Differential expression of subspecies of polyomavirus and murine leukemia virus enhancer core binding protein, PEBP2, in various hematopoietic cells.

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 fibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.     

Oncogene activation in human myeloid leukemia

We have studied by means of DNA-mediated gene transfer the activation of protooncogenes in human myeloid leukemias that represent various stages of myeloid differentiation. DNA from three cell lines, HL-60 (promyelocytic leukemia), Rc2a (myelomonocytic leukemia), and KG-1 (acute myeloblastic leukemia), was capable of transforming NIH/3T3 cells. Hybridization analysis indicated that, in all three tumor cell lines, the N-ras oncogene was activated. The cell lines U-937 ("histiocytic lymphoma") and K-562 (erythroblastic leukemia) yielded no transforming DNA. Fresh leukemia cells derived from an acute myelomonocytic leukemia patient and from a juvenile chronic myelogenous leukemia patient contained an activated N-ras and c-Ki-ras oncogene, respectively. DNA from some other myelogenous leukemia patients was not able to transform NIH/3T3 cells. Our results indicate that hematopoietic tumors of the myeloid lineage may contain oncogenes active in NIH/3T3 cell transformation and that, in particular, the N-ras oncogene may be activated in tumors representing various stages of maturation.     

Mechanism of restriction of murine leukemia viruses varies between different strains of Fv-1n mice.

The mechanism of Fv-1 restriction in DBA/2 (Fv-1ⁿ) mouse cells was investigated by quantitative infectious center assays employing a newly isolated continuous cell line. Titration of B-tropic murine leukemia virus (MuLV) in DBA/2 cells showed two-hit kinetics and, at sufficient multiplicities of infection (MOIs), the entire cell population could be productively infected with the restricted virus. Infection of DBA/2 cells with a single particle of B-tropic (but not with N- or NB-tropic) MuLV which had been inactivated at 43°C rendered the cells permissive to superinfection with B-tropic MuLV. This effect was time-dependent, the cells becoming permissive to B-tropic MuLV by 3 h after the initial infection and regaining their restriction within 18 h. These findings, which are similar to our previous results on the infection of BALB/3T3 (Fv-1^b) cells with N-tropic MuLV, indicate that two B-tropic MuLV particles are required for successful infection of a DBA/2 cell in this non-permissive system. These two particles appear to be functionally different: one temporarily overcomes or “abrogates” Fv-1 restriction but need not replicate, while the second apparently undergoes a normal replication cycle. Two other Fv-1ⁿ cell lines, NIH/3T3 and C3H 10T1/2, gave results which were not analogous to those obtained on DBA/2 cells. Even at high MOIs, only about 10–20% of NIH/3T3 or C3H 10T1/2 cells could be infected with B-tropic MuLV. These quantitative differences in titration patterns indicate that NIH/3T3 and C3H 10T1/2 cells have different mechanisms of Fv-1ⁿ restriction from that of DBA/2 cells, or, more likely, that they possess other restriction systems in addition to Fv-1.     

Retroviral src gene expression in continuous marrow culture increases the self-renewal capacity of multilineage hematopoietic stem cells

To define the action of the retroviral src gene on hematopoietic stem cells, C57BL/6 x DBA/2 (B6D2F1) mouse long-term marrow cultures were infected at initiation with Moloney murine leukemia virus (MuLV) pseudotypes of src-recombinant retroviruses with the src gene inserted in the env region of an amphotropic MuLV (src-Ampho), or in the gag region of Moloney MuLV (src-Mo). Other cultures were infected with Friend spleen focus-forming virus polycythemia-inducing strain (SFFVp), Moloney MuLV, or amphotropic MuLV, or were uninfected controls. Harvested nonadherent cells were tested weekly for multilineage, granulocyte-erythroid-megakaryocyte macrophage (CFU-GEMM) colony formation in vitro in recombinant murine IL-3 and erythropoietin, and individual colonies were removed, split 1:2, with half of each replated for in vitro self-renewal and the other half examined morphologically for number of hematopoietic cellular lineages, or tested for release of MuLV and src virus. Cultures infected with src-Ampho, src-Mo, or SFFVp demonstrated a significant increase in cumulative nonadherent cell and CFU-GEMM production. There was prolonged self-renewal over seven serial transfers of individual CFU-GEMM from src virus-infected cultures over seven serial transfers, and five of 61 individual colonies from the second or third generations contained detectable v-src gene sequences, but none released detectable src virus. Self-renewal of CFU-GEMM was similar to that with permanent IL-3-dependent cell line B6SUtA. In contrast, MuLV-infected or control uninfected cultures produced fewer cells, and self-renewal of CFU-GEMM did not exceed three generations. IL-3-dependent clonal hematopoietic progenitor cell lines, derived from each culture group, formed no detectable tumors in vivo; however, each released the original helper and/or transforming virus. Adherent cell lines, derived from src-Ampho-infected cultures released src virus and formed fibro-sarcomas in vivo. The data support the conclusion that src-recombinant virus expression in long-term marrow cultures increases the self-renewal capacity of multilineage hematopoietic stem cells.     

