Bcl-2 Transgene Research Articles

The tyrosine kinase Lyn is required for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2.

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.

Detection of BCL2 Rearrangements in Follicular Lymphoma

The identification and characterization of recurrent chromosomal translocations has improved tumor classification and yielded numerous insights into tumor pathogenesis, as such events typically lead to the dysregulation of proteins that control critical cellular processes, such as apoptosis, proliferation, differentiation, and immortalization. A prototypical example of such a chromosomal translocation is the (14;18)(q32;q32), which is highly associated with the B cell neoplasm follicular lymphoma. 1 Molecular analyses of DNA isolated from follicular lymphoma cells in the mid-1980’s showed that the t(14;18) creates a derivative chromosome 14 on which the BCL2 gene is juxtaposed to immunoglobulin heavy chain gene (IgH) sequences, 2-5 including enhancer sequences that override normal BCL2 gene control elements and drive inappropriately high levels of BCL2 expression in follicle center B cells. 6,7 Subsequently, several groups noted that enforced expression of BCL2 transgenes in murine hematopoietic cells promoted cell survival, 8,9 the first indication of the anti-apoptotic activity of BCL2. BCL2 transgenic mice also showed increased susceptibility to autoimmune disease 10 and B cell lymphoma, 11 observations that helped to foster the now generally accepted idea that dysregulation of apoptotic pathways is important in the pathogenesis of many forms of cancer and autoimmune disorders. These effects of BCL2 appear to be mediated through sequestration of “BH3 domain only” pro-apoptotic proteins such as BAD, BIM, and NOXA, 12 which resets the apoptotic “rheostat” toward increased resistance to programmed cell death. In addition to its impact on our thinking about cancer pathophysiology, the molecular characterization of the t(14;18) and its downstream consequences has influenced our approach to the diagnosis of hematological malignancy in several ways. The t(14;18) was an early example of an acquired genetic lesion that was strongly associated with a particular neoplasm, and thus of utility in tumor classification. Juxtaposed BCL2 and IgH DNA sequences provide a tumor-specific marker that can be exploited in the detection of minimal residual disease. 13,14 Furthermore, because BCL2 is down-regulated in normal germinal center B cells, 15 its expression (or lack thereof) can help to distinguish reactive and neoplastic follicles. 16 Finally, the presence of the t(14;18) has been suggested to be a marker of poor prognosis in diffuse large B cell lymphoma 17,18 (possibly because some such tumors arise through transformation of unrecognized underlying follicular lymphomas), although it must be said that more recent studies have not detected such a correlation. 19-21 These relationships have prompted the development of multiple methods that aim to detect the t(14;18) in tissue specimens. In this issue of The American Journal of Pathology, Albinger-Hegyi et al 22 report a new set of polymerase chain reaction (PCR)-based tests for detection of the BCL2/IgH fusion genes that offer some significant advantages in sensitivity over other more commonly used PCR-based methods. This commentary will attempt to place this contribution in context by discussing the advantages and limitations of various tests that aim to detect the t(14;18) or its downstream consequences.

The expression of a new variant of the pro-apoptotic molecule Bax, Baxpsi, is correlated with an increased survival of glioblastoma multiforme patients.

Pro- and anti-apoptotic members of the BCL-2 family play a central role in the implementation of apoptosis. Bax, a pro-apoptotic member of this family, has as such been considered as a potential tumor suppressor. Here, we have examined the expression of Bax in 55 patients with glioblastoma multiforme (GBM), the most common and aggressive form of brain tumors. We report on the existence of a new form of Bax, present in 24% of the patients, which we called Baxpsi. Baxpsi is a N-terminal truncated form of Bax which results from a partial deletion of the exon 1 of Bax gene. Baxpsi and the wild-type form, Baxalpha, are encoded by distinct mRNAs, both of which are present in normal tissues. Glial tumors express either Baxalpha or Baxpsi proteins, an apparent consequence of an exclusive transcription of the corresponding mRNAs. The latter feature could be partially linked to distinct methylation profiles of Bax gene in these tumors. The Baxpsi protein is preferentially localized to mitochondria and is a more powerful inducer of apoptosis than Baxalpha. Baxpsi tumors exhibit a slow proliferation in Swiss nude mice and this feature can be circumvented by the co-expression of the Bcl-2 transgene, the functional antagonist of Bax. More importantly, the expression of Baxpsi correlates with a longer survival in patients (18 months versus 10 months for Baxalpha patients). Thus, our results provide the first indication of a beneficial involvement of a variant of the pro-apoptotic protein Bax in tumor progression.

