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Bax-Deficient Mice with Lymphoid Hyperplasia and Male Germ Cell Death
Abstract
BAX, a heterodimeric partner of BCL2, counters BCL2 and promotes apoptosis in gain-of-function experiments. A Bax knockout mouse was generated that proved viable but displayed lineage-specific aberrations in cell death. Thymocytes and
B cells in this mouse displayed hyperplasia, and Bax-deficient ovaries contained unusual atretic follicles with excess granulosa cells. In contrast, Bax-deficient males were infertile as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic
germ cells, but no mature haploid sperm. Multinucleated giant cells and dysplastic cells accompanied massive cell death. Thus,
the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context.
... However, the deletion of the multi-BH domain proteins provides interesting insights into the distinct and overlapping roles of these proteins in the regulation of oocyte apoptosis. For example, Bak knockout mice are healthy and fertile [30], whereas Bax knockout mice exhibit lymphoid hyperplasia [45]. Moreover, Bax-null male mice are infertile due to defective spermatogenesis [45]. ...
... For example, Bak knockout mice are healthy and fertile [30], whereas Bax knockout mice exhibit lymphoid hyperplasia [45]. Moreover, Bax-null male mice are infertile due to defective spermatogenesis [45]. Importantly, Bax knockout mice display a three-fold increase in the number of primordial follicles at 42 days old compared to that in aged-matched wild-type controls, and this follicle surplus appears to contribute to the extended ovarian lifespan in Bax deficient females [32]. ...
Apoptosis is a form of programmed cell death that plays a critical role in cellular homeostasis and development, including in the ovarian reserve. In humans, hundreds of thousands of oocytes are produced in the fetal ovary. However, the majority die by apoptosis before birth. After puberty, primordial follicles develop into mature follicles. While only a large dominant follicle is selected to ovulate, smaller ones undergo apoptosis. Despite numerous studies, the mechanism of oocyte death at the molecular level remains elusive. Over the last two and a half decades, many knockout mouse models disrupting key genes in the apoptosis pathway have been generated. In this review, we highlight some of the phenotypes and discuss distinct and overlapping roles of the apoptosis regulators in oocyte death and survival. We also review how the transcription factor p63 and its family members may trigger oocyte apoptosis in response to DNA damage.
... Bax deficient mice are viable but exhibit lineage−specific peculiarities. In fact, the invalidation of Bax causes lymphoid hyperplasia and male germ cell apoptosis leading to male infertility (Knudson et al., 1995). ...
... Furthemore, the subcellular localization of the effector proteins plays a major role in cancer prognosis as in the case of mitochondrial Bax that was shown to improve apoptosis susceptibility and prognosis in patients suffering from AML. When KO in mice, both proteins do not induce tumor suppression but when KO individually, mice die perinatally (Knudson et al., 1995;Lindsten et al., 2000). Moreover, conditionnal Bax/Bak deletion in the haematopoietic system of mice induces lethal autoimmune diseases that might actually hide tumor development (Mason et al., 2013). ...
Bcl-2 proteins are major regulators of the mitochondrial pathway of apoptosis. Bcl-xL, an anti-apoptotic Bcl-2 homolog, is overexpressed in breast cancer where it increases metastatic potential. This member is present at the mitochondria as well as the endoplasmic reticulum (ER). However, the subsequent function of its ER localization is poorly explored. Our project aims at evaluating the respective functional contributions of the mitochondrial and reticular pools of Bcl-xL. To do so, we generated genetically modified mice expressing exclusively Bcl-xL at the ER referred to as ER-xL or mitochondria referred to as Mt-xL. Subcellular fractionation experiments on mice embryonic fibroblasts (MEFs) validated the exclusive Bcl-xL localization in these mice. By performing cell death assays, we showed that ER-xL MEFs have increased vulnerability to apoptotic stimuli compared to wild-type (WT) and Mt-xL MEFs but are more resistant to ER stress. Moreover, ER-xL MEFs displayed increased calcium sensibility at the ER but a reduced Inositol triphosphate receptor (IP3R)-mediated calcium release. Importantly, the ER-localization of Bcl-xL seems to confer resistance through the Unfolded Protein Response (UPR). We have shown for the first time that upon ER stress, Bcl-xL at the ER negatively regulates IP3R inhibiting ER calcium depletion necessary for UPR initiation and subsequently apoptosis. Overall, this work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliché regulation of apoptosis
... The objective of the current study was to determine if mice lacking the Bax gene are protected from the effects of gastrulation-stage alcohol exposure. Full loss of the Bax gene is not lethal and the mice develop without any major developmental abnormalities, although Bax À/À male mice are infertile (Knudson, Tung, Tourtellotte, Brown, & Korsmeyer, 1995). More subtle effects of Bax deletion have been observed, particularly in the brain and eye, including increased neuronal number (Deckwerth et al., 1996;White, Keller-Peck, Knudson, Korsmeyer, & Snider, 1998), absence of size differences in two sexually dimorphic nuclei (Forger & Peskin, 2005), cerebellar migration defects (Jung et al., 2008), increased retinal cell numbers, glial cell density, and altered microglial shape (Kawai et al., 2009;Mac Nair, Schlamp, Montgomery, Shestopalov, & Nickells, 2016;Mosinger Ogilvie, Deckwerth, Knudson, & Korsmeyer, 1998). ...
... Female and male mice on a C57BL/6 background, heterozygous for a Bax gene deletion were bred in the laboratory from founders generously donated by Mohanish Deshmukh (UNC Chapel Hill, originally obtained from Dr Stanley Korsmeyer (Knudson et al., 1995)). Timed pregnancies were established by housing one or two nulliparous female Bax +/À mice with one Bax +/À male for 1-2 hr. ...
Background:
During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure.
Methods:
Male and female Bax+/- mice mated to produce embryos with full (-/- ) or partial (+/- ) Bax deletions, or Bax+/+ wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features.
Results:
Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax+/+ and Bax-/- embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis.
Conclusions:
Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.
... BAX expression was abundant in GCs of early atresia follicles, but not in atresia follicles, suggesting that BAX protein expression in early atresia promotes GCs apoptosis [78]. Bcl2deficient mice exhibit a reduced number of oocytes and primordial follicles [79], whereas Bax-deficient mice exhibit an excess of abnormal follicles [80,81]. ...
Premature ovarian insufficiency (POI) is a common gynecological endocrine disease, which seriously affects women’s physical and mental health and fertility, and its incidence is increasing year by year. With the development of social economy and technology, psychological stressors such as anxiety and depression caused by social, life and environmental factors may be one of the risk factors for POI. We used PubMed to search peer-reviewed original English manuscripts published over the last 10 years to identify established and experimental studies on the relationship between various types of stress and decreased ovarian function. Oxidative stress, follicular atresia, and excessive activation of oocytes, caused by Stress-associated factors may be the main causes of ovarian function damage. This article reviews the relationship between psychological stressors and hypoovarian function and the possible early intervention measures in order to provide new ideas for future clinical treatment and intervention.
... Knudson et al. [77]. Bax−/− mice exhibit ovarian aberrations. ...
Cell death mediated by genetically defined signaling pathways influences the health and dynamics of all tissues, however the tissue specificity of cell death pathways and the relationships between these pathways and human disease are not well understood. We analyzed the expression profiles of an array of 44 cell death genes involved in apoptosis, necroptosis, and pyroptosis cell death pathways across 49 human tissues from GTEx, to elucidate the landscape of cell death gene expression across human tissues, and the relationship between tissue-specific genetically determined expression and the human phenome. We uncovered unique cell death gene expression profiles across tissue types, suggesting there are physiologically distinct cell death programs in different tissues. Using summary statistics-based transcriptome wide association studies (TWAS) on human traits in the UK Biobank ( n ~ 500,000), we evaluated 513 traits encompassing ICD-10 defined diagnoses and laboratory-derived traits. Our analysis revealed hundreds of significant (FDR < 0.05) associations between genetically regulated cell death gene expression and an array of human phenotypes encompassing both clinical diagnoses and hematologic parameters, which were independently validated in another large-scale DNA biobank (BioVU) at Vanderbilt University Medical Center ( n = 94,474) with matching phenotypes. Cell death genes were highly enriched for significant associations with blood traits versus non-cell-death genes, with apoptosis-associated genes enriched for leukocyte and platelet traits. Our findings are also concordant with independently published studies (e.g. associations between BCL2L11 /BIM expression and platelet & lymphocyte counts). Overall, these results suggest that cell death genes play distinct roles in their contribution to human phenotypes, and that cell death genes influence a diverse array of human traits.
