Evidence for transformation of spleen cells one day after infection of mice with friend leukemia virus.

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.

Human immunodeficiency virus type 1 gp120 mimics a hidden monomorphic epitope borne by class I major histocompatibility complex heavy chains.

Murine monoclonal antibodies (mAbs) M38 and L31 define two epitopes of a surface protein of activated lymphocytes and monocytes. It has been shown that M38 also defines a crossreactive epitope of human immunodeficiency virus type 1 (HIV-1) gp120 (Beretta et al., 1987. Eur. J. Immunol. 17: 1793). The mAb inhibits syncytia formation driven by HIV-1-infected cells. The surface protein was demonstrated to be a class I MHC alpha chain, by sequence analysis of the corresponding cDNA and by immunological means. The epitopes defined by mAbs M38 and L31 are monomorphic and hidden (i.e., inaccessible to antibodies) on native HLA molecules expressed by resting cells, but can be evidenced on denatured proteins by Western blot analysis. The two epitopes become accessible after activation processes have been implemented, likely reflecting a conformational alteration of alpha chains (such as that described by Schnabl et al. 1990. J. Exp. Med. 171:1431). Consistent with molecular data are the results of functional analysis, which indicate that the molecule recognized by M38 and L31 is a gate for pleiotropic negative signals, since the two mAbs were shown to inhibit monocyte antigen presentation and lymphocyte mitogenic proliferation, respectively.

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro.

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.

Lymphomas in mice: failure of induction after a graft-versus-host reaction.

A mild graft-versus-host reaction induced in (BALB/cJ x DBA/2J) F(1) mice by the administration of parental spleen cells that differ at several weak histocompatibility loci did not influence the development of lymphomas in these animals. Rous sarcoma virus also failed to induce tumors in the runt and control animals. Breast carcinomas, presumably due to contamination of the inoculums with mammary tumor virus, occurred in those experimental groups given parental cells, whether or not they were viable or immunologically competent. We found no evidence that the immunologic process-as represented by the graft-versus-host reaction-is causally related to the induction of neoplasia.

Interferons in cell growth and development.

Interferons (IFNs), besides inducing an antiviral state in uninfected cells, are also natural regulatory molecules. They play a key role in the regulation both of cell growth and differentiation, and of development. Up- or down-regulation of oncogenes by IFNs may be one of the mechanisms by which these molecules affect cell physiology. The list of IFN-inducible proteins continues to grow rapidly and future research should identify among these the mediators of the biological effects of IFNs.

Proliferative response and oncogene expression induced by epidermal growth factor in EL2 rat fibroblasts.

Extensive evidence supports a two-step model for the control of fibroblast growth, which includes first the action of a competence factor (e.g., platelet-derived growth factor) followed by the stimulus of a progression factor (e.g., epidermal growth factor [EGF]). We investigated whether this model may be applied to the euploid EL2 fibroblast line recently isolated from rat embryos (E. Liboi, M. Caruso, and C. Basilico, Mol. Cell. Biol. 4:2925-2928, 1984). Our results clearly show that EGF alone leads EL2 cells to proliferate in serum-free conditions at a rate corresponding to 50 to 60% of that observed in the presence of 10% calf serum. It is of interest that, when resting EL2 cells were exposed to EGF, transcription of both c-myc and c-fos was markedly induced. Altogether, these observations suggest that, in contrast with the model of fibroblast growth mentioned above, EL2 cells require the presence of a single growth factor (EGF) for induction of DNA synthesis, and the expression of myc and fos proto-oncogenes may represent an obligatory step in the pathway of commitment of EL2 cells to proliferation. In addition, we showed that EGF may induce EL2 cells to acquire some properties of transformed cells, such as growth in agar and loss of contact inhibition. This suggests that the particular response to EGF of the EL2 line may be strictly connected with the expression of a transformed phenotype.

Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development.

The growth arrest-specific gas5 gene was isolated from mouse genomic DNA and structurally characterized. The transcriptional unit is divided into 12 exons that span around 7 kb. An alternative splicing mechanism gives rise to two mature mRNAs which contain either 11 or 12 exons, and both are found in the cytoplasm of growth-arrested cells. In vivo, the gas5 gene is ubiquitously expressed in mouse tissues during development and adult life. In Friend leukemia and NIH 3T3 cells, the levels of gas5 gene mRNA were high in saturation density-arrested cells and almost undetectable in actively growing cells. Run-on experiments indicated that the gas5 gene is transcribed at the same level in both growing and arrested cells. On the other hand, in dimethyl sulfoxide-induced differentiating cells a sharp decrease in the rate of transcription was observed shortly before the cells reached the postmitotic stage. These results indicate that in density-arrested cells accumulation of gas5 mRNA is controlled at the posttranscriptional level while in differentiating cells expression is regulated transcriptionally.

Inhibitory effect of interferon on multiplication of Friend leukemia cells in vivo.

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.