Interleukin-2-mediated Survival and Proliferative Signals are Uncoupled in T Lymphocytes that Fail to Divide after Activation

T lymphocytes are heterogeneous with respect to their ability to proliferate following activation in vitro and in vivo. Approximately 30% of T lymphocytes fail to progress through the cell cycle, despite showing evidence of an activated state. The population of T lymphocytes that remains undivided during a primary stimulation has been shown to be refractory to restimulation via the TCR and fails to proliferate in response to IL-2. In an in vitro model of T-cell deletion following clonal expansion, we demonstrate that T lymphocytes that do not progress through the cell cycle during primary stimulation have a sevenfold greater survival advantage compared with T lymphocytes that have divided. Progression through multiple division cycles is associated with down-regulation of Bcl-2 during a postactivation period of growth factor withdrawal. However this alone does not account for diminished survival, as constitutive expression of a Bcl-2 transgene did not restore survival to the levels seen in undivided cells. Engagement of the IL-2 receptor on these undivided activated T lymphocytes leads to enhanced survival and up-regulation of Bcl-2 and Bcl-xL. Surprisingly, while IL-2 also induces phosphorylation of Akt, it does not initiate cell cycle progression in this population of primary undivided cells. Our data provide evidence that a T cell's survival capacity is linked to its proliferative behavior. Furthermore, our results provide the first report of a population of T cells, in which the IL-2 receptor-mediated signaling pathways leading to survival and proliferation are naturally uncoupled.

Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic bcl-2 expression.

Independent of apoptosis, dexamethasone induced and a decrease of respiration and citrate synthase activity per cell in cells with and without transgenic Bcl-2 expression. The reduction of respiration, however, was slightly, but statistically more pronounced in apoptotic cells compared to non-apoptotic Bcl-2 over-expressing cells. A slight cytochrome c release was detected in apoptotic cells only. Importantly, the stimulatory effect of FCCP was maintained, indicating that oxidative phosphorylation remained coupled in active mitochondria. Coupled and uncoupled respiration were reduced to almost identical degrees as the activities of the marker enzymes citrate synthase (matrix) and cytochrome c oxidase (respiratory chain). Therefore, the reduction of cellular respiration was mainly caused by a decrease in mitochondrial content per cell. The functional integrity of mitochondria was preserved, apart from the slight degree of cytochrome c release, either through a pore formed by the oligomerisation of BAK in coupled mitochondria or by permeability transition of a small fraction of injured mitochondria.

Xid mice reveal the interplay of homeostasis and Bruton's tyrosine kinase-mediated selection at multiple stages of B cell development

Human X-linked agammaglobulinemia (XLA) and murine X-linked immune defect (XID) are both immunodeficiencies mediated by mutations in Bruton's tyrosine kinase (Btk), yet the developmental stage(s) affected remain controversial. To further refine the placement of the XID defect(s), we used bromodeoxyuridine labeling to determine turnover, production and transition rates of developing B cell subsets in normal, xid and xid mice expressing a human Bcl-2 transgene (xid/bcl-2). We find the xid mutation manifest at two stages of B cell development. The first is early, reducing pre-B cell production by restricting pro-B to pre-B cell transit. Surprisingly, this impairment is offset by increased survival of cells progressing from the pre- to immature B cell pool, suggesting that Btk-independent homeostatic mechanisms act to maintain this compartment. The second point of action is late, substantially reducing mature B cell production. Together, these findings reconcile apparent discrepancies in the developmental stage affected by the murine versus human lesions and suggest previously unappreciated homeostatic processes that act at the pre-B to immature B cell transition. Finally, Btk likely functions differently at these two checkpoints, since ectopic Bcl-2 expression fails to directly complement the early xid lesion, yet reverses the defect impeding final B cell maturation.