... The major histologic abnormalities could be observed from the age of 5 weeks onwards, but the severity of the lesions was unpredictable and was not related to the age of bcl-xL and bcl-2 mice (30). Morphologic alterations of the testes in mice deficient for proapoptotic protein Bax (Bax −/− mice) were very similar to mice overexpressing the apoptosis-protecting Bcl-xL or Bcl-2 proteins, i.e., accumulation of the premeiotic germ cells and enlargement of the testes at an early age followed by degeneration of the accumulated cells and shrinkage of the testes at an advanced age (31). During late puberty, massive degeneration of Bax-deficient germ cells took place through an apoptosis-independent pathway that was triggered by overcrowding of the seminiferous epithelium (32). ...
Laboratory mice (Mus musculus) are preferred animals for biomedical research due to the close relationship with humans in several aspects. Therefore, mice with diverse genetic traits have been generated to mimic human characteristics of interest. Some genetically altered mouse strains, on purpose or by accident, have reproductive phenotypes and/or fertility deviating from wild-type mice. The distinct reproductive phenotypes of genetically altered male mice mentioned in this paper are grouped based on reproductive organs, beginning with the brain (i.e., the hypothalamus and anterior pituitary) that regulates sexual maturity and development, the testis where male gametes and sex steroid hormones are produced, the epididymis, the accessory sex glands, and the penis which involve in sperm maturation, storage, and ejaculation. Also, distinct characteristics of mature sperm from genetically altered mice are described here. This repository will hopefully be a valuable resource for both humans, in terms of future biomedical research, and mice, in the aspect of the establishment of optimal sperm preservation protocols for individual mouse strains.
... BCL2 is located on the outer membrane of mitochondria and inhibits cell apoptosis by inhibiting the release of cytochrome c. BAX is a heterodimer of BCL2, which can induce mitochondrial membrane depolarization, increase mitochondrial outer membrane permeability, promote the release of cytochrome c, and eliminate BCL2's inhibition of cell apoptosis [51,52]. Our experiment showed a positive correlation between the relative protein expression of AMPK and proliferation-related genes and a negative correlation with mTOR. ...
Zearalenone (ZEN), a non-steroidal Fusarium graminearum with an estrogen effect, can cause damage to the gastrointestinal tract, immune organs, liver, and reproductive system. Further analysis of the mechanism of ZEN has become an important scientific issue. We have established in vivo and in vitro models of ZEN intervention, used AMPK/mTOR as a targeted pathway for ZEN reproductive toxicity, and explored the molecular mechanism by which ZEN may induce uterine hypertrophy in weaned piglets. Our study strongly suggested that ZEN can activate the phosphorylation of AMPK in uterine endometrial epithelium cells, affect the phosphorylation level of mTOR through TSC2 and Rheb, induce autophagy, upregulate the expression of proliferative genes PCNA and BCL2, downregulate the expression of apoptotic gene BAX, promote uterine endometrial epithelium cells proliferation, and ultimately lead to thickening of the endometrial and myometrium, increased density of uterine glands, and induce uterine hypertrophy.
... As a next step, we investigated whether apoptotic dysregulation functionally affects tissue homeostasis in vivo. Although it has previously been established that Bax and Bak exhibit some degree of overlapping and compensatory roles, Bak −/− mice appear phenotypically normal while Bax −/− mice exhibit abnormalities including male sterility, mild lymphoid hyperplasia, and an increased number of neurons 22,58,59 . We therefore utilized mice harboring Bak knockout together with K15 promoter-driven CRE-PGR deletion of Bax (BakcBax −/− ) and confetti (XFP) expression ( Fig. 6a; Supplementary Fig. 7a), enabling examination of the role of Bax in HFSCs while preventing compensation by Bak. ...
Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in “loser” cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.
... It has been shown that deletions of the BAX gene may be associated with the occurrence of lymphoid hyperplasia. Hence, this gene is considered to be an important suppressor of hematopoietic malignancies [34]. In the case of polycythemia vera or essential thrombocythemia, there was no significant difference in the frequency of genotypes for the BAX −248G>A (rs4645878) polymorphism between the patients and the control group [33]. ...
Ovarian cancer (OC) is one of the biggest problems in gynecological oncology and is one of the most lethal cancers in women worldwide. Most patients with OC are diagnosed at an advanced stage; therefore, there is an urgent need to find new biomarkers for this disease. Gene expression profiling is proving to be a very effective tool for exploring new molecular markers for OC patients, although the relationship between such markers and patient survival and clinical outcomes is still elusive. Moreover, polymorphisms in genes encoding both apoptosis-associated proteins and oncoproteins may serve as key markers of cancer susceptibility. The aim of our study was to analyze the polymorphisms and expressions of the BCL2, BAX and c-MYC genes in a group of 198 women, including 98 with OC. The polymorphisms and mRNA expressions of the BCL2, BAX and c-MYC genes were analyzed using real-time PCR. The analysis of the BAX (rs4645878; G>A) and c-MYC (rs4645943; C>T) polymorphisms showed no association with ovarian cancer risk. The BCL2 polymorphism (rs2279115; C>A) showed a significant difference in the frequency of genotypes between the studied groups (CC: 23.47% vs. 16.00%, AA: 25.51% vs. 37.00%; p = 0.046; OR = 1.61). Furthermore, the expression levels of the BCL2 and c-MYC genes showed a decrease at the transcript level for OC patients compared to the control group (BCL2: 17.46% ± 3.26 vs. 100% ± 8.32; p < 0.05; c-MYC: 37.56% ± 8.16 vs. 100% ± 9.12; p < 0.05). No significant changes in the mRNA level were observed for the BAX gene (104.36% ± 9.26 vs. 100% ± 9.44; p > 0.05). A similar relationship was demonstrated in the case of the protein expressions of the studied genes. These findings suggest that the CC genotype and C allele of the BCL2 polymorphism could be genetic risk factors for OC development. A gene expression analysis indicated that BCL2 and c-MYC are associated with OC risk.
... Primers used for genotyping were indicated in the key resources table. The following mouse strains were used: Prdm12 LacZ/+ and Prdm12 flox/flox11 ; Rosa26 LSLtdTomato (Ai14, JAX# 007908) 56 ; Advillin-Cre (JAX# 032536) 33 ;Rosa26 CreERT2 (JAX# 008463) 57 ; Bax À/À (JAX#002994) 58 ; Phox2b LacZ/+ and Phox2b-Cre mice. 4,7 Sox10-Venus, 20 Neurog1 GFP,18 Dbx1-Cre 36 and Nav1.8-Cre 43 mice were gifts from S. Shibata, Franç ois Guillemot, A. Pierani and E. Bourinet, respectively. ...
Prdm12 is a transcriptional regulator essential for the emergence of the somatic nociceptive lineage during sensory neurogenesis. The exact mechanisms by which Prdm12 promotes nociceptor development remain, however, poorly understood. Here, we report that the trigeminal and dorsal root ganglia hypoplasia induced by the loss of Prdm12 involves Bax-dependent apoptosis and that it is accompanied by the ectopic expression of the visceral sensory neuron determinants Phox2a and Phox2b, which is, however, not sufficient to impose a complete fate switch in surviving somatosensory neurons. Mechanistically, our data reveal that Prdm12 is required from somatosensory neural precursors to early post-mitotic differentiating nociceptive neurons to repress Phox2a/b and that its repressive function is context dependent. Together, these findings reveal that besides its essential role in nociceptor survival during development, Prdm12 also promotes nociceptor fate via an additional mechanism, by preventing precursors from engaging into an alternate Phox2 driven visceral neuronal type differentiation program.
... Sertoli cells express FasL, which after binding FasR on the germ cell membrane initiates apoptosis [25,42] and that mechanism is probably involved in Sertoli cell control of germ cell/ spermatozoa number. In transgenic models the overexpression of Bcl-2 or Bcl-xl leads to an abundance of spermatogonia and infertility and knockout of Bcl-xl causes a lack of spermatogonia [36,43]. Knockout of p53 results in increased number of defective sperm due to suppression of germ cell apoptosis. ...
... The Bax gene has been involved in the suppression of tumors due to its role in promoting the programmed cell death. The experimental deletions of the Bax gene have been shown to be associated with the incidence of lymphoid hyperplasia and thus it is regarded as an important suppressor against hematopoietic neoplasms [65][66][67][68]. There was no significant difference in the genotype and allelic frequencies of Bax-248 G>A (rs4645878) polymorphisms between healthy controls and PV or ET patients. ...
