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Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.

The levels of serum thymic factor(s) (STF), of Thy-1.2 positivity of splenocytes [as measured by their azathioprine (AZ) sensitivity], and of Thy-1.2-positive "spontaneous" spleen rosette-forming cells (SSRFCs), as well as the presence of infectious virus in the thymus, were assessed as a function of time after virus inoculation in susceptible DBA/2, partially resistant BALB/c, and fully resistant C57BL/6 mice given the polycythemia- or anemia-inducing strain of Friend leukemia virus (FLV-P and FLV-A, respectively). As early as Days 2 to 3, the levels of STF and of AZ sensitivity of splenocytes were profoundly decreased in DBA/2 mice, and, to a lesser extent, in BALB/c mice given FLV-P; however, SSRFCs/spleen were increased in both mouse strains. Conclusive evidence of infectious FLV-P was obtained in the thymuses of DBA/2 mice soon after infection. In mice of the same strains infected with FLV-A, STF levels were similarly decreased, but AZ sensitivity of splenocytes was unaffected, and SSRFCs were decreased. Evidence of early FLV-A infection in the thymus of DBA/2 mice was likewise obtained. In C57BL/6 mice given FLV-A, STF levels, AZ sensitivity of splenocytes, and SSRFC showed changes similar to, but of lower magnitude than, those in BALB/c mice. On the other hand, in C57BL/6 mice given FLV-P, the decrease in STF and AZ sensitivity was almost as pronounced as in susceptible DBA/2 mice in the face of complete absence of infectious virus or viral markers in the thymuses. The observed changes are ascribed to virus infection in view of the following: (a) good temporal correlation between these changes and virus infection; (b) absence of any change in mice given heat-inactivated viruses or spleen homogenate of normal DBA/2 mouse spleen; (c) overall good correlation between mouse genotype and genetic (Fv-1 and Fv-2) restrictions of virus infection on one hand and the magnitude of the observed changes on the other. In particular, the decrease in STF and SSRFC levels is ascribed to the replication-competent (Friend-murine leukemia virus) component of Friend leukemia virus complex, whereas the decrease in AZ sensitivity of splenocytes and the increase of SSRFCs are ascribed to the defective spleen focus-forming virus component of the complex. All changes described so far were transient, since they were not detectable beyond 42 days after virus inoculation in overtly leukemic animals. The observed derangements of thymus-derived immune functions may play an important cofactor role during the onset of leukemia in mice genetically permissive to Friend leukemia virus replication and transformation, but they do not seem relevant to the maintenance of leukemia.

The presence of interferon (IFN) receptors on mouse Friend leukemia cells (FLC) has been investigated in binding experiments with highly purified 125I-labeled mouse alpha/beta IFN. Both IFN-resistant clones and wild-type IFN-sensitive FLC showed a specific saturable binding site for mouse IFN with a similar affinity constant. In contrast to IFN-sensitive FLC, IFN-resistant FLC variants were not inducible by IFN for double-stranded RNA-dependent 2-5A synthetase activity.

The kinetics of both erythroid burst-forming and colony-forming units (BFU-E, CFU-E) and myelomonocytic precursors [myelomacrophage colony-forming unit (CFU-C)] have been evaluated in tibial marrow, peripheral blood, and spleen of DBA/2 mice at time intervals after inoculation of either the anemic (FLV-A) or the polycythemic (FLV-P) strain of Friend leukemia virus. Either one of the viruses induced, at 7-10 days after infection, a massive increase in the number of BFU-Es in peripheral blood, in parallel with their depletion in tibial marrow and increase in spleen. A comparable increase in the blood BFU-E number was observed in splenectomized FLV-infected mice. These results indicate a marrow-spleen migration of BFU-Es. In spleen, the increase of the BFU-E number was associated with an increase in the CFU-E pool. In tibial marrow, a sequence of expansion/depletion waves occurred reciprocally at the level of BFU-E and CFU-E. The cycling of BFU-E([(3)H]thymidine in vitro suicide index) in marrow, blood, and spleen was enhanced, whereas that of CFU-E and CFU-C showed little or no modification. These kinetic data suggest that the main target cell of FLV may be the BFU-E or a closely related element. In plates without added erythropoietin (but containing it in fetal calf serum), expression of CFU-E from FLV-P-treated animals was maximal; that of CFU-E from FLV-A-injected mice was either virtually absent or only slight in marrow or spleen, respectively. BFU-E growth always was fully dependent upon erythropoietin addition. Control studies in FLV-infected resistant mice and in susceptible mice given diluted or heat-inactivated virus provide convincing evidence that the phenomena described are induced by FLV.

