Alan W. Harris

bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship

Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO‐1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl‐2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO‐1‐transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO‐1‐transduced apoptosis, but did not affect cell death induced by gamma‐radiation or serum deprivation. Signalling through Fas/APO‐1 did not down‐regulate Bcl‐2 or induce its antagonists Bax and Bcl‐xS. In Fas/APO‐1‐deficient lpr mice, Bcl‐2 transgenes markedly augmented the survival of antigen‐activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto‐reactive cells in the thymus). These data raise the possibility that Bcl‐2 and Fas/APO‐1 regulate distinct pathways to lymphocyte apoptosis.

DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2

The roles of p53 as an inducer and Bcl-2 as an inhibitor of apoptotic death were explored in lymphoid cells. Lymphocytes from p53-/- mice were radioresistant, but unexpectedly, cycling T lymphoma cells and mitogenically activated T lymphocytes from these animals underwent apoptosis after irradiation or genotoxic drug treatment. Hence, p53 is not the only mediator of apoptosis provoked by DNA damage. Irradiated p53-/- lymphoblasts expressing Bcl-2 were subject to growth arrest but resisted apoptosis. Their accumulation in G1 as well as G2 is suggestive of a p53-independent DNA-damage G1 checkpoint. Since Bcl-2 increased the clonogenic survival of the irradiated cells, expression of survival genes may pose a greater impediment to genotoxic cancer therapy than loss of p53.

Elimination of self-reactive B lymphocytes proceeds in two stages: Arrested development and cell death

In transgenic mice, self-reactive B lymphocytes are eliminated if they encounter membrane-bound self antigens during their development within the bone marrow. We show here that two separate and sequential events, arrested development and cell death, bring about B cell elimination. Developmental arrest is an early outcome of antigen binding in immature B cells, blocks acquisition of adhesion molecules and receptors important for B cell migration and activation, and is rapidly reversible by removal of antigen. Death of the arrested B cells occurs within 1 to 3 days and can be delayed by expression of a bcl-2 transgene, which results in escape of large numbers of self-reactive B cells from the bone marrow but fails to override the developmental arrest. These findings define a novel pathway for B cell elimination, involving an initial stage vulnerable to breakdown in autoimmune disease.

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.

Lymphomas in Eμ-Pim1 transgenic mice exposed to pulsed 900 MHz electromagnetic fields

Whether radiofrequency (RF) fields are carcinogenic is controversial; epidemiological data have been inconclusive and animal tests limited. The aim of the present study was to determine whether long-term exposure to pulse-modulated RF fields similar to those used in digital mobile telecommunications would increase the incidence of lymphoma in E mu-Pim1 transgenic mice, which are moderately predisposed to develop lymphoma spontaneously. One hundred female E mu-Pim1 mice were sham-exposed and 101 were exposed for two 30-min periods per day for up to 18 months to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms. Incident power densities were 2.6-13 W/m2 and specific absorption rates were 0.008-4.2 W/kg, averaging 0.13-1.4 W/kg. Lymphoma risk was found to be significantly higher in the exposed mice than in the controls (OR = 2.4. P = 0.006, 95% CI = 1.3-4.5). Follicular lymphomas were the major contributor to the increased tumor incidence. Thus long-term intermittent exposure to RF fields can enhance the probability that mice carrying a lymphomagenic oncogene will develop lymphomas. We suggest that such genetically cancer-prone mice provide an experimental system for more detailed assessment of dose-response relationships for risk of cancer after RF-field exposure.

The c-myc oncogene perturbs B lymphocyte development in Eμ-myc transgenic mice

Transgenic mice bearing a c-myc oncogene subjugated to the lymphoid-specific immunoglobulin heavy chain enhancer (E mu) develop clonal B lymphoid malignancies, but most young E mu-myc mice lack malignant clones. Their prelymphomatous state has allowed us to examine how constitutive c-myc expression influences B cell development. We find that early stages are overrepresented, even before birth. Pre-B cells of polyclonal origin increase greatly, while B cells develop in reduced number. Both the pre-B and the B cells appear to be in an active state, since they are larger than normal and a greater fraction are in the cell cycle. Enforced myc expression has thus favored proliferation over maturation. Hence, a normal function of c-myc may be to regulate differentiation as well as to promote cell cycling.

FADD/MORT1 regulates the pre‐TCR checkpoint and can function as a tumour suppressor

Productive rearrangement of the T‐cell receptor (TCR) β gene and signalling through the pre‐TCR–CD3 complex are required for survival, proliferation and differentiation of T‐cell progenitors (pro‐T cells). Here we identify a role for death receptor signalling in early T‐cell development using a dominant‐negative mutant of the death receptor signal transducer FADD/MORT1 (FADD‐DN). In rag‐1−/− thymocytes, which are defective in antigen receptor gene rearrangement, FADD‐DN bypassed the requirement for pre‐TCR signalling, promoting pro‐T‐cell survival and differentiation to the more mature pre‐T stage. Surprisingly, differentiation was not accompanied by the proliferation that occurs normally during transition to the pre‐T stage. Consistent with a role for FADD/MORT1 in this cell division, FADD‐DN rag‐1−/− pro‐T cells failed to proliferate in response to CD3ϵ ligation. Concomitant signalling through the pre‐TCR and death receptors appears to trigger pro‐T cell survival, proliferation and differentiation, whereas death receptor signalling in thymocytes that lack a pre‐TCR induces apoptosis. Later in life all FADD‐DN rag‐1−/− mice developed thymic lymphoma, indicating that FADD/MORT1 can act as a tumour suppressor.

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).