The regulation of apoptosis (the programmed cell death) is dependent on the crucial involvement of BCL2 and BAX. The Bax-248G>A and Bcl-2-938 C>A polymorphic variations in the promoter sequences of the Bax and Bcl-2 gene have been recently associated with low Bax expression, progression to advanced stages, treatment resistance, and shortened overall survival rate in some hematological malignancies, including chronic myeloid leukemia (CML) and other myeloproliferative neoplasms. Chronic inflammation has been linked to various stages of carcinogenesis wherein pro-inflammatory cytokines play diverse roles in influencing cancer microenvironment leading to cell invasion and cancer progression. Cytokines such as TNF-α and IL-8 have been implicated in cancer growth in both solid and hematological malignancies with studies showing their elevated levels in patients. Genomic approaches have in recent years provided significant knowledge with the regard to the association of certain SNPs (single nucleotide polymerphisms) either in a gene or its promoter that can influence its expression, with the risk and susceptibility to human diseases including cancer. This study has investigated the consequences of promoter SNPs in apoptosis genes Bax-248G>A (rs4645878)/Bcl-2-938C>A (rs2279115) and pro-inflammatory cytokines TNF-α rs1800629 G>A/IL-8 rs4073 T>A on the risk and susceptibility towards hematological cancers. The study design has 235 individuals both male and female enrolled as subjects that had 113 cases of MPDs (myeloproliferative disorders) and 122 healthy individuals as controls. The genotyping studies were conducted through ARMS PCR (amplification-refractory mutation system PCR). The Bcl-2-938 C>A polymorphism showed up in 22% of patients in the study, while it was observed in only 10% of normal controls. This difference in genotype and allele frequency between the two groups was significant (p = 0.025). Similarly, the Bax-248G>A polymorphism was detected in 6.48% of the patients and 4.54% of the normal controls, with a significant difference in genotype and allele frequency between the groups (p = 0.048). The results suggest that the Bcl-2-938 C>A variant is linked to an elevated risk of MPDs in the codominant, dominant, and recessive inheritance models. Moreover, the study indicated allele A as risk allele which can significantly increase the risk of MPDs unlike the C allele. In case of Bax gene covariants, these were associated with an increased risk of MPDs in the codominant inheritance model and dominant inheritance model. It was found that the allele A significantly enhanced the risk of MPDs unlike the G allele. The frequencies of IL-8 rs4073 T>A in patients was found to be TT (16.39%), AT (36.88%) and AA (46.72%), compared to controls who were more likely to have frequencies of TT (39.34%), AT (37.70%) and AA (22.95%) as such, respectively. There was a notable overrepresentation of the AA genotype and GG homozygotes among patients compared to controls in TNF-α polymorphic variants, with 6.55% of patients having the AA genotype and 84% of patients being GG homozygotes, compared to 1.63% and 69%, respectively in controls. The data from the current study provide partial but important evidence that polymorphisms in apoptotic genes Bcl-2-938C>A and Bax-248G>A and pro-inflammatory cytokines IL-8 rs4073 T>A and TNF-α G>A may help predict the clinical outcomes of patients and determine the significance of such polymorphic variations in the risk of myeloproliferative diseases and their role as prognostic markers in disease management using a case-control study approach.
... If LTMRs of the same subtype compete for territory, we would expect that the receptive fields of individual Aδ-LTMRs would become smaller in Bax −/− animals to accommodate the excess number of neurons, while the receptive fields of C-LTMRs would remain largely unchanged. To test this, we generated triple transgenic a Cre-dependent placental alkaline phosphatase reporter (R26 iAP ), and 3: Bax +/+ , Bax ± , or Bax −/− [16,[46][47][48]. We administered low doses of tamoxifen to timed-pregnant dams (Aδ-LTMRs) or juvenile mice (C-LTMRs) to sparsely label receptive fields and quantified the number of hair follicles in each receptive field of young adult animals (P21). ...
The mammalian somatosensory system is comprised of multiple neuronal populations that form specialized, highly organized sensory endings in the skin. The organization of somatosensory endings is essential to their functions, yet the mechanisms which regulate this organization remain unclear. Using a combination of genetic and molecular labeling approaches, we examined the development of mouse hair follicle-innervating low-threshold mechanoreceptors (LTMRs) and explored competition for innervation targets as a mechanism involved in the patterning of their receptive fields. We show that follicle innervating neurons are present in the skin at birth and that LTMR receptive fields gradually add follicle-innervating endings during the first two postnatal weeks. Using a constitutive Bax knockout to increase the number of neurons in adult animals, we show that two LTMR subtypes have differential responses to an increase in neuronal population size: Aδ-LTMR neurons shrink their receptive fields to accommodate the increased number of neurons innervating the skin, while C-LTMR neurons do not. Our findings suggest that competition for hair follicles to innervate plays a role in the patterning and organization of follicle-innervating LTMR neurons.
... [4][5][6] Mice that have restricted ability to undergo apoptosis by combined genetic deletions of pro-apoptotic Bcl-2 family proteins, including Bax, Bak, and Bok, die perinatally with multiple developmental defects with hyperplasia in some tissues. [7][8][9] These loss-of-function studies indicate the crucial roles of the Bcl-2 family of proteins in cell survival and death with distinct but partially overlapped functional properties, which are essential for normal development and maintaining tissue homeostasis. ...
BCL-2-like protein 1 (BCL2L1) is a key component of cell survival and death mechanisms. Its dysregulation and altered ratio of splicing variants associate with pathologies. However, isoform-specific loss-of-function analysis of BCL2L1 remains unexplored. Here we show the functional impact of genetically inhibiting Bcl-x short-isoform (Bcl-xS) in vivo. Bcl-xS is expressed in most tissues with predominant expression in the spleen and blood cells in mice. Bcl-xS knockout (KO) mice show no overt abnormality until 3 months of age. Thereafter, KO mice develop cardiac hypertrophy with contractile dysfunction and splenomegaly by 6 months. Cardiac fibrosis significantly increases in KO, but the frequency of apoptosis is indistinguishable despite cardiomyopathy. The Akt/mTOR and JNK/cJun signaling are upregulated in male KO heart, and the JNK/cJun is activated with increased Bax expression in KO spleen. These results suggest that Bcl-xS may be dispensable for development but is essential for maintaining the homeostasis of multiple organs.
... Those genes were mostly upregulated in the supplemented blastocysts and involved in various developmental processes (Figure 4 and Table S9). These include BAK1 which functions as a pro-apoptotic regulator involved in a wide variety of cellular activities and shaping tissue and organ morphologies via apoptosis [48,68,69]. In addition, PDGFRA is a receptor kinase binding to PDGF that stimulates the growth and migration of vascular smooth muscle cells, fibroblasts, and glial cells [46,70,71]. ...
Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell division. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.
... All mice were on a C57BL/6 background and bred at WEHI. Mice deficient for BAX [56] or BAK [44] were crossed with Eµ-Myc mice [17]. Offspring were monitored for lymphoma and euthanised when they reached the ethical endpoint as determined by trained animal technicians according to the WEHI Animal Ethics Committee guidelines. ...
BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.
... Knudson et al. [172] demonstrated that, in mice, BAX deletion resulted in the appearance of unusual atretic follicles with their granulosa cells seemed unable to activatee apoptosis. Evidence has been recently obrained that death mechanisms such as caspase-independent cell death and autophagy may be acting in the mammalian ovary [173]. ...
In eukaryotic cells, many macromolecules are organized as membraneless biomolecular condensates (or biocondensates). Liquid-liquid and liquid-solid phase transitions are the drivers of the condensation process. The absence of membrane borders makes biocondensates very flexible in their composition and functions, which vary in different cells and tissues. Some biocondensates are specific for germ line cells and are, thus, termed germ granules. This review summarizes the recent data on the composition of germ granules and their functions in gametes. According to these data, germ granules are involved in the determination of germline cells in some animals, such as Amphibia. In other animals, such as Mammalia, germ granules are involved in the processes of transposons inactivation and sequestration of mRNA and proteins to temporarily decrease their activity. The new data on germ granules composition and functions sheds light on germ cell differentiation and maturation properties.
... KO mouse models of various downstream targets of Trp53 (Bax, Apaf1, and Cdkn1a) show spermatogenic abnormalities. However, Bax KO mice accumulate spermatogonia that cannot differentiate into sperm (Knudson et al., 1995). Apaf1 KO mice show degeneration of spermatogenesis (Honarpour et al., 2000). ...
A small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT⁺ differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern.
... Similar results were previously reported in rats (Yan et al., 2000) and mice (Knudson et al., 1995). Additionally, in the present investigation, COX2 protein expression was upregulated in testicular tissue of aged donkeys when compared with young (negative expression) or adult animals. ...
