Multiple steps are required for the induction of tumors by Abelson murine leukemia virus.

Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.     

Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.     

NK activity in C57BL/Ka mice during the development of radiation-induced lymphomas

Treatment of C57BL/Ka mice with a split dose wholebody irradiation (four weekly irradiations of 1,75 Gy) induces the development of thymic lymphomas. NK activity of spleen cells has been determined at several intervals after leukemogenic treatment. Two days after irradiations. NK activity is normal and decreases strongly after one week. This period of decline persists during about one month. Then, NK activity restores and reaches control values. Lymphomas appear in spite of NK activity restoration. The diminution of NK activity during the preleukemic period could favour preleukemic cells apparition.     

Immunologic mechanisms in the pathogenesis of virus-induced murine leukemia. III. Target cell specificity of autoreactive thymocytes.

Thymocytes from preleukemic mice persistently infected with Moloney murine leukemia virus (MuLV-M-carriers) were vigorously autoaggressive toward normal syngeneic target cells; they exhibited a graded response to allogeneic cells, but they spared xenogeneic cells or syngeneic cells infected with MuLV-M or MuLV-G (Gross). Syngeneic target cells infected with nononcogenic lymphocytic choriomeningitis virus (LCMV), or transformed by the chemical carcinogen 3-methylcholanthrene were not similarly spared. This phenomenon, apparently induced by MuLV-M, is not associated with all persistent virus carrier states. Thymocytes from mice persistently infected with LCMV or with the lactic dehydrogenase virus (LDHV) failed to demonstrate an autoaggressive behavior. That transplantable lymphoma cells (derived from MuLV-M-carriers) were autoreactive in a pattern similar to thymocytes from preleukemic mice suggests a unique role for MuLV in the events leading from altered recognition of "self" to lymphoma.     

H-2I-linked control of immunological resistance to viral leukemogenesis as a response to preleukemic cells

The mechanism of resistance to leukemogenesis by two radiation leukemia virus variants, A-RadLV and D-RadLV, was investigated. Resistance to these viruses is linked toH-2I in both B10.S and C57BL/10 mice. The resistance of virus-infected mice to transplantation of syngeneic, A- or D-RadLV-induced lymphoma cells was similar to their resistance to leukemogenesis by the same viruses. This resistance could be transferred by lymphoid cells from immune donors to normal recipients, and it was specific for RadLV lymphomas. Virus-primed (responder x sensitive)F₁ hybrids rejected only resistant-type parental lymphoma cells. Hence, it appears thatH-2I-linked resistance to RadLV leukemogenesis is regulated byIr genes. Resistant mice immunized by A- or D-RadLV rejected syngeneic lymphoma cells, irrespective of whether they were sensitive or resistant to the RadLV variant used for the induction of the lymphoma cells. It follows that resistant and sensitive type lymphomas are antigenically similar for the effector mechanism, and that theIr genes may be expressed in the sensitization phase of the reaction. In virus-infected mice which are resistant to A- or D-RadLV we were able to demonstrate the presence of preleukemic lymphocytes. Normal mice could be immunized by these preleukemic cells against lymphoma challenge. These data are interpreted to suggest that mice havingH-2I-linked resistance to RadLV infection may be sensitized by their preleukemic cells, and that these preleukemic cells are then arrested in their development as a result of the immune response.     

H-21-linked control of immunological resistance to viral leukemogenesis as a response to preleukemic cells

The mechanism of resistance to leukemogenesis by two radiation leukemia virus variants, A-RadLV and D-RadLV, was investigated. Resistance to these viruses is linked to H-21 in both B10.S and C57BL/10 mice. The resistance of virus-infected mice to transplantation of syngeneic. A- or D-RadLV-induced lymphoma cells was similar to their resistance to leukemogenesis by the same viruses. This resistance could be transferred by lymphoid cells from immune donors to normal recipients, and it was specific for RadLV lymphomas. Virus-primed (responder x sensitive)F1 hybrids rejected only resistant-type parental lymphoma cells. Hence, it appears that H-21-linked resistance to RadLV leukemogenesis is regulated by Ir genes. Resistant mice immunized by A- or D-RadLV rejected syngeneic lymphoma cells, irrespective of whether they were sensitive or resistant to the RadLV variant used for the induction of the lymphoma cells. It follows that resistant and sensitive type lymphomas are antigenically similar for the effector mechanism, and that the Ir genes may be expressed in the sensitization phase of the reaction. In virus-infected mice which are resistant to A- or D-RadLV we were able to demonstrate the presence of preleukemic lymphocytes. Normal mice could be immunized by these preleukemic cells against lymphoma challenge. These data are interpreted to suggest that mice having H-21-linked resistance to RadLV infection may be sensitized by their preleukemic cells, and that these preleukemic cells are then arrested in their development as a result of the immune response.     