Interferon alpha-2b and retinoic acid combined treatment affects proliferation and gene expression of human cervical carcinoma cells.

The in vivo and in vitro antitumor effectiveness of IFNs is well documented. Their combination with differentiating agents, such as retinoic acid, has been demonstrated to be a promising therapy for patients with advanced squamous cell cancer of the skin and the cervix. However, the mechanisms that mediate these antitumor responses are not yet known. We studied the epidermoid cell line ME 180 derived from human cervical carcinoma to test its responsiveness to IFN-alpha-2b (INTRON A) and all-trans-retinoic acid (RA). Both agents have demonstrated ability to inhibit the growth of ME 180 cells in a dose- and time-dependent manner. The antiproliferative effect was further increased by the treatment with IFN-alpha-2b and RA combined. In accordance with this result, we found that the combination of the two agents has the effect of increasing the expression of the 2-5A synthetase gene, which is thought to play a key role in antigrowth responses to IFNs. At increased levels of 2-5A synthetase mRNA corresponds a significant increase in 2-5A synthetase activity. Although RA per se has no effect on the 2-5A synthetase expression, when it is combined with IFN-alpha-2b it appears to be able to potentiate the IFN-induced 2-5A synthetase expression. Moreover, the combination of IFN-alpha-2b and RA produces a similar effect also on the expression of the HLA-A2 gene, which has been shown to be induced in ME 180 cells both by IFN-alpha-2b and RA alone. In view of the possible mechanisms of action of the two agents, it is interesting to note that their combination increases, although transiently, the expression of IRF1, which codes for a transcription factor that regulates IFN gene expression and is thought to be involved in the regulation of IFN-induced effects and in mediating cell death or apoptosis.

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia.

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.

Effects of in vivo Friend leukemia virus infection on levels of serum thymic factors and on selected T-cell functions in mice.

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.

Three submicroscopic deletions at the APC locus and their rapid detection by quantitative-PCR analysis.

We describe three unrelated kindreds, affected by familial adenomatous polyposis (FAP), with 5q submicroscopic deletions that encompass the entire adenomatous polyposis coli (APC) gene and the adjacent DP1 gene. In one family the deletion encompasses also the MCC (mutated in colon cancer) gene. Affected members of these families had dysplastic adenomatous polyps and congenital hypertrophy of the retinal pigment epithelium (CHRPE); no individual was affected by mental retardation or facial dysmorphism. The deletions were detected by linkage analysis with several intragenic and closely flanking polymorphic markers and confirmed by a quantitative PCR analysis. This procedure could have an impact on the detection of the molecular defect in FAP patients in whom mutational analysis fails to identify the specific mutation.

Interaction of HIV-1 with susceptible lymphoblastoid cells. 1H NMR studies.

Different strains of HIV susceptible lymphoblastoid cells have been infected by HIV-1 and examined by means of 1H NMR spectroscopy at different times after infection, taking advantage of the presence of high resolution lipid signals from the plasma membrane of tumor cells. A transient decrease in intensity of fatty acid signals, originated by changes in membrane structure, has been observed early after viral infection. Marked alterations in membrane-dependent steps of phospholipid synthesis can also be inferred by the observed transient depression in peaks from choline-based metabolites. Spectral modifications deriving from changes in lipid metabolism are also produced both in infected cells a few days after infection and in permanently infected cells. 1H NMR can, therefore, monitor structural and metabolic effects induced by HIV infection.

Hemin inhibits the interferon-beta-induced antiviral state in established cell lines.

Hemin and other metalloporphyrins are known as very versatile compounds in nature, because they are able to carry out numerous functions in a free state or in association with specific proteins. When Friend murine erythroleukemia cells are treated with IFN-beta plus 100 microM hemin, the antiviral state is not observed, whereas the antiviral effect of IFN-gamma is unaffected by hemin treatment. This inhibitory effect of hemin is not restricted to erythroid cells. In fact, it is also observed in murine L929 and in human cell lines treated with IFN-beta. Neither trivalent iron in other forms nor hemin analogs (such as protoporphyrin IX or Sn(2+)-protoporphyrine IX) mimic this effect. Conversely, Co(3+)-protoporphyrin IX was as effective as hemin. At the transcriptional level, results obtained by run-on assays on nuclei from IFN-treated cells indicate that hemin does not completely inhibit IFN-beta induction of 2-5A synthetase gene(s) at 6 h of treatment but abolishes it at 24 h. In addition, hemin is able to inhibit the accumulation of IFN-induced 2-5A synthetase mRNAs. Experiments carried out to investigate the hemin effect on the early steps of the IFN signaling pathway indicate that hemin interferes with the ability of type I IFN to bind to its receptor, probably by a direct action on the IFN molecule.

Metabolic and structural effects of HIV infection in human peripheral blood mononuclear cells can be monitored with 1H NMR spectroscopy.