This study aimed to assess the effects of age on testicular morphometry and function in donkeys. Testes and epididymides of 57 donkeys were harvested immediately after slaughtering. The donkeys were grouped: young (1‐4 years‐old, n=13); adult (5‐15 years‐old, n=25) and aged (>15 years‐old, n=19). Each testis and epididymis were weighted separated. Testicular volume was calculated. Epididymal sperm was harvested by retrograde flushing method, and sperm parameters were evaluated. The testicular parenchyma was immunolabelled for BAX and COX2. Adult and aged donkeys had greater testicular weight and volume than young (P<0.05). Epididymal sperm concentration, motility, and viability were greater (P<0.05) in adults and aged (931.8±39.3 and 858.2±33.2× 106/mL) than in young animals (316.3±72.8× 106/mL). Aged donkeys had higher percentage of morphologic sperm defects than the other categories (P<0.05). Histological examination revealed the presence of age‐related degenerative changes in testicular tissue of donkeys. Aged donkeys had higher COX2 protein expression than adult and young donkeys. BAX protein was overly expressed in adults than aged or young animals. In conclusion, advancement of age affects the testicular morphometry and function in donkeys.
... Constant spontaneous GCD in the adult testis has also been reported in several mammalian species (12)(13)(14). Morphological features of germ cell debris and the phenotypes of mouse mutants demonstrated that also in mammals, the process of GCD deviates from the conventional apoptotic program (13,58). Moreover, phagocytic Sertoli cells that line the seminiferous tubules of mammalian testes display the double-edged sword function of both supporting the developing germ cells and clearing the resulting debris (59). ...
Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) “eat-me” signals on target cells initiate the process, the precise sequence of events is largely unknown. Here, we show that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, we demonstrate that cyst cell–derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors.
... 13 Bax 'nakavt edilmiş' ve Bcl-2-Beh'yi aşırı eksprese eden transgenik farelerde, spermatogonianın çoğalması aşamasındaki erken dönemde yine apoptozis şekillendiği ve hayvanların infertilisine neden olduğu görülmüştür. 17,18 Yine J3c/-2 eksikliği olan farelerde normal spermatogenezis sergilenirken, Bcl-xL seviyelerinin daha düşük Bax seviyelerinin daha yüksek olduğu ve prolifere olan germ hücrelerinde ölüm şekillendiği gözlenmiştir. 13 Bcl-2 ailesi içerisinde protein ekspresyonunundaki değişiklikler sonucu p53 proteini üzerinden Bax ile birlikte Bad'in da germ hücrelerinin apoptozisine katıldığı belirtilmiştir. ...
G erek insan gerekse omurgalı canlılarda çeşitli görev ve fonksiyonlarda olan ve canlılığın temel yapıtaşını oluşturan sayısız hücreler bulunmaktadır. Her bir hücrede anabolik reaksiyonlar ile canlılık faaliyetlerinin sürdürülmesi için üre-tim ve katabolik reaksiyonlar ile yıkım ürünlerinin uzaklaştırılması için işlemler bir iç denge içerisinde sürdürülmektedir. Hücredeki yıkım fazla olduğunda katabolik ürünler hücreye zarar vererek, önce hücre ardından ilgili dokuda homeostaz (denge) durumu or-tadan kaldırmaktadır. Her gün milyonlarca hücre iç dengenin sağlanması amacıyla bir takım özel mekanizmalar doğrultusunda hücre ölümü şekillenmektedir. Hücre ölümü, hücrenin yaşamsal faaliyetlerinin son noktasıdır. Hücreler, kaza sonucu hücre ölümü (Accidentally Cell Death-ACD) veya düzenlenmiş hücre ölümü (Regularly Cell Death-35 ÖZET Hücre ölümü fizyolojik ve patolojik etkiler altında yaşam boyu süren bir olaydır. Erkek üreme sisteminde apoptozis-otofaji gibi iyi bilinenler dışında yakın zamanda nekroptozis, ferroptozis ve pi-roptozis gibi çok çeşitli mekanizmalarla gelişmektedir. Apoptozis, erkek üreme sisteminde germ hücre-lerinden gelişim sırasında spermatogenezde görülür. Sertoli hücrelerinin spermatogenezi destekleme kapasitesini sınırlandırmak ve anormal gelişen spermatozoonların elimine edilmesinde yararlanılır. Bu şekilde spermatozoanın olgunlaşmayı tamamlaması ve fertiliteyi gerçekleştirebilmesi için bir fırsat oluş-turulur. Ayrıca bozuk genetik yapıda olan ve onarım şansı olmayan spermatozoalardan kusurlu embriyo gelişimine engel olunur. Çeşitli fizyolojik ve patolojik etkiler altında otofaji de gelişmektedir. Kök hüc-reden olgun bir sperm hücresi gelişinceye kadar spermatogonium, spermatosit ve spermatozoa dahil olmak üzere çeşitli aşamalarda otofaji etkili olabilmektedir. Bu tür programlanmış hücre ölüm meka-nizmalarının iyi bilinmesi; erkek üreme sisteminde gelişiminin kavranmasıyla suni tohumlamada olu-şabilecek hatalı uygulamaların engellenmesine, çeşitli hastalıkların ve kusurlu embriyonel gelişiminin engellenmesine, hücre ölümüne karşı ilaçlar ve tedavi yaklaşımlarının tespit edilmesine katkı sağlaya-caktır. Anah tar Ke li me ler: Hücre ölümü; erkek üreme sistemi; spermatozoa; patogenetik mekanizmalar ABS TRACT Cell death is a lifelong event going on under physiologic and pathological effects. In the male reproductive system, apart from the well-known ones such as apoptosis-autophagy, it has recently developed by various mechanisms such as necroptosis,ferroptosis and pyroptosis. In the apoptosis, it occurs in spermatogenesis during development from germ cells in the male reproductive system. It is used to limit the capacity of Sertoli cells to support spermatogenesis and to eliminate abnormally developing spermatozoa. In this way, an opportunity is created for the spermatozoa to complete maturation and achieve fertility. In addition, defective embryo development is prevented from spermatozoa with defective genetic structure and no chance of repair. Autophagy also develops under various physiological and pathological effects. Autophagy can be effective at various stages, including spermatogonia, spermato-cyte and spermatozoa, until a mature sperm cell develops from the stem cell. To the best knowledge of such kind of programmed cell death mechanisms, by understanding development of the male reproductive system, it will contribute to the prevention of malpractices that may occur in artificial insemination, the prevention of various diseases and defective embryonic development, the determination of drugs and treatment approaches against cell death.
... Myh9 lox/lox (MGI: 4838521) and Myh10 lox/lox (MGI: 4443039) mice have been previously reported (Jacobelli et al., 2010;Ma et al., 2009) and were maintained in a 129/Sv and C57BL/6J mixed genetic background. The following mouse alleles were backcrossed to, and maintained on, a congenic C57BL/6J genetic background: Crect (MGI: 4887352) (Reid et al., 2011), Bax lox/lox (MGI: 99702) (Knudson et al., 1995), Bak −/− (MGI: 1097161) (Lindsten et al., 2000), Rosa26 mTmG/mTmG (MGI: 3716464) (Muzumdar et al., 2007) and Rosa26 nTnG/nTnG (MGI: 5504463) (Prigge et al., 2013). For genotyping, tail biopsies were collected at postnatal day 10 and either sent to Transnetyx or lysed for in-house PCR. ...
Tissue fusion frequently requires the removal of an epithelium intervening distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES, results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static and live imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to also express periderm markers, suggesting migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis and reveal a crucial and unique form of collective epithelial migration during tissue fusion.
... Bax is a member of Bcl2 family proteins and mediates neuronal cell death 8 , including physiological retinal apoptosis 9,10 and neuronal death in the Rb/p107 double knockout brain 11 . Bax-null males are infertile 12 . To generate α-Cre; Rb f/f ;Bax −/− mice, we first generated α-Cre;Rb f/f ;Bax +/− males and Rb f/f ;Bax −/− females, and inter-bred them. ...
The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα0⁺ olfactory neurons. Most α-Cre⁺ olfactory neurons do not express Pax6, implicating the influence of neighboring regulatory elements. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.
... Mitochondria are at the crossroads of cell death and metabolism (Rastogi et al., 2019). The BCL-2 family of proteins regulates cell death at the mitochondria (Knudson et al., 1995;Hsu et al., 1997;Kluck et al., 1997Kluck et al., , 1999Vander Heiden et al., 1997;Inohara et al., 1998;Jürgensmeier et al., 1998;Green, 2000;Wei et al., 2000;Ke et al., 2018) and has been implicated in maintaining mitochondrial homeostasis in the absence of a cell death signal (Li et al., , 2013Rasmussen et al., 2018Rasmussen et al., , 2020Salisbury-Ruf et al., 2018;Joshi et al., 2020). Programmed cell death (apoptosis) is an integral part of brain development and maturation (Kuan et al., 2000). ...