B-tropic oncornavirus production by BALB/c methylcholanthrene-induced sarcoma cells.

Six oncogenic cell lines were established from primary sarcomas that had been induced in thymectomized, aged, BALB/cSt mice by the injection of 3-methylcholanthrene. Of these lines, three produced B-tropic murine leukemia virus (MuLV), i.e. MuLV that would patently infect fibroblasts of BALB/c origin but not those from NIH Swiss mice. One of these lines, M-138, produced large quantities of virus, permitting the isolation of milligram quantities of B-tropic MuLV. Neonatal BALB/c mice injected with the M-138 MuLV developed high plasma levels of the species-specific virion antigen p30, but during 9 months of observation failed to manifest clinical signs of leukemia. Plasma p30 levels of NIH Swiss mice (non-permissive host) that were inoculated also as neonates with the same virus preparation, remained undetectable by radioimmunoassay. Xenotropic MuLV, i.e. that which infects cells of species other than the species of origin, was released by one of the three remaining cell lines. Two of the six original lines have remained free of detectable oncornavirus production. Our results suggest that cell lines that are stable sources of large quantities of B-tropic MuLV may be easily obtainable by the described manipulations. Equally important, however, is the fact that these data re-emphasize the danger that is inherent in assuming that a cell line is virus-free simply because it was isolated from a chemical carcinogen-induced neoplasm     

Genetic heterogeneity in acute myeloid leukemia: maximizing information flow from MuLV mutagenesis studies.

The study of myeloid leukemia induced by slow transforming murine leukemia viruses (MuLV) in the laboratory mouse has led to discovery of many important genes with critical roles in regulating the growth, death, lineage determination and development of hematopoietic precursor cells. This review provides an overview of the susceptible strains and virus isolates that cause acute myeloid leukemia (AML) in mice. In addition, newer methodologies, involving the use of the polymerase chain reaction, that have been used to identify cancer genes mutated by proviral insertion in mouse models, will be discussed. As cancer is a multi-gene disease, a system in which pairs of oncogenic mutations are classified as redundant, neutral or synergistic is described. The potential to combine MuLV mutagenesis with recent advances in mouse transgenesis in order to model specific forms of myeloid leukemia or genetic pathways common in human AML will be discussed. Finally, a general strategy for maximizing these genetically rich models to foster a better understanding of AML physiology and developing therapies is proposed.     

Properties of flat variants of murine sarcoma virus transformed non-producer cells isolated after high-temperature passage

High-temperature passage (40.5°C) increased the incidence of flat variants obtained from Kirsten sarcoma virus transformed BALB/3T3 non-producer cells from less than 0.1% to 4%. The flat variants had growth properties similar to those of the BALB/3T3 line. The variant lines did not release detectable type C virus, but some contained intracisternal type-A particles. Super-infection of the variants with murine leukemia virus (MuLV) resulted in retransformation and release of sarcoma virus. Some of the variant lines may be suitable indicator cells for the assay of MuLV. Modal chromosome numbers of the variant lines were considerably higher than those of their progenitor cell line.     

Functional Consequences of APO-1/Fas (CD95) Antigen Expression by Normal and Neoplastic Hematopoietic Cells