Inactivated cellular preparations and cell wall materials of Candida albicans (CA) were tested for their capacity to suppress the growth of Friend Leukemia Cell (FLC)-induced tumours and the infection by Friend Leukemia Virus (FLV) in histocompatible mice. Factors affecting the inhibition of tumor growth by CA cellular preparations were: i) the schedule of agent administration; ii) the method of cell inactivation; iii) the FLC load. In particular, mice given 10(7) yeast cells, inactivated by cold alkali, on days -14 and +1 with respect to 10(4) FLC challenge on day 0 did not develop tumors. A crude cell wall fraction derived from cells extracted with hot alkali was still effective in reducing (but not suppressing) tumour growth whereas a purified, particulate glucan fraction (glucan "ghosts", essentially consisting of beta 1,3-1,6 glucan) was ineffective. No cellular preparation or cell wall fraction exerted anti-FLV effects (as shown by splenomegaly measurements) nor did any CA material induce interferon-like activity in the serum of animals injected with either FLC or FLV. Therefore, the observed antitumor activity by CA was not mediated by antiviral effects but possibly due to an "adjuvant-type", nonspecific, immunopotentiation of host antitumor response, as documented in other animal tumor models.

Low molecular weight chromatin peptides exert a dose-dependent inhibition of Dimethylsulfoxide (DMSO)-induced erythroid differentiation of murine Friend Leukemia Cells (FLC). This effect correlates with the degree of purification of the peptide fractions. Crot analysis of globin mRNA amounts in DMSO-treated FLC given the peptides showed a 4-5-fold decrease of messenger RNA in the cytoplasma with no nuclear storage of globin transcripts. Spectrin accumulation in "induced" FLC is inhibited as well. The effects of the peptides on erythroid markers are reversible upon removal of the compounds. They also appear to be specific for induced gene expression as (1) no effects are observed on cell growth and RNA synthesis in normal non-differentiating cell lines; and (2) no changes have been detected with regard to the expression of integrated viral genes coding for continuous shedding of viral particles.

We analyzed the effects of a polycythemic substrain of Friend leukemia virus, i.e. the FLV-P virus, on splenic NK activity of DBA/2 susceptible mice. One day after virus injection a significant increase of NK activity was found, which persisted until day 10. On the other hand, 14-21 days after virus injection a marked and significant depression of activity was measured. This depression was associated with the appearance of suppressor cells able to inhibit the lytic activity of untreated splenocytes when mixed in vitro in the 4 h 51Cr-release assay. The suppressor cell population was insensitive to treatment with anti-Thy 1.2 plus complement, was adherent to Sephadex G-10 and nylon, but did not adhere to plastic, suggesting it is neither a T-cell nor a typical macrophage. The possible relevance of NK activity modulation in relation to the induction of leukemia is discussed.

The multiplication of Friend leukemia cells in the spleens of irradiated or nonirradiated histocompatible DBA/2 mice was determined by the uptake of 125-5-iodo-2'-deoxyuridine. The multiplication was inhibited by daily treatment with mouse interferon preparations or the interferon inducer, Newcastle disease virus. Since, under the experimental conditions used, the proliferation of the Friend leukemia cells was independent of the replication of the Friend virus, the inhibitory effect of interferon might not be mediated by an antiviral effect but by some other mechanism.

A synthetic medium allowing indefinite optimal growth of Friend erythroleukemia cells (FLC) is described. It consists of Iscove's modified Dulbecco's medium supplemented with bovine serum albumin, transferrin, and a lipid mixture. Transferrin and lipids are essential for Friend cells growth. Under these conditions, FLC erythroid differentiation, promoted by a number of inducers, is less efficient than in cultures with serum-rich medium, suggesting that unknown serum factors may play an additional role in this phenomenon. Conversely, the enhancement of erythroid differentiation induced by low doses of Interferon is superimposable in both types of cultures.

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

It has been suggested that the interferon (IFN)-induced 2',5'-oligoadenylate (2-5A) synthetase, which polymerizes ATP into a series of 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, plays a general role in cell growth and terminal differentiation. For instance, an increase in 2-5A synthetase activity has been described during dimethyl sulfoxide (Me2SO)-induced erythroid differentiation of Friend leukemia cells (FLC). 2-5A synthetase has been measured in two Friend leukemia cell sublines by various techniques including a radioimmunoassay of its products which would detect 10(-16) mol of 2-5A cores. Although cells of both sublines fully differentiate (as measured by benzidine staining), only in one subline was there an increase in 2-5A synthetase activity upon treatment with Me2SO. Hexamethylenebisacetamide, another potent agent of differentiation in this system, did not increase 2-5A synthetase activity in either of these two sublines. An IFN-resistant FLC variant differentiated normally upon treatment with Me2SO or hexamethylenebisacetamide while it was noninducible for 2-5A synthetase activity by exogenous IFN or by the inducers themselves. A similar situation has been observed with regard to the level of phosphorylation of the IFN-induced Mr = 67,000 protein band. In addition, treatment of IFN-sensitive and resistant FLC sublines with mouse alpha beta IFN antiserum did not affect differentiation. Even though we have duplicated previous findings on the increase of 2-5A synthetase activity in Me2SO-induced FLC, the lack of any such increase with other inducers or other sublines indicates that there is no causal relationship between the enzyme activation and FLC differentiation.