Mitochondrial homeostasis -including function, morphology, and inter-organelle communication- provides guidance to the intrinsic developmental programs of corticogenesis, while also being responsive to environmental and intercellular signals. Two- and three-dimensional platforms have become useful tools to interrogate the capacity of cells to generate neuronal and glia progeny in a background of metabolic dysregulation, but the mechanistic underpinnings underlying the role of mitochondria during human neurogenesis remain unexplored. Here we provide a concise overview of cortical development and the use of pluripotent stem cell models that have contributed to our understanding of mitochondrial and metabolic regulation of early human brain development. We finally discuss the effects of mitochondrial fitness dysregulation seen under stress conditions such as metabolic dysregulation, absence of developmental apoptosis, and hypoxia; and the avenues of research that can be explored with the use of brain organoids.
... The B6.129P2 (Cg)-Cx3cr1 tm1Litt /J mice were a gift from Richard Lang with permission from Dr. Steffen Jung (Jung et al., 2000). B6.129 × 1-Bax tm1Sjk /J mice (JAX 002994) (Knudson et al., 1995) and B6.129S4-Itgam tm1Myd /J (JAX 003991) (Coxon et al., 1996) were purchased from the Jackson Laboratory and both were crossed with the B6.129P2 (Cg)-Cx3cr1 tm1Litt /J strain. The B6.129-Mertk tm1Grl /J and B6.129-Axl tm1Grl /J strains (Lu et al., 1999) were a kind gift from Dr. Greg Lemke and double knockouts were generated in house. ...
Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drives multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia towards distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.
... In the first wave, an increased number of germ cell apoptosis occurs in testes. The deletion of Bax and the overexpression of Bcl-2 or Bclx eliminates the apoptosis during the first wave, resulting in the accumulation of spermatogonia and spermatocytes in the transgenic mice, which are infertile (Knudson et al. 1995;Damavandi et al. 2002). Also, caspase 2, 3, 8, and 9 are involved in germ cell apoptosis of the first wave. ...
The male reproductive system consists of testes, a series of ducts connecting the testes to the external urethral orifice, accessory sex glands, and the penis. Spermatogonial stem cells differentiate and mature in testes and epididymides, and spermatozoa are ejaculated with exocrine fluids secreted by accessory sex glands. Many studies have clarified the detailed structure and function of the male reproductive system, and have shown that various biologic controls, including genomics, epigenetics, and the neuroendocrine–immune system regulate proliferation, differentiation, and maturation of germ cells. In other words (1) genetic deletion or abnormalities, (2) aberration of DNA methylation and histone modifications, as well as small RNA dysfunction, and (3) neuroendocrine–immune disorders are involved in functional failure of the male reproductive system. In this article, we review these three factors for germ cell microcircumstance, especially focused on the immunoendocrine environment. In particular, the relation between factors protecting germ cells with strong auto-immunogenicity and opposite factors compromising this protection are discussed. Reductions in sperm count, concentration, and semen quality are serious problems in developed countries, although the causes are complex and remain unclear. The accumulation of basic knowledge regarding the structure, function, and regulation of the male reproductive system under various experimental conditions will be important to resolve these problems.
... We obtained 6-to 8-week-old male C57Bl/6 (WT) mice from Charles River Laboratory (Wilmington, MA). We obtained 4-to 10-week-old male mice with a targeted null mutation in the Bax gene (Bax -/-, 002994 -B6.129X1-Bax tm1Sjk ) from Jackson Laboratory (Bar Harbor, ME, [25]), We confirmed Bax -/by PCR according to the vendor's protocol, using the following primers: GTT GAC CAG AGT GGC GTA GG (Common), CCG CTT CCA TTG CTC AGC GG (Mutant forward), and GAG CTG ATC AGA ACC ATC ATG (WT forward). We obtained primers from Integrated DNA Technologies (Coralville, IA). ...
The BCL-2 (B-cell lymphoma-2) family of proteins contributes to mitochondrial-based apoptosis in models of neurode-generation, including glaucomatous optic neuropathy (glaucoma), which degrades the retinal ganglion cell (RGC) axonal projection to the visual brain. Glaucoma is commonly associated with increased sensitivity to intraocular pressure (IOP) and involves a proximal program that leads to RGC dendritic pruning and a distal program that underlies axonopathy in the optic projection. While genetic deletion of the Bcl2-associated X protein (Bax-/-) prolongs RGC body survival in models of glaucoma and optic nerve trauma, axonopathy persists, thus raising the question of whether dendrites and the RGC light response are protected. Here, we used an inducible model of glaucoma in Bax-/-mice to determine if Bax contributes to RGC dendritic degeneration. We performed whole-cell recordings and dye filling in RGCs signaling light onset (αON-Sustained) and offset (αOFF-Sustained). We recovered RGC dendritic morphologies by confocal microscopy and analyzed dendritic arbor complexity and size. Additionally, we assessed RGC axon function by measuring anterograde axon transport of cholera toxin subunit B to the superior colliculus and behavioral spatial frequency threshold (i.e., spatial acuity). We found 1 month of IOP elevation did not cause significant RGC death in either WT or Bax-/-retinas. However, IOP elevation reduced dendritic arbor complexity of WT αON-Sustained and αOFF-Sustained RGCs. In the absence of Bax, αON-and αOFF-Sustained RGC dendritic arbors remained intact following IOP elevation. In addition to dendrites, neuroprotection by Bax-/-generalized to αON-and αOFF-Sustained RGC light-and current-evoked responses. Both anterograde axon transport and spatial acuity declined during IOP elevation in WT and Bax-/-mice. Collectively, our results indicate Bax contributes to RGC dendritic degeneration and distinguishes the proximal and distal neurodegenerative programs involved during the progression of glaucoma.
... Apoptosis is imperative for proper development, homeostasis, and cancer prevention in vertebrates [21,22]. A classic example of the importance of apoptosis in an organism's crucial development is a widely known example of the resorption (absorption into the circulation of cells and tissues) of a tadpole tail during its metamorphosis process [23]. ...
One of the vital aspects of a cell is cell death to continue their normal cell turnover, propagation, proper development, and the maintenance of the immune system. Cell death is an essential process in the body as it promotes the removal of unwanted cells. It is the programmed culling of cells in entire eukaryotic development processes to survive and progress for the next generation. Molecular aberration in the process of apoptosis may have pathological manifestations, including cancer, neurodegenerative disorders, autoimmune disease, and ischemic damage. Classically, cell death is categorized primarily into four different types: apoptosis, autophagy, necrosis, and entosis; depending on cellular and molecular signatures governing the pathway involved. The purpose of this review is to compare and contrast the recent literature on cell death and to familiarize with the current state of knowledge on this topic. In summary, the hallmarks of various modes of cell death are thoroughly explained along with the other types of cell death such as ferroptosis, pyroptosis, necroptosis, and lysosomal-dependent cell death.
... BAK and BAX have been suggested to function redundantly [18,19], despite potential differences in the levels of tissue expression and subcellular localisation (Figure 1) [20]. BAX is in a dynamic equilibrium between the cytosol and mitochondria, [21] and is predominantly found in the cytosol in its inactive state, as its α9-helix transmembrane (TM) domain is sequestered in its hydrophobic groove ( Figure 1) [8]. ...
The BCL-2 protein family govern whether a cell dies or survives by controlling mitochondrial apoptosis. As dysregulation of mitochondrial apoptosis is a common feature of cancer cells, targeting protein–protein interactions within the BCL-2 protein family is a key strategy to seize control of apoptosis and provide favourable outcomes for cancer patients. Non-BCL-2 family proteins are emerging as novel regulators of apoptosis and are potential drug targets. Voltage dependent anion channel 2 (VDAC2) can regulate apoptosis. However, it is unclear how this occurs at the molecular level, with conflicting evidence in the literature for its role in regulating the BCL-2 effector proteins, BAK and BAX. Notably, VDAC2 is required for efficient BAX-mediated apoptosis, but conversely inhibits BAK-mediated apoptosis. This review focuses on the role of VDAC2 in apoptosis, discussing the current knowledge of the interaction between VDAC2 and BCL-2 family proteins and the recent development of an apoptosis inhibitor that targets the VDAC2–BAK interaction.
... Bax is a member of Bcl2 family proteins and mediates neuronal cell death 8 , including physiological retinal apoptosis 9,10 and neuronal death in the Rb/p107 double knockout brain 11 . Bax-null males are infertile 12 . To generate α-Cre; ...