Transgenic Bcl-2 Expressed in Photoreceptor Cells Confers Both Death-sparing and Death-inducing Effects

To examine its potential role within the retina as a modulator of cell death and photoreceptor degeneration, bcl-2 expression was targeted to the photoreceptors of transgenic mice by the human IRBP promoter. Three transgenic families were established, with levels of transgene expression between 0.2 and two-fold relative to that of endogenous bcl-2. The effect of bcl-2 expression on genetically programmed photoreceptor degeneration was evaluated by crossing these transgenic mice with mice that develop a rapid degeneration of rod photoreceptors due to expression of a distinct transgene, SV40 T antigen (Tag). Transgenic Bcl-2 was localized to photoreceptor inner segments and was capable of abrogating the activation of caspase activity and the resulting cell death associated with ectopic expression of Tag. However, Bcl-2 itself ultimately caused photoreceptor cell death and retinal degeneration. Several proteins not expressed normally in Tag or other transgenic retinas undergoing photoreceptor degeneration were induced in the Bcl-2 transgenic retinas. Analysis by mass spectroscopy identified one of these proteins as αA-crystallin, a member of a protein family that associates with cellular stress. Since Bcl-2 can promote as well as spare cell death in the same photoreceptor population, its potential utility in ameliorating photoreceptor death in human hereditary blinding disorders is compromised.

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

Differential roles of cytokine receptors in the development of epidermal gamma delta T cells.

IL-7 and IL-15 play important roles in gammadelta T cell development. These receptors transmit proliferation and/or survival signals in gammadelta T cells. In addition, the IL-7R promotes recombination and transcription in the TCR gamma locus. To clarify the role of the cytokine receptors in the development of epidermal gammadelta T cells, we introduced a Vgamma3/Vdelta1 TCR transgene, derived from Thy-1+ dendritic epidermal T cells (DETC), into IL-7Ralpha-deficient mice, and we found that they partly rescued gammadelta T cells in the adult thymus but not in the spleen. Introduction of an additional Bcl-2 transgene had a minimal effect on gammadelta T cells in the adult thymus of these mice. In contrast to the adult thymus, the introduction of the Vgamma3/Vdelta1 TCR transgene into IL-7Ralpha-/- mice completely restored Vgamma3+ T cells in the fetal thymus and DETC in the adult skin. On the contrary, the same Vgamma3/Vdelta1 TCR transgene failed to rescue DETC in the skin of IL-2Rbeta-deficient mice, even with the additional Bcl-2 transgene. These results suggest that the IL-2/IL-15R, rather than the IL-7R, plays an essential role in proliferation and survival of DETC in the fetal thymus and the skin. In contrast, the IL-7R is probably essential in the induction of V-J recombination of TCRgamma genes. Thus, this study proves that IL-7R and IL-2/IL-15R serve differential functions in epidermal gammadelta T cell development.

The beta-catenin--TCF-1 pathway ensures CD4(+)CD8(+) thymocyte survival.

The association of trans-acting T cell factors (TCFs) or lymphoid enhancer factor 1 (LEF-1) with their coactivator beta-catenin mediates transient transcriptional responses to extracellular Wnt signals. We show here that T cell maturation depends on the presence of the beta-catenin--binding domain in TCF-1. This domain is necessary to mediate the survival of immature CD4(+)CD8(+) double-positive (DP) thymocytes. Accelerated spontaneous thymocyte death in the absence of TCF-1 correlates with aberrantly low expression of the anti-apoptotic protein Bcl-x(L). Increasing anti-apoptotic effectors in thymocytes by the use of a Bcl-2 transgene rescued TCF-1-deficient DP thymocytes from apoptosis. Thus, TCF-1, upon association with beta-catenin, transiently ensures the survival of immature T cells, which enables them to generate and edit T cell receptor (TCR) alpha chains and attempt TCR-mediated positive selection.

Essential requirement for c-kit and common gamma chain in thymocyte development cannot be overruled by enforced expression of Bcl-2.

The thymus in mice lacking both the receptor tyrosine kinase c-kit and the common cytokine receptor gamma chain (gamma(c)) is alymphoid because these receptors provide essential signals at the earliest stages of thymocyte development. The signals transduced by these receptors potentially regulate proliferation, survival, or differentiation, but the contribution of each receptor to distinct intracellular signaling cascades is only poorly defined. Here, we have examined whether enforced expression of Bcl-2 can rescue thymocyte development in c-kit and gamma(c) single or double mutant mice. A bcl-2 transgene (E(mu)-bcl-2-25; expressed in the T cell lineage) was introduced into (a) c-kit and gamma(c) wild-type (c-kit+gamma(c)+bcl+), (b) c-kit-deficient (c-kit(-)gamma(c)+bcl+), (c) gamma(c)-deficient (c-kit+gamma(c)-bcl+), or (d) c-kit and gamma(c) double-deficient mice (c-kit-gamma(c)-bcl+). The bcl-2 transgene was functionally active in wild-type and c-kit or gamma(c) single mutants, as it promoted survival of ex vivo isolated thymocytes, including pro-T cells. In vivo, however, transgenic Bcl-2 did not release T cell precursors from their phenotypic block and failed to increase progenitor or total thymocyte cellularity in c-kit or gamma(c) single or double mutants. These data argue strongly against a role for Bcl-2 as a key mediator in signaling pathways linked to cytokine and growth factor receptors driving early thymocyte development.

Elimination of bax expression in mice increases cerebellar purkinje cell numbers but not the number of granule cells

Cerebellar Purkinje cells and granule cells have been studied extensively as models for investigating neuron-target interactions and the regulation of cell numbers in the developing central nervous system. Recent studies of transgenic mice that overexpress a human Bcl-2 transgene in Purkinje cells suggest both that programmed cell death plays an unexpected role in regulating Purkinje cell number and that Purkinje cells influence the number of granule cells. The role of cell death-related proteins and Purkinje-granule cell interactions in cerebellar development was investigated further in this study by counting the number of Purkinje and granule cells in knockout mutants with a deletion in the proapoptotic gene, Bax. The total number of Purkinje cells was estimated using stereological counting principles in six adult wild type mice, four hemizygous Bax +/- controls, and six Bax -/- knockout mutants. The total number of granule cells per cerebellum was estimated in three adult wild type mice, three hemizygous Bax +/- controls, and three Bax -/- knockout mutants. The number of Purkinje cells increased significantly by over 30% in the Bax -/- knockout mutants compared with wild type and hemizygote controls, whereas the number of granule cells was unchanged in the Bax -/- mutants. There was no change in the volume of the cerebellar cortex or in the size of Purkinje cell bodies in the Bax -/- mutants, implying that Purkinje cell density was increased in the Bax -/- mutants. The increase in Purkinje cell numbers in the Bax -/- knockout mice supports previous evidence that Purkinje cells undergo a period of naturally occurring cell death that is mediated at least in part by the cell death proteins Bcl-2 and Bax. The lack of an effect of Bax gene expression on granule cell numbers indicates that Bax is not an obligate participant in naturally occurring cell death in granule cells.

Elevated Bcl-2 is not a causal event in the positive selection of T cells.

T cell development is characterized by the induction of apoptosis in most immature thymocytes and the rescue from apoptosis of a small proportion of cells by the process of positive selection.Up-regulation of the anti-apoptotic molecule Bcl-2 is associated with thymocytes undergoing positive selection and a bcl-2 transgene promotes the generation of mature T cells. In contrast,mice transgenic for the pro-apoptotic molecule Bax show impaired T cell maturation. We have used fetal thymic organ culture to determine the action of Bcl-2 and Bax on positive selection of thymocytes. Our data show that Bcl-2 and Bax do not alter the number of thymocytes positively selected by a defined peptide ligand. This implies that Bcl-2 and Bax alter the production of mature T cells in vivo by influencing thymocyte viability rather than by direct action on positive selection. It also presents a solution to the 'chicken-and-egg' scenario relating to Bcl-2 up-regulation and positive selection. The data suggest that the up-regulation of Bcl-2 associated with T cell maturation is a consequence of positive selection rather than a cause of it.