Murine monoclonal antibody (mAb) 7C11 binds to the same cell surface epitope as anti-APO-1 and anti-Fas and reacts specifically with cells transfected with a cDNA encoding the human Fas antigen. Furthermore, incubation with 7C11 causes death of hematopoietic cell lines that express APO-1/Fas but not APO-1/Fas-negative cell lines. 7C11 therefore recognizes the human APO- 1/Fas (CD95) antigen, a 40 to 50 kDa cell surface glycoprotein that can trigger apoptosis or programmed cell death. Expression of APO- 1/Fas antigen by normal and neoplastic hematopoietic cells was determined by flow cytometry using 7C11. APO-1/Fas is expressed by 30 to 40% of resting peripheral blood T cells, B cells, and monocytes and by -5% of resting NK cells and thymocytes. It was not detected on granulocytes, erythrocytes, or platelets. Approximately 80 to 90% of activated T cells, B cells, and thymocytes express APO-1/Fas, as do the majority of activated NK cells. Perturbation of APO-1/Fas by 7C11 does not affect the viability of resting lymphocytes or monocytes. In contrast, activated T cells and NK cells undergo apoptosis within 3 hours of exposure to 7C11. Other mAb that stimulate T cells or NK cells do not cause rapid induction of programmed cell death. APO-1/Fas antigen is expressed by many cell lines of lymphoid and myeloid lineage. However, this antigen was detected on neoplastic cells from only one of 69 patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or multiple myeloma. Only 3 out of 25 tumor samples from patients with non-Hodgkin's lymphoma were found to express APO-1/Fas. All three of these lymphomas harbored the bcl-2-Ig fusion gene associated with the chromosomal translocation t (14; 18). Conversely, only 27% of lymphomas that possessed the bcl-2-Ig gene were found to express the APO-1/Fas antigen. Like normal activated lymphocytes, leukemia and lymphoma cells that expressed APO-1/Fas antigen were found to undergo apoptosis in vitro after incubation with 7C11. The APO-1/Fas antigen appears to regulate the growth of normal hematopoietic cells, and the marked upregulation of this antigen on activated normal lymphocytes contrasts sharply with the absence of APO- 1/Fas on neoplastic cells of hematopoietic lineage. Defects in the apoptotic signal delivered through this antigen might contribute to the pathogenesis of hematopoietic neoplasms. Thus, the gene encoding APO- 1/Fas can be considered a novel type of tumor suppressor gene, just as bcl-2 can be considered a cellular proto-oncogene.     

Phenotypic attributes of the malignant cell population in Hodgkin's disease indicate a monocyte/macrophage origin

The cellular origin of the malignant cells in Hodgkin's disease (HD) has been discussed for several decades. Previous investigations on fresh biopsy material and cultured cells from lesions of Hodgkin's disease have led to various suggestions such as a T lymphocyte, B lymphocyte or monocyte/macrophage origin of HD cells. However, all these studies have been hampered by uncertainty in the identification of the truly malignant cells. It has therefore been desirable to establish in vitro lines of the malignant cell population in HD. A number of cell lines from HD lesions has been described, but most of these studies can, upon a critical appraisal, be dismissed as not being representative for Hodgkin's and Reed-Sternberg cells. We report on the phenotypes of established cell lines of Hodgkin's cells and conclude that it is most likely that the malignant Hodgkin's cells are derived from cells of the monocyte/macrophage lineage.     

The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation

The AGR2 gene encodes a secretory protein that is highly expressed in adenocarcinomas of the esophagus, pancreas, breast, and prostate. This study explores the effect of AGR2 expression with well-established in vitro and in vivo assays that screen for cellular transformation and tumor growth. AGR2 expression in SEG-1 esophageal adenocarcinoma cells was reduced with RNA interference. Cellular transformation was examined using NIH3T3 cells that express AGR2 after stable transfection. The cell lines were studied in vitro with assays for density-dependent and anchorage-independent growth, and in vivo as tumor xenografts in nude mice. SEG-1 cells with reduced AGR2 expression showed an 82% decrease in anchorage-independent colony growth and a 60% reduction in tumor xenograft size. In vitro assays of AGR2-expressing NIH3T3 cells displayed enhanced foci formation and anchorage-independent growth. In vivo, AGR2-expressing NIH3T3 cells established tumors in nude mice. Thus, AGR2 expression promotes tumor growth in esophageal adenocarcinoma cells and is able to transform NIH3T3 cells. Immunohistochemistry of the normal mouse intestine detected AGR2 expression in proliferating and differentiated intestinal cells of secretory lineage. AGR2 may be important for the growth and development of the intestine as well as esophageal adenocarcinomas.     

Induction of B-tropic and N-tropic murine leukemia virus from B10.BR/SgLi mouse embryo cell lines by 5-iodo-2'-deoxyuridine.

In contrast to the original B10.BR/SgSn congenic mouse strain, adult mice of the B10.BR/SgLi subline showed a high level of expression of B-tropic ecotropic murine leukemia virus (MuLV). Both B-tropic and N-tropic ecotropic MuLV could be included in cultures of virus-free cell lines derived from embryos of B10.BR/SgLi mice. Both various were also inducible from each of several clonal cell lines and from cultures of F1 embryos of matings of B10.BR/SgLi males with females of strains NFS/N and A/J, which are negative for B-tropic virus. Thus the information for B-tropic MuLV as well as that for N-tropic MuLV was transmitted as a genetic element in the B10.BR/SgLi subline.     

