Mutagenesis associated with nitric oxide production in transgenic SJL mice

We recently reported development of an experimental model for the study of nitric oxide (NO·) toxicology in vivo. SJL mice were injected with superantigen-bearing RcsX (pre-B-cell lymphoma) cells, which migrated to the spleen and lymph nodes, where their rapid growth induced activation of macrophages to produce large amounts of NO· over a period of several weeks. In the experiments described here, we used this model to investigate mutagenesis in splenocytes exposed to NO· during RcsX cell growth. Transgenic mice were produced by crossbreeding animals of the pUR288 transgenic C57BL/6 and SJL strains. RcsX cells were injected into F1 mice and NO· production was confirmed by quantification of urinary nitrate, the ultimate metabolite of NO·. Mutant frequency in the lacZ gene of the pUR288 plasmid was determined in DNA isolated from spleen (target) and kidney (nontarget) tissues. A significant elevation in mutant frequency was found in the spleen, but not in the kidney, of tumor-bearing mice. Furthermore, increases in mutant frequency in the spleen as well as NO· production were abrogated by administration of N-methylarginine, a NO· inhibitor, to mice following injection of RcsX cells. These results indicate that NO· had mutagenic activity in RcsX tumor-bearing mice and thus support a possible role for its involvement in the carcinogenic process.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

A human myeloma cell line suitable for the generation of human monoclonal antibodies

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine–aminopterin–thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein–Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-β1 (TGF-β1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-β production. Here, we examine the role of TGF-β in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-β production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-β does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-β1−/−, and Smad3−/− T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-β by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-β production and that CTLA-4-mediated inhibition can occur independently of TGF-β. Collectively, these data demonstrate that CTLA-4 and TGF-β represent distinct mechanisms for regulation of T cell responses.

Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Antiviral activities of the soluble extracellular domains of type I interferon receptors

Alternative splicing leads to the expression of multiple isoforms of the subunits (IFNAR1 and IFNAR2) of the type I IFN receptor. Here we describe two transcripts representing extracellular forms of ovine IFNAR1 and show that soluble extracellular forms of both IFNAR2 and IFNAR1, prepared in recombinant form in Escherichia coli, have antiviral (AV) activity in the absence of IFN. Exposure of Madin-Darby bovine kidney cells to the extracellular domain (R2E) of IFNAR2 at concentrations as low as 10 nM afforded complete protection against vesicular stomatitis virus and led to the rapid activation of the transcription factors ISGF3 and GAF. Although R2E can bind IFN (Kd ≈70 nM), activity was observed irrespective of whether or not ligand was present. R2E was inactive on mouse L929 cells but active on L929 cells expressing a membraneanchored, ovine/human chimeric IFNAR2 with an ovine extracellular domain. The data suggest that AV activity is conferred by the ability of soluble R2E to associate with the transfected IFNAR2 subunit rather than resident murine IFNAR1. Soluble extracellular forms of IFNAR1 have lower AV activity than R2E on Madin-Darby bovine kidney cells but are less species-specific and protect wild-type L929 cells as efficiently as the transfected cell line, presumably by interacting with one of the murine receptor subunits.

Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.

4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation.

Oncogenic potential of human mycoplasmas was studied using cultured mouse embryo cells, C3H/10T1/2 (C3H). Mycoplasma fermentans and Mycoplasma penetrans, mycoplasmas found in unusually high frequencies among patients with AIDS, were examined. Instead of acute transformation, a multistage process in promotion and progression of malignant cell transformation with long latency was noted; after 6 passages (1 wk per passage) of persistent infection with M. fermentans, C3H cells exhibited phenotypic changes with malignant characteristics that became progressively more prominent with further prolonged infection. Up to at least the 11th passage, all malignant changes were reversible if mycoplasmas were eradicated by antibiotic treatment. Further persistent infection with the mycoplasmas until 18 passages resulted in an irreversible form of transformation that included the ability to form tumors in animals and high soft agar cloning efficiency. Whereas chromosomal loss and translocational changes in C3H cells infected by either mycoplasma during the reversible stage were not prominent, the onset of the irreversible phase of transformation coincided with such karyotypic alteration. Genetic instability--i.e., prominent chromosomal alteration of permanently transformed cells--was most likely caused by mutation of a gene(s) responsible for fidelity of DNA replication or repair. Once induced, chromosomal alterations continued to accumulate both in cultured cells and in animals without the continued presence of the transforming microbes. Mycoplasma-mediated multistage oncogenesis exhibited here shares many characteristics found in the development of human cancer.

Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity.

In the tumor-bearing host, T cells invariably fail to induce a clinically significant antitumor immune response. Although model systems support the existence of tumor peptide antigens, the molecular interactions critical for antigen presentation by the tumor cell remain unresolved. Here, we demonstrate that human follicular lymphoma cells are highly inefficient at presenting alloantigen despite their strong expression of major histocompatibility complex and low-to-intermediate expression of some adhesion and B7 costimulatory molecules. Activation of follicular lymphoma cells via CD40 induces or up-regulates both adhesion and B7 costimulatory molecules essential to repair this defect. More importantly, once primed, alloreactive T cells efficiently recognize unstimulated follicular lymphoma cells. Thus, correction of defective tumor immunity requires not only expression of major histocompatibility complex but also sufficient expression of multiple adhesion and costimulatory molecules.

Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.

Minisatellite instability in severe combined immunodeficiency mouse cells

We have recently found that okadaic acid, which shows strong inhibitory activity on protein serine/threonine phosphatases and tumor-promoting activity in vivo and in vitro, induces minisatellite mutation (MSM). Human tumors and chemically induced counterparts in experimental animals are also sometimes associated with MSM. In the present study, we demonstrated minisatellite (MS) instability in severe combined immunodeficiency (SCID) cells in which the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is impaired. Cells from a SCID fibroblast cell line transformed by simian virus 40 large tumor antigen, SC3VA2, and from an embryonal SCID fibroblast cell line, SC1K, were cloned and propagated to 107 to 108 cells, and then subjected to subcloning. After propagation of each subclone to 107 to 108 cells, DNA samples were digested with HinfI and analyzed by Southern blotting using the Pc-1 MS sequence as a probe. Under low-stringency conditions, about 40 MS bands were detected, with 45% ± 6% and 37% ± 3% of SC3VA2 and SC1K cells, respectively, having MSM. In contrast, cells from the RD13B2 cell line, which was established from SCVA2 by introducing human chromosome 8q fragments, on which DNA-PKcs is known to reside, to complement the SCID phenotype, showed a very low frequency of MSM (3% ± 3%). The high frequencies of MSM in SC3VA2 and SC1K were significant, with no difference between the two. The present study clearly demonstrates that MS instability exists in SCID fibroblasts, suggesting that DNA-PKcs might be involved in the stable maintenance of MS sequences in the genome.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp+/+) ES cells and Parp+/− and Parp−/− ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp−/− clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp+/− clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp−/− clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.