Infection of human peripheral blood lymphocytes by human immunodeficiency virus type 1 (HIV-1) was investigated by means of 1H nuclear magnetic resonance spectroscopy, taking advantage of the presence of signals from fluid lipid domains in the membrane of stimulated lymphocytes. A transient decrease of the lipid methylene signal intensity was observed at the time of HIV internalization, monitoring a general rearrangement of membrane structure associated with virus entry. A similar effect was also observed a few days after infection, when HIV particles are released by infected cells as demonstrated by high reverse transcriptase activity in cell supernatant. Signals arising from choline-based metabolites were also affected by HIV infection, indicating a possible slowing down of phospholipid synthesis.

HIV, HTLV-1, and HBV infections in a cohort of Italian intravenous drug abusers: analysis of risk factors.

A seroepidemiological survey of a group of 291 intravenous drug abusers (IVDAs), 45 household contacts of IVDAs, and 39 laboratory workers has been carried out to determine the prevalence of HIV-1, HIV-2, HTLV-1, and HBV antibodies in the sera, as well as to evaluate the role of various risk factors. Among i.v. drug abusers, the prevalence was 32.3% for HIV-1 and 6.6% for HTLV-1. For both viruses, the total figures did not significantly change from 1985 through 1987, accounting for a slow viral circulation in this group. No seropositivity (HIV-1, HTLV-1) was found among laboratory workers, whereas one subject was found seropositive for HIV-1 among household contacts. From 1985 to 1986, 5 out of 58 subjects seronegative for HIV-1 and 5 out of 82 seronegative for HTLV-1 seroconverted (incidence rates of 8.6 and 6.1%, respectively). From 1986 to 1987, none out of 11 seronegatives for HIV and 1 out of 16 seronegatives for HTLV-1 seroconverted. The total figures for hepatitis B markers were 79.2% among IVDAs, 24.4% among household contacts, and 25.6% among laboratory workers. A significant correlation was found between presence of HBV markers and seropositivity for HIV and HTLV-1. A significant association with HIV-1 seropositivity was found for history of sexual intercourse with HIV-1 seropositive partners and for sexual promiscuity. These data emphasize the important role played by sexual behavior in addition to needle-sharing in the spreading of multiple infections among drug abusers.

Action of lysosomotropic amines on spontaneous and interferon enhanced NK and CTL cytolysis.

Cell-mediated cytotoxicities, such as natural killer (NK) and T-dependent cytotoxic (CTL) activities, are inhibited to the same extent by lysosomotropic amines, even when interferon is added to cultures to enhance lysis. It is postulated that the amines block steps common to spontaneous and IFN-enhanced NK and CTL lytic mechanisms.

In vitro spontaneous production of anti-SIV antibodies is a reliable tool in the follow-up of protection of SIV-vaccinated monkeys.

To assess the reliability of the spontaneous in vitro synthesis of simian immunodeficiency virus (SIV)-specific antibodies as a marker in the monitoring of protection in SIV-vaccinated animals, Macaca fascicularis monkeys were immunized with formalin-inactivated SIVmac251 or SIVmac251/32H, and challenged with human-derived (SIVmac251/32H) or monkey-derived live SIV. As judged by virus isolation and polymerase chain reaction (PCR) techniques, immunized animals were protected against human-derived SIV challenge, and no spontaneous in vitro synthesis of anti-SIV antibody was observed in nonstimulated peripheral blood mononuclear cell cultures over a 4-month follow-up. On the contrary, human cell-grown SIVmac251 immunization did not afford protection against monkey-derived SIV, and all the animals became infected and showed spontaneous in vitro synthesis of anti-SIV antibodies. These data demonstrate that lack of protection in SIV-vaccinated monkeys is strictly associated with PBMC ability of spontaneously produce anti-SIV antibodies in vitro following challenge, and suggest that this parameter might also constitute a reliable marker for monitoring protection in large-scale HIV vaccination and immunotherapy programs.

Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate.

HUT-78 cells were infected with a reverse transcriptase (RT)-positive supernatant of a culture of peripheral blood lymphocytes (PBL) from an AIDS patient and then cloned. Of these clones, two have been isolated and characterized. Clone D10 is persistently and productively infected with an HIV variant. The clone F12, in spite of the presence of an integrated full-length HIV provirus, does not release virus particles in the medium. D10 and F12 clones substantially differ in terms of protein pattern; that is, D10 is super-imposable to infected HUT-78 cells, whereas F12 exhibits a decreased uncleaved p55 gag precursor and the presence of uncleaved gp160 and of a unique p19, although they do not show qualitative or quantitative differences in viral RNA synthesis. Restriction patterns of F12 proviral DNA do not show major genomic deletions. These results indicate that F12 clone cells carry an HIV genome with minor mutations that probably affect the correct production of viral proteins at a posttranscriptional level. In addition, the F12 clone is resistant to high-multiplicity superinfection with HIV-1 or HIV-2.

Growth and differentiation of Friend erythroleukemia cells in serum-free culture.

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.