The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse (MGI:3052661) is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in olfactory neurons, implicating the influence of neighbouring regulatory elements. RT-PCR and the buried food pellet (BFP) test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.
TP53 encodes a transcription factor that is centrally-involved in several pathways, including the control of metabolism, the stress response, DNA repair, cell cycle arrest, senescence, programmed cell death, and others. Since the discovery of TP53 as the most frequently-mutated tumor suppressor gene in cancer over four decades ago, the field has focused on uncovering target genes of this transcription factor that are essential for tumor suppression. This search has been fraught with red herrings, however. Dozens of p53 target genes were discovered that had logical roles in tumor suppression, but subsequent data showed that most were not tumor suppressive, and were dispensable for p53-mediated tumor suppression. In this review, we focus on p53 transcriptional targets in two categories: (1) canonical targets like CDKN1A (p21) and BBC3 (PUMA), which clearly play critical roles in p53-mediated cell cycle arrest/senescence and cell death, but which are not mutated in cancer, and for which knockout mice fail to develop spontaneous tumors; and (2) a smaller category of recently-described p53 target genes that are mutated in human cancer, and which appear to be critical for tumor suppression by p53. Interestingly, many of these genes encode proteins that control broad cellular pathways, like splicing and protein degradation, and several of them encode proteins that feed back to regulate p53. These include ZMAT3, GLS2, PADI4, ZBXW7, RFX7, and BTG2. The findings from these studies provide a more complex, but exciting, potential framework for understanding the role of p53 in tumor suppression.
Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.
The male reproductive toxicity of microplastics (MPs) and nanoplastics (NPs) has attracted great attention, but the latent mechanisms remain fragmented. This review performed the adverse outcome pathway (AOP) analysis and meta-analysis in 39 relevant studies, with the AOP analysis to reveal the cause-and-effect relationships of MPs/NPs-induced male reproductive toxicity and the meta-analysis to quantify the toxic effects. In the AOP framework, increased reactive oxygen species (ROS) is the molecular initiating event (MIE), which triggered several key events (KEs) at different levels. At the cellular level, the KEs included oxidative stress, mitochondrial dysfunction, sperm DNA damage, endoplasmic reticulum stress, apoptosis and autophagy of testicular cells, repressed expression of steroidogenic enzymes and steroidogenic acute regulatory protein, disrupted hypothalamic-pituitary-testicular (HPT) axis, and gut microbiota alteration. These KEs further induced the reduction of testosterone, impaired blood-testis barrier (BTB), testicular inflammation, and impaired spermatogenesis at tissue/organ levels. Ultimately, decreased sperm quality or quantity was noted and proved by meta-analysis, which demonstrated that MPs/NPs led to a decrease of 5.99 million/mL in sperm concentration, 14.62% in sperm motility, and 23.56% in sperm viability, while causing an increase of 10.65% in sperm abnormality rate. Overall, this is the first AOP for MPs/NPs-mediated male reproductive toxicity in mammals. The innovative integration of meta-analysis into the AOP analysis increases the rigorism of the results.
Hypoxia has become an unfavorable factor affecting the sustainable development of the large yellow croaker Larimichthys crocea, an economically important mariculture fish in China. Apoptosis is a consequence of hypoxia on fish. However, the effects of hypoxia stress on apoptosis in L. crocea remain largely unknown. We investigated the effect of environmental hypoxia on apoptosis in L. crocea. Results show that hypoxia induced apoptosis in L. crocea both in vivo and in vitro. The mitochondrial membrane potential was significantly reduced in large yellow croaker fry (LYCF) cells. The expression levels of B-cell lymphoma/leukemia-2 (Bcl-2) mRNA and protein were also significantly decreased in the liver and LYCF cells during 96 h and 48 h of hypoxia stress, respectively, whereas the expression level of Bcl-2 associated X (Bax) mRNA, Casp3 mRNA, and activity of caspase-3/7/9 were significantly increased, indicating that hypoxia induced caspase-dependent intrinsic apoptosis in L. crocea. The expression level of the apoptosis-inducing factor (AIF) protein was significantly increased in the liver and LYCF cells. The level of AIF protein was significantly decreased in the cytoplasm but increased in the nuclei of L. crocea, demonstrating that hypoxia induced the AIF-mediated caspase-independent intrinsic apoptosis. In addition, the activity of caspase-8 was significantly increased, indicating that hypoxia stress induced extrinsic apoptosis in L. crocea. Therefore, hypoxia induced apoptosis in L. crocea through both the intrinsic and extrinsic pathways. The present study accumulated basic biological information to help elucidate the mechanism of hypoxia response in marine fish.
Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one‐cell stage to the blastocyst stage in vitro; however, long‐term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long‐term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax , which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis‐dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax , but not Bak1 , was responsible for these effects. These findings suggest that long‐term in vitro culture induces Bax ‐dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long‐term culture of rat preimplantation embryos for genetic engineering research.
Although external beam radiotherapy (xRT) is commonly used to treat central nervous system (CNS) tumors in patients of all ages, young children treated with xRT frequently experience life-altering and dose-limiting neurocognitive impairment (NI) while adults do not. The lack of understanding of mechanisms responsible for these differences has impeded the development of neuroprotective treatments. Using a newly developed mouse model of xRT-induced NI, we found that neurocognitive function is impaired by ionizing radiation in a dose- and age-dependent manner, with the youngest animals being most affected. Histologic analysis revealed xRT-driven neuronal degeneration and cell death in neurogenic brain regions in young animals but not adults. BH3 profiling showed that neural stem and progenitor cells, neurons, and astrocytes in young mice are highly primed for apoptosis, rendering them hypersensitive to genotoxic damage. Analysis of single-cell RNA sequencing data revealed that neural cell vulnerability stems from heightened expression of proapoptotic genes including BAX, which is associated with developmental and mitogenic signaling by MYC. xRT induced apoptosis in primed neural cells by triggering a p53- and PUMA-initiated, proapoptotic feedback loop requiring cleavage of BID and culminating in BAX oligomerization and caspase activation. Notably, loss of BAX protected against apoptosis induced by proapoptotic signaling in vitro and prevented xRT-induced apoptosis in neural cells in vivo as well as neurocognitive sequelae. On the basis of these findings, preventing xRT-induced apoptosis specifically in immature neural cells by blocking BAX, BIM, or BID via direct or upstream mechanisms is expected to ameliorate NI in pediatric patients with CNS tumor.
Significance
Age- and differentiation-dependent apoptotic priming plays a pivotal role in driving radiotherapy-induced neurocognitive impairment and can be targeted for neuroprotection in pediatric patients.
Circular RNA (circRNA) molecules are noncoding RNAs with ring-like structures formed by covalent bonds and are characterized by no 5′caps or 3′polyadenylated tails. Increasing evidence shows that circRNAs may play an important role in tumorigenesis and cancer metastasis. Circ-SHPRH originates from exons 26–29 of the SHPRH gene, and it is closely associated with human cancers. We searched PubMed, Web of Science, and Embase databases for relevant literatures until 24 December 2022. Eighteen research papers were included in this review, and 11 papers were selected for meta-analysis after screening. Three eligible published studies about circ-SHPRH were enrolled based on their tumor diagnosis aspect, 7 eligible published studies were related to overall survival (OS), and 3 eligible published studies were related to tumor grade. Many studies have shown that circ-SHPRH acts as a miRNA sponge or encodes a protein to regulate downstream genes or signal pathways, and exerts specific biological functions that affect the proliferation, invasion, and apoptosis of cancer cells. Meta-analysis showed that patients with high expression of circ-SHPRH had better OS (HR = 0.53, 95% CI 0.38–0.74, p-value <0.05) and lower TNM stage (HR = 0.33, 95% CI 0.18–0.62, p-value = 0.001). In addition, circ-SHPRH has potential diagnostic value (AUC = 0.8357). This review will help enrich our understanding of the role and mechanism of circ-SHPRH in human cancers. Circ-SHPRH has the potential to be a novel diagnostic and prognostic biomarker for various solid cancers.
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
HIVAN is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, we used a transgenic (Tg) mouse model (CD4C/HIV-Nef) in which HIV-1 nef expression is under control of regulatory sequences (CD4C) of the human CD4 gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis (FSGS) associated with microcystic dilatation, similar to human HIVAN. Proliferation of tubular and glomerular Tg cells is enhanced. To identify kidney cells permissive to the CD4C promoter, CD4C/GFP reporter Tg mice were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on ten different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (p53, TRAIL, TNF-α, TNF-R2, Bax), in immune cell recruitment (MIP-1α, MCP-1, CCR-2, CCR-5 and CX3CR-1), in NO formation (eNOS, iNOS) or in cell signaling (Fyn, Lck, Hck/Fgr). However, deletion of Src partially and that of Hck/Lyn largely abrogated its development. Our data suggest that Nef expression in mesangial cells through Hck/Lyn represents important cellular and molecular events for the development of HIVAN in these Tg mice.
Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.
This study aimed to determine the regulatory mechanism of bone morphogenetic protein 4 (BMP4) gene in the testes of Tibetan sheep and its role in the blood-testis barrier (BTB). First, we cloned BMP4 gene for bioinformatics analysis, and detected the mRNA and protein expression levels of BMP4 in the testes of Tibetan sheep pre-puberty (3 months old), during sexual maturity (1 year old), and in adulthood (3 years old) by qRT-PCR and Western blot. In addition, the subcellular localization of BMP4 was analyzed by immunohistochemical staining. Next, BMP4 overexpression and silencing vectors were constructed and transfected into primary Sertoli cells (SCs) to promote and inhibit the proliferation of BMP4, respectively. Then, CCK-8 was used to detect the proliferation effect of SCs. The expression of BMP4 and downstream genes, pathway receptors, tight junction-related proteins, and cell proliferation and apoptosis-related genes in SCs were studied using qRT-PCR and Western blot. The results revealed that the relative expression of BMP4 mRNA and protein in testicular tissues of 1Y group and 3Y group was dramatically higher than that of 3M group (P < 0.01), and BMP4 protein is mainly located in SCs and Leydig cells at different development stages. The CDS region of the Tibetan sheep BMP4 gene was 1229 bp. CCK-8 results demonstrated that the proliferation rate of BMP4 was significantly increased in the overexpression group (pc-DNA-3.1(+)-BMP4) (P<0.05). In addition, the mRNA and protein expressions of SMAD5, BMPR1A, BMPR1B and tight junction related proteins Claudin11, Occludin and ZO1 were significantly increased (P<0.05). The mRNA expression of cell proliferation-related gene Bcl2 was significantly enhanced (P<0.05), and the expression of GDNF was enhanced (P>0.05); The mRNA expression of apoptosis-related genes Caspase3 and Bax decreased significantly (P<0.05), while the mRNA expression of cell cycle-related genes CyclinA2 and CDK2 increased significantly (P<0.05). It is worth noting that the opposite results were observed after transfection with si-BMP4.In summary, what should be clear from the results reported here is that BMP4 affects testicular development by regulating the Sertoli cells and blood testes barrier, thereby modulating the spermatogenesis of Tibetan sheep.
The post-ganglionic sympathetic neurons play an important role in modulating visceral functions and maintaining homeostasis through complex and reproducible axonal and dendritic connections between individual neurons and with their target tissues. Disruptions in these connections and in sympathetic nervous system function are observed in several neurological, cardiac and immune-related disorders, which underscores the need for understanding the mechanisms underlying neuronal polarity, axonal growth and dendritic growth in these neurons. The goals of this chapter are to explore our current understanding of the various growth factors, their signaling pathways, downstream effectors and interplay between these pathways to regulate different stages of axonal and dendritic growth in sympathetic neurons.
Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK / BAX and for subsequent steps involving, as yet, undefined roles in kidney development.
Microplastics are widely distributed, such as oceans, rivers and the atmosphere, with many opportunities for human exposure and potential health risks. Polystyrene microplastic (PS-MPS) exposure has been found to cause sperm damage to mice; however, the mechanism by which this happens remains unclear. Here, GC-2 cells, a mouse spermatocyte line, were exposed to 5 µm PS-MPS to investigate mitochondrial damage. The results showed that 5 µm PS-MPS decreased ATP content, reduced the mitochondrial membrane potential, damaged the integrity of the mitochondrial genome, and caused an imbalance of homoeostasis between mitochondrial division and fusion. The mitochondrial PINK1/Parkin autophagy pathway was activated. Time-series analysis revealed that PS-MPS damaged the mitochondrial structure through cellular oxidative stress, and mitochondrial function was maintained to some extent after PS-MPS damage. This study revealed the mitochondrial toxicity of polystyrene microplastics, thus providing a basis for understanding the causes of sperm damage by polystyrene microplastics.
The effect of Bcl-xL upon the developmental death of T cells was assessed by generating transgenic mice that expressed Bcl-xL within all thymocyte subsets. Bcl-xL protected thymocytes from a variety of apoptotic stimuli, including gamma irradiation, glucocorticoids, and anti-CD3 treatment. Bcl-xL altered thymocyte maturation, resulting in increased numbers of CD3int/hi and CD4-8+ thymocytes. Overall, the phenotype of Bcl-xL transgenics was essentially indistinguishable from a Bcl-2 transgenic model. Overexpression of Bcl-xL or Bcl-2 resulted in the down-regulation of the other molecule, providing further evidence of their reciprocal regulation. In a genetic test of redundancy, the Bcl-xL transgene rescued mature T cells in Bcl-2 null mice. Immunoprecipitation indicated that Bcl-xL, like Bcl-2, heterodimerized with the death-promoting molecule Bax in thymocytes. This in vivo model argues that Bcl-xL, like Bcl-2, functions in a common pathway to repress cell death.
Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.
Most examples of cell death in animals are controlled by a genetic program that is activated within the dying cell. The apoptotic process is further regulated by a set of genes that act as repressors of cell death. Of these, bcl-2 is expressed in a variety of embryonic and postnatal tissues which suggests a critical role for bcl-2 in organogenesis and tissue homeostasis. Surprisingly, mutant mice with targeted disruption of bcl-2 appear normal at birth and complete maturation of lymphoid tissues before succumbing to fulminant lymphopenia and polycystic renal disease by 2-5 weeks of age. This suggests that there may be genes other than bcl-2 that can regulate apoptosis during development. To begin to investigate this possibility, we have cloned and characterized the murine bcl-x gene, whose human counterpart displays striking homology to bcl-2. The predicted murine bcl-xL gene product exhibits a high level of amino acid identity (97%) to its human counterpart. Just like Bcl-2, the murine bcl-xL gene product can act as a dominant inhibitor of cell death upon growth factor withdrawal. In addition, the bulk of the bcl-xL product localizes to the periphery of mitochondria as assessed by a bcl-xL-tag expression system, suggesting that both Bcl-2 and Bcl-xL proteins prevent cell death by a similar mechanism. bcl-xL is the most abundant bcl-x mRNA species expressed in embryonic and adult tissues. The levels of bcl-xL mRNA appear higher than those of bcl-2 during embryonal development and in several adult organs including bone marrow, brain, kidney and thymus. In addition to bcl-xL, we have identified another form of bcl-x mRNA, bcl-x beta, that results from an unspliced bcl-x transcript. bcl-x beta mRNA is expressed in various embryonic and postnatal tissues. Surprisingly, the expression of bcl-xS (a negative regulator of programmed cell death) was undetectable by a sensitive S1-nuclease assay and polymerase chain reaction analysis of mouse tissues. Based on its tissue and developmental patterns of expression, it appears that bcl-x may play an important role in the regulation of cell death during development and tissue homeostasis.
Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ (beta-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S. Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2. Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2, Bcl-X-L, or Mcl-1 but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.
