Immunogenicity of viable tumor cells

Summary The immunogenicity of KMT-17 fibrosarcoma cells which had been xenogenized by infection with FV was compared to that of KMT-17 cells which had been admixed with BCG. We report here that 10⁵ and 10⁶ KMT-17 cells also grew progressively to kill rats, but when 10⁵ KMT-17 cells were administered with BCG the tumor cells did not grow in the majority of rats. The strength of immunogenicity (ETD₅₀), as measured by the number of immunizing cells required for a suppression of growth of 10⁷ KMT-17 cells in 50% of the rats, was 2.1×10³ for FV-KMT-17 and 36.3×10³ for BCG+KMT-17. The tumor cell dose (LTD₅₀) which was required to kill 50% of the rats immunized with 10⁵ FV-KMT-17 was more than 10,000 times higher than that found in normal rats, whereas the number of tumor cells required to kill 50% of the rats immunized with the same number of BCG+KMT-17 was only 3,680 times higher than the amount found in normal rats. Thus the immunogenicity of FV-KMT-17 is much stronger than that of BCG+KMT-17.The difference in immunogenicity between the two vaccines was also observed in the tumor-neutralizing activities of spleen cells obtained from rats which had been immunized with both vaccines, as measured by a Winn assay. Moreover, the antitumor activity of spleen cells from rats immunized with FV-KMT-17 was concentrated in the carrageenan-resistant and plastic nonadherent cells, while that of spleen cells from rats immunized with BCG+KMT-17 was observed in carrageenan-sensitive and plastic adherent cells as well as in nonadherent cells. The involvement of different effector cells indicates that different mechanisms operate in the antitumor resistance in rats immunized with either FV-KMT-17 or BCG+KMT-17. Abbreviations used: FV, Friend leukemia virus; FV-KMT-17, Friend leukemia virus infected KMT-17 cells; EDT₅₀, a 50% effective tumor dose; LTD₅₀, a 50% lethal tumor dose     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Development of highly immunogenic variants of a rat fibrosarcoma line during in vitro cultivation

Summary Rat firosarcoma KMT-17 cells descreased in tumorigenicity when cultured in vitro. Eight clones derived from cultured KMT-17 cell lines (c-KMT-17) were examined for their tumorigenicity, immunosensitivity, and immunogenicity. All the clones were less or nontumorigenic in normal syngeneic rats than KMT-17 cells maintained in vivo. All eight clones produced tumors in rats immuno-suppressed with 600 rad ⁶⁰Co; differences in degree of tumorigenicity were seen among clones in rats irradiated with 250 rad ⁶⁰Co. Although immunosensitivity of the eight clones to complement-dependent and cell-mediated cytotoxicity was the same or less than that of KMT-17 cells, al leight clones induced greater transplantation resistance to KMT-17 than KMT-17 itself. Cold target inhibition tests demonstrated new antigens in a highly immunogenic variant in addition to the original tumor associated antigen (TAA). New glycolipids, not observed in KMT-17 cells, were demonstrated in the clones by thin layer chromatography. These results suggest that new antigens appearing during culture of KMT-17 may act as helper antigens for TAA, increasing the immunogenicity and decreasing the tumorigenicity of the cultured cells.This work was partially supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science und Culture, Japan.     

Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.     

Immunological regression of metastatic cancer in the liver as a result of “in vivo xenogenization”

We attempted to induce the regression of liver metastatic tumor cellsin vivo by the administration to rats of Friend leukemia virus (FV) (in vivo xenogenization). The virus which was used in this experiment, FV, is highly immunogenic and does not normally cause disease in an adult rat. At first, we induced a FV viremia in tumor bearing rats in order to deliver the virus to the site of the tumor cells. FV viremia was induced by injecting 60 mg/kg cyclophosphamide (CY) i.v. after the administration of FV, and by transferring syngeneic bone marrow cells so that FV would be able to infect them and then replicate.In order that the tumor cells which were infected with virus should regress, it was necessary to break down their tolerance to FV antigens. As adoptive immunotherapy we therefore, transferred syngeneic spleen cells from rats which had been immunized with FV to tumor bearing rats. The result of this experiment was that these tumor bearing rats infected with FV which had received either normal syngeneic spleen cells or no spleen cells as controls died from liver metastasis (8 out of 9 rats (89%) and 15 out of 17 (88%) respectively). On the other hand, only 4 out of the 15 (27%) tumor bearing rats which were infected with FV and which received FV-immune spleen cells died from liver metastasis.These sets of data indicate that thein vivo xenogenization of tumor cells are indeed able to induce the regression of metastic tumor cells.     

Enhancement of anti-murine colon cancer immunity by fusion of a SARS fragment to a low-immunogenic carcinoembryonic antigen

Background  

It is widely understood that tumor cells express tumor-associated antigens (TAAs), of which many are usually in low immunogenicity; for example, carcinoembryonic antigen (CEA) is specifically expressed on human colon cancer cells and is viewed as a low-immunogenic TAA. How to activate host immunity against specific TAAs and to suppress tumor growth therefore becomes important in cancer therapy development.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

Differential Requirements of Cellular and Humoral Immune Responses for Fv2-Associated Resistance to Erythroleukemia and for Regulation of Retrovirus-Induced Myeloid Leukemia Development

To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2^r B6 mice lacking CD4⁺ T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8⁺ T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8⁺ T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2^r mice through cellular immune responses.     

Multistage Friend erythroleukemia: independent origin of tumor clones with normal or rearranged p53 cellular oncogenes.

The erythroleukemia induced by Friend virus complex in adult mice is a multistage malignancy characterized by the emergence, late in the disease, of tumorigenic cell clones. We have previously shown that a significant proportion of these clones have unique rearrangements in their cellular p53 oncogene. The clonal relationships among Friend tumor cells isolated in the late stages of Friend erythroleukemia were analyzed by examining the unique integration site of Friend murine leukemia virus and the unique rearrangement in their cellular p53 oncogene. The majority of clones isolated from individual mice infected with Friend virus were clonally related as judged by the site of Friend murine leukemia virus integration. However, Southern gel analysis of DNA from individual Friend cell clones indicated that all of the clones with a normal p53 gene from the same mice were clonally related, but were unrelated to the Friend cell lines with a rearranged p53 gene. These results suggest that Friend tumor cells with rearrangements in their p53 gene arise as the result of a unique transformation event, rather than by progression from already existing tumor cells with a normal p53 gene. They also suggest that such rearrangements in the p53 gene confer a strong selective advantage to these cells in vivo.     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

Expression of a tolerizing tumor antigen in peripheral tissue does not preclude recovery of high-affinity CD8+ T cells or CTL immunotherapy of tumors expressing the antigen

Transgenic (TG) mice were generated selectively expressing the gag protein of Friend murine leukemia virus (FMuLV) in the liver. FMuLV(gag) is also expressed by the FBL leukemia, and is the immunodominant tumor Ag of the CD8(+) T cell response in C57BL/6 mice. gag-TG mice expressing FMuLV(gag) in the liver were tolerant to the protein and failed to generate a CTL response to either FBL or FMuLV(gag). This tolerance reflected anergy rather than deletion, as CTL responsiveness could be recovered after four cycles of in vitro stimulation. Adoptively transferred gag-specific T cells were not anergized in gag-TG recipients, as revealed by antitumor activity in vivo. Also, such T cells did not induce detectable autoimmune injury in gag-TG liver cells. These results suggest that the requirements for a tissue Ag to provide a tolerizing stimulus are distinct from those for being the target of a T cell-mediated autoimmune response and that the requirements for induction and maintenance of peripheral tolerance are distinct for naive and primed T cells. That anergic T cells reactive with tumor-associated Ags can be recovered by repetitive in vitro stimulation and can mediate tumor therapy suggests strategies that use such Ags to generate CTL for adoptive immunotherapy should be further developed.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.     

Nonspecific inhibition of tumor growth in vivo by admixed allogeneic tumor-sensitized lymphoid cells and identical inactivated allogeneic tumor cells.

With the in vivo tumor neutralization test (Winn test), growth of a transplanted (KMT-17) from Wistar-King-Aptekman rats was inhibited by allogeneic tumor (AH-66 from Donryu rats)-sensitized syngeneic lymphoid cells admixed with mitomycin C (MMC)-treated AH-66 cells. The observed tumor inhibition may be immunologically nonspecific, since no cross-antigens were detected by membrane immunofluorescence on the surfaces of KMT-17 and AH-66 cells. Close contact among KMT-17, AH-66-sensitized lymphoid cells and MMC-treated AH-66 cells was required for the inhibition of KMT-17 growth. AH-66 cells pretreated with formalin or ultrasonication lost tumor inhibitory activity when they were admixed with AH-66-sensitized lymphoid cells, and only MMC-treatment effectively preserved the tumor inhibitory activity of AH-66 cells. The sensitized spleen cells, draining lymph node, or peripheral blood cells inhibited tumor growth when they were admixed with MMC-treated AH-66 cells, whereas nucleated cells from bone marrow, thymus, or distal lymph node did not. Growths of KMT-17 were inhibited by admixed sensitized spleen cells and MMC-treated AH-66 even when pre-irradiated rats were used as recipients.     

Suppression of Established Friend Virus Leukemia by Statolon: I. Demonstration of a Latent Infection in Clinically Normal Mice

A single inoculation of statolon into mice with established Friend virus (FV) leukemia can suppress the viral infection and produce a clinical remission lasting many months. Eventually, however, most of the mice develop characteristic FV leukemia. Persistence of FV activity in the spleens of mice during clinical remission can be demonstrated by cell transfer and histopathologic studies. Transfer to normal mice of a large number of spleen cells (10⁷) from mice in remission produces FV leukemia, and transfer of a small number of cells (10²) produces immunity to FV challenge. Histopathologic examination reveals clusters of abnormal FV leukemia-like cells directly beneath the capsules of the spleens of mice in clinical remission.     

Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Summary Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90–100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90–100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90–100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.     

Spleen focus-forming virus: specific neutralization by antisera to certain gag gene-encoded proteins.

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.     

Inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and in vivo.

Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.     

Expression of a transformation-related protein (p53) in the malignant stage of Friend virus-induced diseases.

Stage 1 (pre-malignant) and stage 2 (malignant) cells derived from mice infected with Friend murine leukemia virus or polycythemia-inducing Friend virus complex were examined and compared for the expression of a transformation-related cellular protein, p53. Stage 2 cells were found to express high levels of p53, whereas stage 1 cells did not express detectable levels of this protein. These results indicate that p53 may be a marker for transformed cells present in the second stage of diseases induced by Friend murine leukemia virus or polycythemia-inducing Friend virus complex.