The influence of in vivo infection with the polycythemic substrain of Friend leukemia virus on noninducible ('natural') resistance against allogeneic normal or malignant grafts was studied in lethally irradiated mice. Parallel studies were performed on the NK system in the same experimental conditions. The results indicate that FLV-P infection of mice with full (DBA/2) vs partial (BALB/c and CD2F1) susceptibility did not suppress their in vivo natural resistance against bone marrow or El-4 leukemia cells. On the other hand, a decline in NK activity paralleled the progression of leukemic disease in the more susceptible DBA/2 hosts.

A number of Friend leukemia cell variants with a interferon-gamma (IFN-gamma)-resistant phenotype have been isolated. They appear resistant to the antiproliferative action of IFN-gamma and to the induction of the antiviral state assessed by Friend leukemia virus release and vesicular stomatitis virus yield. Selection was performed via a prolonged exposure to increasing amounts of highly purified recombinant IFN-gamma of wild-type Friend cells or of variant clones thereof already resistant to IFN-alpha/beta (Affabris et al., 1982, Virology 120, 441-452). Only the clones derived from IFN-alpha/beta-resistant variants showed a phenotype fully resistant to IFN-gamma treatment while keeping their previously acquired resistance to IFN-alpha/beta. These cells are not deficient in high-affinity receptors for IFN-gamma so that their resistant phenotype appears to be mediated by events distal to binding of IFN-gamma to its receptors. Furthermore, analysis of IFN-induced dsRNA-dependent 2-5A synthetase and 67K protein kinase enzymatic activities, biochemical markers for cellular responses to IFN, showed that both these activities were not induced in IFN-alpha/beta and IFN-gamma-resistant clones when treated with either type of IFN. Accordingly, no increased expression of 2-5A synthetase mRNA(s) could be detected by probing poly(A)+-enriched RNA from cells exposed to IFN-alpha/beta or IFN-gamma treatment with murine or human specific cDNAs. On the other hand, no major changes in restriction patterns of 2-5A synthetase gene(s) were observed in these variant cells by restriction endonuclease digestion and Southern blotting. In addition, analysis of 2-5A synthetase mRNA induction, performed on wild-type cells, showed that the kinetic of induction due to IFN-gamma treatment is slower than that obtained with IFN-alpha/beta.

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon-treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.

Proliferation and erythroid differentiation of transplanted DBA/2 marrow cells and Friend virus-induced leukemic cells were assessed in syngeneic, allogeneic (H-2 compatible), and (BALB/c x DBA/2)F(1) hybrid mice (CDF(1)). Measurements were made 5 days after transplantation of donor cells into nonirradiated or X-irradiated mice by the spleen colony or the (125)IUdR-(59)Fe uptake methods. Growth of DBA/2J (Jackson subline) marrow grafts was poor in irradiated CDF(1)J hybrids as compared with growth in syngeneic and allogeneic hosts. The DBA/2J transplants proliferated, however, without impairment in irradiated CDF(1) hybrids which were the progeny of DBA/2 male parents of other sublines, e.g. DBA/2Ha, DBA/2Cr, and DBA/2Cum. In contrast, tissue-cultured Friend leukemic cells of DBA/2J origin grew deficiently in all CDF(1) hybrids tested, regardless of irradiation and of the DBA/2 parent's subline. The growth pattern of transplanted DBA/2J cells was a manifestation of hybrid resistance. The results with DBA/2J and other DBA/2 subline grafts suggested that hybrid histocompatibility alleles were expressed to a greater extent in leukemic than in normal marrow cells, for the former were consistently recognized as "nonself" by CDF(1) mice, but not the latter cells. The property of deficient growth in irradiated CDF(1)Ha hybrids was acquired by DBA/2J hemopoietic cells within 6 hr from infection in vivo with Friend leukemia virus, and persisted during the following 8 days. It was ascribed to enhanced expression of hybrid histocompatibility gene(s) (Hh) induced by the virus. Autonomous growth potential of hemopoietic cells, manifested by proliferation in nonirradiated recipients, was first detected 24 hr from infection, and likewise persisted at the later intervals. At the same time, the infected cells grew deficiently also in nonirradiated CDF(1)Ha mice. The two irreversible cellular changes were regarded as the earliest signals of virus-induced transformation.