Susan E. Bear

Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype.

The Gfi-1B Proto-Oncoprotein Represses p21WAF1 and Inhibits Myeloid Cell Differentiation

ABSTRACT Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21 WAF1 . Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at −1518 to −1530 in thep21WAF1 promoter. Forced expression ofGfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21 WAF1 and inhibited G1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of thep21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21 WAF1 induction by IL-6.

Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via a JmjC domain-dependent process

A common integration site, cloned from MoMuLV-induced rat T cell lymphomas, was mapped immediately upstream of Not dead yet-1 (Ndy1)/KDM2B, a gene expressed primarily in testis, spleen, and thymus, that is also known as FBXL10 or JHDM1B. Ndy1 encodes a nuclear, chromatin-associated protein that harbors Jumonji C (JmjC), CXXC, PHD, proline-rich, F-box, and leucine-rich repeat domains. Ndy1 and its homolog Ndy2/KDM2A (FBXL11 or JHDM1A), which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess Jumonji C-dependent histone H3 K36 dimethyl-demethylase or histone H3 K4 trimethyl-demethylase activities. Here, we show that mouse embryo fibroblasts engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process that targets the Rb and p53 pathways. Knockdown of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promote senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells and that inhibition of senescence depends on histone H3 demethylation.

Unequal Contribution of Akt Isoforms in the Double-Negative to Double-Positive Thymocyte Transition

Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.

The immune response of mice repeatedly injected with allogeneic tumor cells.

The development and kinetics of cell-mediated cytotoxicity (CMC), antibody-mediated complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) were studied in an allogeneic model. Using microcytotoxic assays of 51Cr release from labeled EL-4 tumor cells, CDC, ADCC, and CMC were measured at 14 intervals during the 77-day course of the experiment. The results obtained demonstrate the oscillating nature of the immune response. The rise and fall of activity was almost synchronous for the three functions studied. A generalized trend of increasing antibody-dependent functions and a simultaneous dampening of CMC was noted.

H-2-mediated resistance to an ecotropic type c retrovirus: localization of controlling genes and ontogenic studies of resistance.

H-2 recombinant mice of the C57BL/10 series were used to study resistance to infection by an ecotropic (B-tropic) type C retrovirus. The gene(s) responsible for resistance was mapped to theI- E/I-C subregions. Ontogenic studies indicated that thisI-region mediated resistance is not present in the neonate but develops with age, and is firmly established within 2 weeks after birth. This temporary neonatal susceptibility in mice of resistant genotype is not the result of active suppression of the immune response.

The role of effector cells and antiserum in the inhibition of cell-mediated cytotoxicity of allogeneic tumor cells.

SUMMARY Previous experiments in this laboratory demonstrated a progressive decrease in cell-mediated cytotoxicity (CMC) against allogeneic tumor cells by immune spleen cells from mice repeatedly immunized with those tumor cells. In the present study, immune spleen cells, obtained at specified intervals during the course of multiple immunizations of BALB/c mice with EL-4 lymphoma cells, were tested for CMC against EL-4 target cells pretreated with anti-EL-4 serum which had been obtained from singly or repeatedly immunized animals. Cytolysis of EL-4 cells was measured by a 51Cr-release assay. The results indicate that blocking of CMC in an allogeneic tumor model may occur by two pathways. First, antigen or antigen-antibody complexes present in the immunized animal may bind in vivo to the antigen receptor sites of sensitized effector cells that are used in the in vitro CMC assay, thereby blocking their interaction with tumor cells. Second, immune serum that is added to the in vitro CMC assay may contain highly avid antibodies, as well as antigen-antibody complexes, that bind to tumor cells and thereby block interaction with sensitized effector cells. The identification of these elements may be of prognostic significance in certain clinical situations.