Introduction of v-abl oncogene induces monocytic differentiation of an IL-3-dependent myeloid progenitor cell line

There are a variety of murine hematopoietic progenitor cell lines which are differentiation arrested, but still require growth factors such as interleukin-3 for their continued growth and survival. While oncogenes such as v-myc and v-abl have been demonstrated to abrogate the requirement for exogenous growth factors, none have been shown to have an effect on the differentiation of these cell lines. In this report, we demonstrate that the introduction and expression of Abelson murine leukemia virus into a myeloblast progenitor cell line can promote further differentiation along the monocytic lineage. There is a marked alteration in cell morphology, the acquisition of Mac-1 antigen expression, the induction of nonspecific esterase expression and the induced ability to phagocytize opsonized zymosan. Thus, the expression of Abelson murine leukemia virus protein in interleukin-3-dependent hematopoietic progenitors can provide differentiation-inducing signals in cells which are arrested in differentiation. The potential role of Abelson murine leukemia virus gene products in normal hematopoietic cell differentiation and in transformation is discussed.     

Effect of X-irradiation dose rate on the clonagenic survival of human and experimental animal hematopoietic tumor cell lines: evidence for heterogeneity

It is a generally accepted principle of radiation biology that hematopoietic progenitor cells demonstrate dose rate independent killing by x-irradiation over the clinically relevant range for total body irradiation (TBI) (5-25 rad/min). To determine whether low dose rate (5 rad/min, or 20 rad/min) compared to conventional dose rate (200 rad/min) x-irradiation altered the clonagenic survival of leukemia and lymphoma cell lines, several permanent cell lines were studied. These included: bg/bg cl 1, mouse basophillic leukemia; LW12, [W/fu rat acute myelogenous leukemia (AML)]; and human cell lines: JY and Daudi (B-cell lymphomas); K45, (T-cell leukemia); K562, (erythroleukemia); HL60 and KG1 (monomyeloid leukemias), and U937 (human histiocytic/monocytic lymphoma). Dose rate independent killing was demonstrated at several plating densities with mouse and rat leukemia lines and all human leukemia lines tested except lines HL60 and U937. With HL60, increased plating density increased the D0 at each dose rate. This effect was not attributable to an increased plating efficiency. With line U937 there was a clear dose-rate effect with increase in D0 from 88 rad, n 4.6 at 200 rad/min, to D0 = 166, n 2.3 at 5 rad/min. The data demonstrate that some human hematopoietic tumor derived cell lines of myeloid/monocyte/macrophage lineage can exhibit atypical repair of irradiation damage in vitro. This repair may be enhanced by conditions relevant to clinical TBI including low irradiation dose-rate and cell to cell interactions by tumor cells in close proximity.     

Retroviruses in radiation-induced lymphomas

The nucleotide sequence of RadLV/VL3 (T+L+), the thymotropic and leukemogenic entity of the in-vitro propagated radiation leukemia virus complex (RadLV/VL3), is that of a recombinant retrovirus. The gag, pol and most of the env gene are very similar to the homologous regions of Akv MuLV. The 3' end of the env gene and the LTR appear to have derived from a xenotropic MuLV. However, the LTR has acquired a feature shared by other lymphomagenic MuLVs. This feature consists in sequence rearrangements resulting in the generation of presumed enhancer elements. RadLV/VL3(T+L+)-specific proviral sequences were found adjacent to the c-myc gene in several virus-induced thymic lymphomas of the rat, suggesting that the enhancer elements might play a role in lymphomagenesis. However, we found that the presence of a provirus at a specific DNA site can lead to an in-vitro growth advantage and to clonal cell selection independently of a lymphomagenic process. We conclude from this observation that clonal appearance of an integrated provirus in cultured radiogenic lymphoma cells does not necessarily reflect a viral induction of radiation-induced leukemogenesis.     

Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens

Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.     

A tumor promoting phorbol ester, TPA, enhances polyomavirus DNA replication by activating the function of the viral enhancer

The effect of tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) on enhancer dependent polyomavirus (Py) DNA replication was examined in cells expressing Py large T antigen. The results showed that TPA enhanced Py DNA replication by stimulating the activity of the A element, one of the two cores of Py enhancer. Of the three subdomains of the A element, the biding sites of PEBP1 (PEA1), a member of AP1 family, and of PEBP5 were by themselves able to activate Py DNA replication. Furthermore, each binding site of PEBP1 and PEBP5 responded to TPA to enhance Py DNA replication. The results suggest that growth promoting signals could activate DNA replication directly via enhancer binding proteins.     

Efficient induction of focus formation in a subclone of NIH3T3 cells by c-myc and its inhibition by serum and by growth factors

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested.