intravascular invasion.     

Combination of interferon alpha with either Ara-C or ATRA in vitro reduces the selective action of interferon against CML CFU-GM.

Although interferon (IFN)-alpha has no specific inhibitory effect on the plating efficiency of granulocyte-macrophage colony-forming cells (CFU-GM) from patients with chronic myeloid leukaemia (CML), it does selectively inhibit the replating ability (secondary colony formation) of CML CFU-GM. Thus, amplification of CFU-GM may be a target for IFN-alpha and other agents used in the treatment of CML. Here we examined whether cytarabine (Ara-C) or all-trans retinoic acid (ATRA) exert similar effects and whether they might in combination with IFN-alpha enhance its efficacy. We found that Ara-C preferentially inhibits the formation of CML CFU-GM compared to normal CFU-GM, but this inhibition was not increased by addition of IFN-alpha. When Ara-C was added to cultures containing IFN-alpha, the inhibition of replating by CML progenitors was abrogated. ATRA increased significantly the plating efficiency of normal CFU-GM. The addition of IFN-alpha to ATRA had no effect on CML or normal colony numbers. However, addition of ATRA to cultures containing IFN-alpha reversed the selective inhibition of CML CFU-GM replating seen in cultures containing IFN-alpha alone. In four IFN-alpha/Ara-C experiments, secondary CML patient-derived colonies were examined by fluorescence in situ hybridisation (FISH). All of them were Ph chromosome positive. No significant effects on CFU-GM production were observed when CML primitive haemopoietic progenitor cells were investigated in a delta (delta) assay. Thus we conclude that combining IFN-alpha with Ara-C or ATRA neutralises the effect of IFN-alpha on CML CFU-GM. This observation provides a rationale for treating patients with alternating courses of IFN-alpha and Ara-C or ATRA, rather than giving either of these two agents in combination with IFN-alpha.     

Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors

We have studied the anti-tumor effects of human recombinant IL-2, alone or in association with LAK cells, in mice transplanted subcutaneously (s.c.) with the following syngeneic tumors: highly metastatic Friend leukemia cells (FLC), nonmetastatic FLC, lymphoma RBL-5 cells and HeJ16 fibrosarcoma cells. In these tumor models, peri-tumoral injections of IL-2 were more effective in inhibiting tumor growth than a systemic treatment. Although s.c. IL-2 treatment resulted in marked inhibition of tumor growth in mice injected s.c. with highly metastatic FLC, it was not effective in inhibiting growth of FLC in the liver and spleen. IL-2 therapy was more effective at increasing survival time in mice transplanted with non-metastatic FLC or with RBL-5 cells. In mice transplanted with HeJ16 fibrosarcomas, s.c. IL-2 treatment resulted in highly significant anti-tumor effect and survival of 70% of tumor-injected mice. No general correlation was found between in vitro sensitivity or resistance to the cytolytic activity of LAK cells and the anti-tumor effects observed in vivo. Subcutaneous injection of IL-1 beta in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Combined treatment of IL-1 beta and IL-2 produced a synergistic anti-tumor effect: 60% of mice injected with highly metastatic FLC survived. Combined IL-1/IL-2 treatments exerted no anti-tumor activity either in DBA/2 mice injected with antibody to Thy 1.2 antigen or in nude mice, indicating that T cells play important roles during IL-1/IL-2 therapy. In vitro treatment of FLC with IL-1 beta resulted in a slight inhibition of cell multiplication, whereas even high doses of IL-2 did not affect FLC multiplication. Our results indicate that local combined treatments with IL-1 and IL-2 can induce potent, host-dependent (T cell-mediated) anti-tumor effects against highly malignant tumors.     

Proliferative response of hairy cells to B cell growth factor (BCGF): in vivo inhibition by interferon-alpha and in vitro effects of interferon-alpha, -beta, and -gamma

Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administration of single doses of IFN-alpha. When hairy cells were maintained in culture for 1 week, they recovered their capacity to be stimulated by BCGF. This reversion was also shown in vivo in hairy cells isolated 1 week after IFN administration. Since in vivo growth of hairy cells could possibly result from the autocrine secretion of BCGF, we propose that the therapeutic effect of IFN-alpha on HCL may be due in part to an inhibition of such autocrine loop.     

Characterization of growth inhibitory factors for mouse monocytic leukemia cells.

Growth inhibitory (GI) factors for mouse monocytic leukemia cells were previously found in conditioned medium (CM) of a clone of mouse myeloblastic leukemia Ml cells (R1-GI factor) and in CM of mouse lung tissue (L-GI factor). In the present study, the effects of these GI factors on the growth of variant cell lines (Mm-A, Mm-P and Mm-S2) of mouse monocytic line Mm-1 cells were examined. Mm-A are highly leukemogenic to syngeneic SL mice, Mm-P cells are moderately leukemogenic, while Mm-S2 cells have little or no leukemogenicity. The R1-GI factor markedly suppressed the growth of Mm-A cells, whereas it inhibited the growth of Mm-P and Mm-S2 cells only moderately. Recombinant mouse interferon beta (IFN beta) had similar target specificities to those of the R1-GI factor on these variant cells. Moreover, anti-mouse IFN beta antibody completely neutralized the GI activity of the R1-GI factor on Mm-A cells. These results show that the R1-GI factor and mouse IFN beta are very similar and probably identical proteins. The L-GI factor had different target specificities from the R1-GI factor: it showed strongest GI activity on Mm-P cells, moderate GI activity on Mm-S2 cells and weak GI activity on Mm-A cells. Recombinant human interleukin 6 (IL-6) had similar target specificities to the L-GI factor on these Mm-1 variant cells. Furthermore, the L-GI factor could support the proliferation of IL-6-dependent MH60.BSF2 cells. Anti-mouse IL-6 antiserum neutralized the GI activity of the L-GI factor on Mm-P cells. Thus the L-GI factor and mouse IL-6 seem to be closely related and possibly to be identical proteins.     

The mechanism of interferon effect on cell transformation by murine sarcoma virus

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.