Induction of endoplasmic reticulum stress in thymic lymphocytes by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period

Infection of thymic lymphocytes by a mink cell focus-forming murine leukemia virus induces apoptosis during the preleukemic period of lymphomagenesis. In this study, we observed that during this period, the viral envelope precursor polyprotein accumulated to high levels in thymic lymphocytes from mice inoculated with virus. Envelope accumulation occurred with the same kinetics as the induction of endoplasmic reticulum (ER) stress, which resulted in the upregulation of the 78-kDa glucose-regulated protein (GRP78). In thymic lymphomas, GRP78 levels were higher than those in virus-infected preleukemic cells, and GRP58 was upregulated. These results suggest that Env precursor accumulation induces ER stress, which participates in thymic lymphocyte apoptosis. The subsequent upregulation of ER chaperone proteins GRP78 and GRP58 may contribute to rescuing cells from virus-induced apoptosis.     

The nucleolar apparatus of the lymphoid cells in AKR mice at different stages of leukemogenesis

Morphological features of nuclear apparatus in mice leukemic cells of the peripheral blood, lymph nodes, thymus, bone marrow and spleen were studied. These cells were taken from 12-months-old AKR mice. Enlarged percentage of cells with nucleolar lipid component in thymus characterizes preleukemic state for 6-months-old AKR line mice.     

N-acetyl-neuraminic acid (NANA) serum level in the diagnostics of preleukemic states, acute micromyeloblastic leukemias and pancytopenias.

The levels of N-acetyl-neuraminic acid were determined in patients with preleukemic states, acute micromyeloblastic leukemias and pancytopenias. A statistically significant increase of NANA was found in patients with micromyeloblastic leukemia in comparison with preleukemic states and pancytopenias. A significant rise in the NANA level was observed in preleukemic states in comparison with pancytopenia of other origins. The assay of the NANA level may be employed as a sensitive biochemical test for differential diagnostics of these diseases.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

A hypothesis: radiation-related leukemia is mainly attributable to the small number of people who carry pre-existing clonally expanded preleukemic cells

Human leukemia frequently involves recurrent translocations. Since radiation is a well-known inducer of both leukemia and chromosomal translocations, it has long been suspected that radiation might cause leukemia by inducing specific translocations. However, recent studies clearly indicate that spontaneous translocations specific to acute lymphocytic leukemia (ALL) actually occur much more frequently than do leukemia cases with the same translocations. Moreover, the ALL-associated translocation-bearing cells are often found to have clonally expanded in individuals who do not develop ALL. Since radiation-induced DNA damage is generated essentially randomly in the genome, it does not seem likely that radiation could ever be responsible for the induction of identical translocations of relevance to ALL in multiple cells of an individual and hence be the primary cause of radiation-related leukemia. An alternative hypothesis described here is that the radiation-related ALL risk for a population is almost entirely attributable to a small number of predisposed individuals in whom relatively large numbers of translocation-carrying pre-ALL cells have accumulated. This preleukemic clone hypothesis explains various known characteristics of radiation-related ALL and implies that people who do not have substantial numbers of preleukemic cells (i.e. the great majority) are likely at low risk of developing leukemia. The hypothesis can also be applied to chronic myelogenous leukemia and to young-at-exposure cases of acute myelogenous leukemia.     

Thymic epithelium controls thymocyte expression of preleukemic phenotype and leukemogenic retroviruses

