Mary L. Bath

A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes

Members of the tumour necrosis factor receptor family that contain a death domain have pleiotropic activities. They induce apoptosis via interaction with intracellular FADD/MORT1 and trigger cell growth or differentiation via TRADD and TRAF molecules. The impact of FADD/MORT1‐transduced signals on T lymphocyte development was investigated in transgenic mice expressing a dominant negative mutant protein, FADD‐DN. Unexpectedly, FADD‐DN enhanced negative selection of self‐reactive thymic lymphocytes and inhibited T cell activation by increasing apoptosis. Thus signalling through FADD/MORT1 does not lead exclusively to cell death, but under certain circumstances can promote cell survival and proliferation.

Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.

Cyclin D1 is the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle. Deregulated cyclin D1 expression has been implicated in several human neoplasms, most consistently in centrocytic B lymphoma, where the cyclin D1 gene usually has been translocated to an immunoglobulin locus. To determine directly whether constitutive cyclin D1 expression is lymphomagenic, transgenic mice were generated having the cyclin D1 gene linked to an immunoglobulin enhancer. Despite abundant transgene expression, their lymphocytes were normal in cell cycle activity, size and mitogen responsiveness, but young transgenic animals contained fewer mature B‐ and T‐cells. Although spontaneous tumours were infrequent, lymphomagenesis was much more rapid in mice that co‐expressed the cyclin D1 transgene and a myc transgene than in mice expressing either transgene alone. Moreover, the spontaneous lymphomas of myc transgenic animals often ectopically expressed the endogenous cyclin D1 gene. These findings indicate that this G1 cyclin can modulate differentiation and collaborate with myc‐like genes in oncogenesis.

The Proapoptotic BH3-Only Protein Bim Is Expressed in Hematopoietic, Epithelial, Neuronal, and Germ Cells

Proapoptotic Bcl-2 family members activate cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. Bim belongs to a distinct subgroup of proapoptotic proteins that only resemble other Bcl-2 family members within the short BH3 domain. Gene targeting experiments in mice have shown that Bim is essential for the execution of some but not all apoptotic stimuli, for hematopoietic cell homeostasis, and as a barrier against autoimmunity. There are three Bim isoforms, Bim(S), Bim(L), and Bim(EL), which have different proapoptotic potencies due at least in part to differences in interaction with the dynein motor complex. The expression pattern of Bim was investigated by immunohistochemical staining, immunoprecipitation followed by Western blotting, and in situ hybridization. Bim was found in hematopoietic, epithelial, neuronal, and germ cells. Bim(L) and Bim(EL) were coexpressed at similar levels in many cell types, but Bim(S) was not detected. Microscopic examination revealed a punctate pattern of Bim(L) and Bim(EL) immunostaining, indicating association with cytoplasmic structures. These results are discussed in the context of the phenotype of Bim-deficient mice and the post-translational regulation of Bims pro-apoptotic activity.

Abnormalities of the immune system induced by dysregulated bcl-2 expression in transgenic mice.

Dysregulated expression of the putative cellular oncogene bcl-2 by chromosomal translocation has been strongly implicated in follicular center B cell lymphoma, one of the most common hematologic malignancies in humans. These tumors usually contain a 14;18 chromosomal translocation (Fukuhara et al 1979) that juxtaposes bcl-2 with the immunoglobulin heavy chain (Igh) locus (Tsujimoto et al 1984; Cleary and Sklar 1985; Bakshi et al 1985). The recombination presumably subjects bcl-2 to the control of Igh regulatory sequences which enforce its constitutive expression in B lymphoid cells. The bcl-2 gene encodes a 24 kD non-glycosylated protein located on the cytoplasmic face of the plasma membrane (Tsujimoto et al 1987; Chen-Levy et al 1989). It is normally expressed in pre-B cells, quiescent in resting B cells, expressed again in activated B cells and then downregulated upon their terminal differentiation (Reed et al 1987; Gurfinkel et al 1987; Chen-Levy et al 1989). The first indication of the function of this putative oncogene was revealed by our previous studies on interleukin-3-dependent cell lines infected with a bcl-2 retrovirus. The cells survived withdrawal of growth factor, but entered a Go state, suggesting that constitutive bcl-2 expression promotes cell survival rather than proliferation (Vaux et al 1988).

Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

Gata-3 Negatively Regulates the Tumor-Initiating Capacity of Mammary Luminal Progenitor Cells and Targets the Putative Tumor Suppressor Caspase-14

ABSTRACT The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of “luminal-like” cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v‐abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu‐v‐abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre‐B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c‐myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu‐myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v‐abl with c‐myc is stage specific, efficiently transforming plasma cells but not pre‐B cells or B cells.

Inhibition of In Vitro Fertilizing Capacity of Cryopreserved Mouse Sperm by Factors Released by Damaged Sperm, and Stimulation by Glutathione

Background In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide. Methodology/Principal Findings The proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells®) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6±2.4% versus 27.3%±2.8% in wells alone). On the assumption that the soluble factor could be H2O2, reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice. Conclusions/Significance IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.

Simple and Efficient In Vitro Fertilization with Cryopreserved C57BL/6J Mouse Sperm

Abstract Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%–12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%–88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%–63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.

A test of lymphoma induction by long-term exposure of Eμ-Pim1 transgenic mice to 50 Hz magnetic fields

E mu-Pim1 transgenic mice expressing a dysregulated Pim1 oncogene in their lymphoid cells were used to test whether exposure to 50 Hz magnetic fields can increase the frequency of malignant lymphoma in mice of a strain predisposed to develop such tumors spontaneously at low incidence. Specific-pathogen-free female mice were allocated randomly into groups of approximately 100 at 6-8 weeks of age and then exposed for 20 h/day for up to 18 months to sinusoidal magnetic fields of 0, 1, 100 or 1000 microT, or 1000 microT pulsed 15 min on and 15 min off. Additional E mu-Pim1 mice were injected with ethylnitrosourea (50 mg/kg body weight) as positive controls for enhanced lymphomagenesis; these yielded a cumulative incidence of lymphoma of 60% in 9 months. A lethal, transgene-dependent renal glomerular disease occurred at a frequency that varied from 9% to 19% among the groups, but the increase was statistically significant only at the 1000-microT exposure. Lymphoblastic and non-lymphoblastic (predominantly follicular) lymphomas were seen in 26 to 35% of the exposed mice, but at no significantly higher incidence than the 29% found in the sham-exposed mice. Hence we conclude that the lymphoma-prone mice did not reveal any tumorigenic effect of long-term exposure to 50 Hz magnetic fields.