Overexpression of Bcl-2 differentially restores development of thymus-derived CD4-8+ T cells and intestinal intraepithelial T cells in IFN-regulatory factor-1-deficient mice.

Mice lacking IFN-regulatory factor (IRF)-1 have reduced numbers of mature CD8+ T cells within the thymus and peripheral lymphoid organs, suggesting a critical role of IRF-1 in CD8(+) T cell differentiation. Here we show that endogenous Bcl-2 expression is substantially reduced in IRF-1(-/-)CD8+ thymocytes and that introduction of a human Bcl-2 transgene driven by Emu or lck promoter in IRF-1(-/-) mice restores the CD8(+) T cell development. Restored CD8+ T cells are functionally mature in terms of allogeneic MLR and cytokine production. In contrast to thymus-derived CD8+ T cells, other lymphocyte subsets including NK, NK T, and TCR-gammadelta(+) intestinal intraepithelial lymphocytes, which are also impaired in IRF-1(-/-) mice, are not rescued by expressing human Bcl-2. Our results indicate that IRF-1 differentially regulates the development of these lymphocyte subsets and that survival signals involving Bcl-2 are critical for the development of thymus-dependent CD8+ T cells.

Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice

To test whether the antiapoptotic protein Bcl-2 prevents apoptosis and injury of cardiomyocytes after ischemia-reperfusion (I/R), we generated a line of transgenic mice that carried a human Bcl-2 transgene under the control of a mouse alpha-myosin heavy chain promoter. High levels of human Bcl-2 transcripts and 26-kDa Bcl-2 protein were expressed in the hearts of transgenic mice. Functional recovery of the transgenic hearts significantly improved when they were perfused as Langendorff preparations. This protection was accompanied by a threefold decrease in lactate dehydrogenase (LDH) released from the transgenic hearts. The transgenic mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by reperfusion. The infarct sizes, expressed as a percentage of the area at risk, were significantly smaller in the transgenic mice than in the nontransgenic mice (36.6 +/- 5 vs 69.9 +/- 7.3%, respectively). In hearts subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion, Bcl-2 transgenic hearts had significantly fewer terminal deoxynucleodidyl-transferase nick-end labeling-positive or in situ oligo ligation-positive myocytes and a less prominent DNA fragmentation pattern. Our results demonstrate that overexpression of Bcl-2 renders the heart more resistant to apoptosis and I/R injury.

The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression.

Rel and RelA, individually dispensable for lymphopoiesis, serve unique functions in activated B and T cells. Here their combined roles in lymphocyte development were examined in chimeric mice repopulated with c-rel(-/-) rela(-/-) fetal liver hemopoietic stem cells. Mice engrafted with double-mutant cells lacked mature IgM(lo)IgD(hi) B cells, and numbers of peripheral CD4(+) and CD8(+) T cells were markedly reduced. The absence of mature B cells was associated with impaired survival that coincided with reduced expression of bcl-2 and A1. bcl-2 transgene expression not only prevented apoptosis and increased peripheral B-cell numbers, but also induced further maturation to an IgM(lo)IgD(hi) phenotype. In contrast, the survival of double-mutant T cells was normal and the bcl-2 transgene could not rectify the peripheral T-cell deficit. These findings indicate that Rel and RelA serve essential, albeit redundant, functions during the later antigen-independent stages of B- and T-cell maturation, with these transcription factors promoting the survival of peripheral B cells in part by upregulating Bcl-2.

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase-deficient fetal thymic organ cultures.

Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2'-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vbeta gene rearrangements and pre-TCR-alpha expression were normal in ADA-deficient cultures, the production of alphabeta TCR(+) thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of beta selection. In contrast, the production of gammadelta TCR(+) thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as beta selection.

Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca 2+), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed.

Differential Effects of Diethylstilbestrol and 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Thymocyte Differentiation, Proliferation, and Apoptosis in bcl-2 Transgenic Mouse Fetal Thymus Organ Culture

Both the estrogenic drug diethylstilbestrol (DES) and the pervasive environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibit thymocyte development. The mechanisms by which either agent induces thymic atrophy are still undetermined. We previously found that TCDD and DES inhibited C57BL/6 murine fetal thymocyte organ cultures (FTOC) at different stages of development. Now, using bcl-2 transgenic (TG) mice, we have further investigated their effects on FTOC proliferation, differentiation, maturation, and apoptosis. As with C57BL/6 mice, thymocyte development in C3H/bcl-2 FTOCs was inhibited by either TCDD (10 nM) or DES (20 μM) in both bcl-2 TG− and TG+ littermates. However, the percentage reduction of cell number induced by DES in bcl-2 TG+ FTOCs was significantly less than the level of inhibition in TG− FTOCs. There was no difference in the level of reduction from TCDD-exposed TG+ or TG− FTOC. Whereas TCDD increased production of mature CD8 cells in either strain, DES mainly yielded cells in the CD4−CD8− (DN) stage in TG− mice. The anti-apoptotic bcl-2 transgene overcame some DES blocking of DN thymocyte development, allowing more cells to differentiate into CD4 single-positive cells. Analysis of cell cycle showed that TCDD inhibited entry into S phase, whereas DES blocked cell cycling in the G2/M phase. TCDD did not induce detectable apoptosis in FTOC. However, unlike the effects of 17β-estradiol (E2) in vivo, DES induced apoptosis in the TG− FTOC, and these apoptotic cells were mainly in the DN subpopulation. This apoptosis could be prevented by the overexpression of bcl-2 in the TG+ mice. Our results demonstrate that, in addition to inhibition of fetal thymocytes at different stages of development by TCDD and DES, DES also induces thymic atrophy by both bcl-2-inhibitable apoptosis and by inducing cell cycle arrest in G2/M in the latest stage in the stem cell compartment. TCDD, on the other hand, does not induce apoptosis, but inhibits entry into cell cycle in the earliest stage in the stem cell compartment.

Nicotinic acetylcholine receptors in neonatal motoneurons are regulated by axotomy: an electrophysiological and immunohistochemical study in human Bcl-2 transgenic mice

Motoneuron axotomy was exploited as a model system for studying functional and morphological changes caused in motoneuron cell bodies by peripheral axon injury. Rodent facial motoneurons express functional nicotinic acetylcholine receptors. We have determined the effect of neonatal unilateral facial nerve transection on these receptors by using electrophysiological and immunohistochemical techniques. To avoid rapid apoptotic cell death of axotomized motoneurons, the study was done in mice overexpressing the human bcl-2 transgene. Intact motoneurons responded to acetylcholine by generating a rapidly rising inward current, which was insensitive to methyllycaconitine, a selective antagonist of α7-containing nicotinic receptors, but was suppressed by dihydro-β-erythroidine, a broad-spectrum antagonist. This indicates that mouse facial motoneurons possess nicotinic receptors which are probably devoid of the α7 subunit. In striking contrast, axotomized motoneurons displayed little or no sensitivity to acetylcholine. Axotomy did not affect the sensitivity of facial motoneurons to the selective glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxaxolepropionic acid. Immunohistochemical studies revealed that the α4 nicotinic receptor subunit was present in intact motoneurons but was undetectable in axotomized motoneurons. By contrast, the β2 subunit was comparable in intact and axotomized motoneurons. α3 immunoreactivity was undetectable, both in intact and in axotomized motoneurons. Thus, mouse facial nicotinic receptors are possibly of the α4β2 type and axotomy interferes negatively with the expression of the α4 subunit. By down-regulating nicotinic receptors, peripheral nerve injury may facilitate motoneuron degeneration. Alternatively, nicotinic receptor downregulation and motoneuron degeneration may be independent consequences of peripheral axotomy.