Congenitally athymic AKR-streaker (nustr/nustr) mice were grafted separately with syngeneic or allogeneic, irradiated (1200 R) thymic reticuloepithelial (TRE) elements (stroma) or nonirradiated whole thymus grafts (control group) from N-tropic murine leukemia virus (MuLV) infection-susceptible (Fv-1n/n) or N-tropic-MuLV-infection-resistant (Fv-1b/n) murine strains. From 3 to 13 mo after grafting, the mononuclear cells repopulating the thymus grafts were stained with fluorescent monoclonal antibodies to thymocyte differentiation antigens, peanut agglutinin, and an antibody to MuLV antigens and were then analyzed by flow cytometry. Irradiated TRE of the Fv-1n/n genotype, whether from high or low leukemia-incidence strains, contained lymphoid cells of host (nustr/nustr) origin with alterations in thymocyte differentiation and MuLV antigen expression consistent with preleukemic changes. In contrast, transplanted TRE of the low leukemia-incidence Fv-1b/n genotype restricted preleukemic changes in thymocyte differentiation and MuLV antigen expression by lymphoid cells derived from the nustr/nustr host. Thus, nustr/nustr lymphocytes must infect susceptible TRE (Fv-1n/n) with N-tropic-MuLV before preleukemic changes occur in the mustr/nustr lymphocytes that later migrate to the thymus. Therefore, it was the radiation-resistant cells in the thymus that amplified or suppressed expression of AKR MCF retroviruses and the preleukemic phenotype, not the thymic lymphocytes. Thy-1.1+ splenocytes of ungrafted nustr/nustr mice were comparable in percentage to nustr/+ but were deficient in the Lyt-1+2- subpopulation and unresponsive to mitogens or alloantigens in vitro. Analysis of splenocyte cell surface markers, mitogen, MLC, and CML responses of Fv-1n/n-thymus-grafted nustr/nustr mice showed restoration of Lyt-1+2- cells to levels comparable to nustr/+ and reconstitution of in vitro proliferative and cytotoxic responses.     

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

Interaction of radiation-induced preleukemia cells and bone marrow grafts in C57BL/Ka mice

The repopulation of thymus inoculated with radiation-induced preleukemia cells was studied in 400 R irradiated mice grafted with normal bone marrow cells. These marrow cells gave rise to an actively regenerating thymic progeny, as well as in 400 R treated mice receiving only a bone marrow graft. Moreover, the marrow graft did not prevent the progression of inoculated preleukemic cells towards lymphoma growth.     

Genetic imbalances in preleukemic thymuses

To understand the molecular mechanisms involved in preleukemia, the suppression subtractive hybridization method was used in a murine radiation-induced thymic lymphoma model. Seventeen mRNAs overexpressed in preleukemic thymuses were identified: mouse laminin binding protein (p40/37LBP), E25 protein, Rattus norvegicus clone BB.1.4.1, profilin, poly(A) binding protein (PABP), mouse high mobility group protein 1, topoisomerase I, clusterin, proteasome RC1 subunit, rat prostatein C3 and C1 subunits; two ESTs and four unknown genes. The overexpression of PABP, clusterin, profilin, and the p40/37LBP mRNAs was confirmed in preleukemic thymuses and can be related to some cellular events observed during the preleukemic period, i.e., alterations of cell cycle and apoptosis properties. The p40/37LBP and 67-kDa laminin receptor proteins were upregulated during the preleukemic period. The data suggest that additional studies on p40/37LBP and 67-kDa laminin receptor regulation are required to evaluate their potential role in the lymphoma prevention by TNF-alpha and IFN-gamma.     

Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis.

We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.     

Sequences between the enhancer and promoter in the long terminal repeat affect murine leukemia virus pathogenicity and replication in the thymus

We previously showed that the 93-bp region between the enhancer and promoter (named DEN for downstream of enhancer) of the long terminal repeat (LTR) of the MCF13 murine leukemia virus is an important determinant of the ability of this virus to induce thymic lymphoma. In this study we observed that DEN plays a role in the regulation of virus replication in the thymus during the preleukemic period. A NF-kappaB site in the DEN region partially contributes to the effect of DEN on both lymphomagenicity and virus replication. To further study the effects of DEN and the NF-kappaB site on viral pathogenicity during the preleukemic period, we examined replication of wild-type and mutant viruses with a deletion of the NF-kappaB site or the entire DEN region in the thymus. Thymic lymphocytes which were infected with wild-type and mutant viruses were predominantly the CD3(-) CD4(+) CD8(+) and CD3(+) CD4(+) CD8(+) cells. The increase in infection by wild-type virus and both mutant viruses of these two subpopulations during the preleukemic period ranged from 9- to 84-fold, depending upon the time point and virus. The major difference between the wild-type and both mutant viruses was the lower rate and lower level of mutant virus replication in these thymic subpopulations. Significant differences in replication between wild-type and both mutant viruses were seen in the CD3(-) CD4(+) CD8(+) and CD3(-) CD4(-) CD8(-) subpopulations, suggesting that these thymic cell types are important targets for viral transformation.     

AKR leukemogenesis: identification and biological significance of thymic lymphoma receptors for AKR retroviruses.

We have previously demonstrated that in vitro cell lines of mouse thymic lymphomas express surface receptors specific for the retrovirus that induced them. This study extends these observations to an analysis of receptor-bearing cells in the preleukemic and leukemic phases of spontaneous AKR thymic lymphomagenesis. AKR mice regularly begin expressing N-tropic retroviruses (as assayed on NIH fibroblasts by the XC plaque assay) in several tissues early in life; thymic lymphocytes also express these viruses, but are not autonomously transformed. Later thymic lymphomas emerge which are capable of metastasizing in the host of origin or transplanting leukemias into syngeneic hosts. Just prior to the appearance of thymic lymphomas, these mice also begin producing xenotropic retroviruses [as assayed in xenogeneic (For example, mink) fibroblasts], and concomitant with the appearance of the leukemias is the appearance of "recombinant" retroviruses which cause mink fibroblast foci (MCF); these viruses express elements of both N- and X-tropic virus envelopes and N-tropic viral gene products in their cores. Spontaneous AKR leukemias also produce other retroviruses which do not cause XC plaques or mink fibroblast foci; these are called SL viruses. The subject of this study was to test whether in vivo thymocytes in the preleukemic and leukemic periods also bear receptors specific for N-tropic, recombinant MCF and SL AKR retroviruses. We demonstrated that each spontaneous thymic lymphoma does bear receptors that bind viruses produced by the lymphomas and MCF-247 to a high degree and that bind N-ecotropic AKR retroviruses less well. Thymic lymphocytes predominating in the preleukemic period do not express detectable levels of receptors for either of the viruses. In some mice, receptor-positive cells co-exist with receptor-negative cells; only the receptor-positive cells are capable of transplanting leukemia to syngeneic hosts. We conclude that the presence of specific cell surface receptors for lymphoma cell-produced and recombinant AKR retroviruses is a marker for leukemia in these hosts.     

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.     

Cellular changes in the thymuses of preleukemic AKR mice: correlation with changes in the expression of murine leukemia viruses.

Thymus cells of preleukemic and leukemic AKR mice express on their cell surface elevated levels of antigens associated with the murine leukemia virus (MuLV) proteins gp70 and p30. The gp70 antigenicity is contained in a 70,000 dalton polypeptide that corresponds to the viral envelope protein, while the p30 antigenicity is contained in two polypeptides of 85,000 and 95,000 daltons that correspond to glycosylated forms of the polyprotein product of the gag gene.The expression of these viral coded proteins on the cell surface of thymocytes varies both quantitatively with the age of the mouse and qualitatively with the cellular populations that express these antigens. Four discrete stages in the leukemic pathway can be identified. First, low numbers of cells from the thymuses of young (2 month old) AKR mice express p30 (<0.25%) and gp70 (2–7%) antigens. Expression of gp70 antigen is restricted to large cells in the subcapsular region of the thymus. Second, thymuses of 6 month old AKR mice show a selective depletion of cortical thymocytes with a concomitant increase in the medullary region of the thymus. Thymus cells of these mice contain elevated numbers of cells that express an increased concentration of p30 and gp70 antigens. Viral antigens are found on the surface of all large cells of the subcapsular region of the thymus, and in variable numbers (2–85%) of small cells of the cortical and medullary regions. Third, the thymuses of some 8 month old AKR mice demonstrate selective hypertrophy of a single thymic lobe. The enlarged lobe contains a population of cells that are intermediate in size between the small cortical cells and leukemic blast cells. This new cell population expresses elevated levels of p30 and gp70 viral antigens. These cells, which are not leukemic (since transfer of high numbers of these cells to syngeneic hosts does not induce transplantable disease), may represent preleukemic thymocytes. Fourth, thymuses of mice with overt leukemia contain primarily leukemic blast cells. These cells express extremely high levels of viral antigens on their cell surfaces, and upon transfer of these cells to syngeneic hosts, they rapidly induce transplantable leukemias.The increased expression of viral antigens on the surface of thymus cells is correlated with an increased production of infectious ecotropic and xenotropic MuLV in the thymus. During aging, the percentage of cells producing ecotropic MuLV increases 10-fold, while the percentage of cells producing xenotropic MuLV increases 100 fold.