The protein encoded by the bcl-2 gene is a regulator of programmed cell death and apoptosis. The cell survival-promoting activity of this protein is opposed by Bax, a homologous protein that forms heterodimers with Bcl-2 and accelerates rates of cell death. In this report, the in vivo patterns of bax gene expression were immunohistochemically assessed in the mouse, with a polyclonal antibody raised against a synthetic peptide corresponding to a unique region in the murine Bax protein. Direct comparisons were made with Bcl-2 by using anti-peptide antisera specific for the mouse Bcl-2 protein. The expression of bax was more widespread than bcl-2. For example, Bax immunoreactivity was present in the hepatocytes of the liver, the exocrine pancreas, and the renal tubule epithelial cells whereas Bcl-2 was absent from these tissues. Both the Bax and Bcl-2 proteins were present in several epithelia examined, including the small intestines, colon, breast, prostate, respiratory tract, and skin. The most intense Bax immunostaining was seen in cells located in the base of the crypts of the small intestinal mucosa, consistent with reports of high rates of spontaneous and inducible apoptosis in this region. Bcl-2 immunostaining was completely absent from these cells but was present in the absorptive epithelial cells of the small intestine. In contrast, Bax immunostaining in the colon tended to be stronger in the surface epithelial cells that had advanced up the crypts towards the lumen and that are destined for programmed cell death, whereas Bcl-2 immunoreactivity generally was stronger in the base of the colonic crypts. Similarly, bax expression in the gastric pits of the stomach occurred in a gradient such that higher levels of Bax immunostaining were found in the upper layers of gastric glands than in the lower regions. In addition, strong Bax immunostaining was detected in the androgen-dependent secretory epithelial cells of the prostate, whereas Bcl-2 was limited to the androgen-independent basal cells. Like Bcl-2, Bax was found in the thymic medulla but not the cortex, despite the propensity for immature cortical thymocytes to undergo apoptosis. Unlike Bcl-2, however, Bax immunostaining tended to be more intense in the germinal center lymphocytes of lymph nodes than in the interfollicular lymphocytes, consistent with the high rate of apoptotic cell death in the former.(ABSTRACT TRUNCATED AT 400 WORDS)
Transgenic mice which carry hybrid p53 promoter-chloramphenicol acetyltransferase (CAT) transgenes were found to express CAT enzymatic activity predominantly in the testes. Endogenous levels of p53 mRNA and protein were lower than in the nontransgenic control mice. The various p53 promoter-CAT transgenic mice exhibited in their testes multinucleated giant cells, a degenerative syndrome resulting presumably from the inability of the tetraploid primary spermatocytes to complete meiotic division. The giant-cell degenerative syndrome was also observed in some genetic strains of homozygous p53 null mice. In view of the hypothesis that p53 plays a role in DNA repair mechanisms, it is tempting to speculate that the physiological function of p53 that is specifically expressed in the meiotic pachytene phase of spermatogenesis is to allow adequate time for the DNA reshuffling and repair events which occur at this phase to be properly completed. Primary spermatocytes which have reduced p53 levels are probably impaired with respect to DNA repair, thus leading to the development of genetically defective giant cells that do not mature.
Death by apoptosis is characteristic of cells undergoing deletion during embryonic development, T- and B-cell maturation and endocrine-induced atrophy. Apoptosis can be initiated by various agents and may be a result of expression of the oncosuppressor gene p53 (refs 6-8). Here we study the dependence of apoptosis on p53 expression in cells from the thymus cortex. Short-term thymocyte cultures were prepared from mice constitutively heterozygous or homozygous for a deletion in the p53 gene introduced into the germ line after gene targeting. Wild-type thymocytes readily undergo apoptosis after treatment with ionizing radiation, the glucocorticoid methylprednisolone, or etoposide (an inhibitor of topoisomerase II), or after Ca(2+)-dependent activation by phorbol ester and a calcium ionophore. In contrast, homozygous null p53 thymocytes are resistant to induction of apoptosis by radiation or etoposide, but retain normal sensitivity to glucocorticoid and calcium. The time-dependent apoptosis that occurs in untreated cultures is unaffected by p53 status. Cells heterozygous for p53 deletion are partially resistant to radiation and etoposide. Our results show that p53 exerts a significant and dose-dependent effect in the initiation of apoptosis, but only when it is induced by agents that cause DNA-strand breakage.
Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.
Testis-specific lactate dehydrogenase, LDH-X, was localized spatially and temporally in the germinal epithelium of the mouse by the indirect fluorescent antibody technique. The enzyme was first detected in midpachytene primary spermatocytes and appeared to increase in concentration as spermatogenesis progressed to the spermatid. LDH-X synthesis, therefore, is initiated in the primary spermatocyte and continues at least until sometime during spermiogenesis. The uniform fluorescence of the cells containing LDH-X suggests that it is distributed throughout the cytoplasm rather than restricted to organelles such as mitochondria.
The phenomena of spermatogonial degeneration have been studied in normal adult rat testes using a simplified classification of the germinal epi-thelium based upon the six types of differentiating spermatogonia. The following features distinguished this from schemes based on acrosome development. Rather than 14 stages of unequal duration, there are only six stages, five of which are of the same length. The classification starts at the beginning of spermatogenesis with A¹ spermatogonia rather than at the onset of spermiogenesis. The classification is derived from actual biological events in spermatogenesis, namely generation times of spermatogonia, rather than upon arbitrary events in acrosome development. Most importantly, this new classification can be used with most types of preparations and in most experimental conditions.
The progressive movement of primary spermatocytes from the basal to the adluminal compartment of the seminiferous tubule was studies after testes were fixed with standard and hypertonic solutions. In stages VI, VII and VIII of the cycle (classification of Leblond and Clermont, '52), preleptotene spermatocytes were observed within the basal compartment of the seminiferous tubule. Resting on the basal lamina, these cells were bound tightly to neighboring Sertoli cells by desmosome-like junctions. In late stage VIII and early stage IX, basal processes of Sertoli cells were observed between the newly formed leptotene cells and the basal lamina, and in stage IX, the Sertoli processes met to form a junction of the zonula adherens type. This junction formed a permeability barrier which restricted the free access of fixative into the spaces around leptotene cells. Evidence for this was found in the absence of the shrinkage artifact produced with hypertonic solutions in earlier stages. In longitudinal sections, the permeability barrier was first observed in an area of the tubule in which sperm release was also taking place. In mid- stage IX and in stage X, Sertoli-Sertoli junctional specializations formed de novo below the leptotene spermatocyte, while those from the preceding stages, present above the leptotene spermatocytes, remained intact. Thus, tight junctions were in evidence for a considerable period of the time, both above and below the leptotene spermatocytes. At no time in the process of germ cell movement toward the lumen did these cells exhibit evidence of amoeboid movement or lose desmosome-like contacts with the surrounding Sertoli cells.
The BCL2 protooncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death. BCL2 was isolated from the chromosomal breakpoint of follicular B-cell lymphoma. Transgenic mice that overexpress BCL2 display extended survival of resting B cells. In this study we use a monospecific anti-human BCL2 antibody to define the distribution of BCL2 protein within organized tissues. BCL2 is restricted within germinal centers to the follicular mantle and to portions of the light zone implicated in the selection and maintenance of plasma cells and memory B cells. BCL2 is present in the surviving T cells in the thymic medulla. All hematopoietic lineages that derive from a renewing stem cell also display BCL2. A limited number of nonlymphoid tissues demonstrate BCL2 and can be grouped as (i) glandular epithelium in which hormones or growth factors regulate hyperplasia and involution, (ii) complex differentiating epithelium such as skin and intestine characterized by long-lived stem cells, and (iii) long-lived postmitotic cells such as neurons. Within these tissues that demonstrate apoptotic cell turnover, BCL2 is often topographically restricted to long-lived or proliferating cell zones. BCL2's function as an antidote to apoptosis may confer longevity to progenitor and effector cells in these tissues.
The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.
Human follicular B cell lymphomas possess a t(14;18) interchromosomal translocation that juxtaposes the putative proto-oncogene bcl-2 with the immunoglobulin (Ig) heavy chain locus. We generated minigene constructs representing the bcl-2-Ig fusion gene found at this chromosomal breakpoint. These constructs were placed into the germ line of mice to assess the effects of the t(14;18) during development. The transgene demonstrates a lymphoid pattern of expression and uniformly results in an expanded follicular center cell population. Hyperplastic splenic follicles coalesce to form massive regions of splenic white pulp. Mice over 15 weeks of age demonstrate regional lymphadenopathy with abnormal cellular infiltrates. The expanded lymphoid compartment is composed predominantly of polyclonal B220-positive, IgM/IgD-positive B cells. Provocatively, the bcl-2-Ig transgene confers a survival advantage to a population of mature B cells assessed in vitro. bcl-2-Ig transgenic mice document a prospective role for the t(14;18) in B cell growth and the pathogenesis of follicular lymphoma.
A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
T cell activation through the TCR can result in either cell proliferation or cell death. The role of costimulatory receptors in regulating T cell survival has not been defined. Here, we present data demonstrating that CD28 costimulation enhances the in vitro survival of activated T cells. One mechanism for this enhancement is the ability of CD28 costimulation to augment the production of IL-2, which acts as an extrinsic survival factor for T cells. In addition, CD28 costimulation augments the intrinsic ability of T cells to resist apoptosis. Although CD28 signal transduction had no effect on Bcl-2 expression, CD28 costimulation was found to augment the expression of Bcl-XL substantially. Transfection experiments demonstrated that this level of Bcl-XL could prevent T cell death in response to TCR cross-linking, Fas cross-linking, or IL-2 withdrawal. These data suggest that an important role of CD28 costimulation is to augment T cell survival during antigen activation.
A family of Bcl-2-related proteins regulates cell death and shares highly conserved BH1 and BH2 domains. BH1 and BH2 domains of Bcl-2 were required for it to heterodimerize with Bax and to repress apoptosis. A yeast two-hybrid assay accurately reproduced this interaction and defined a selectivity and hierarchy of further dimerizations. Bax also heterodimerizes with Bcl-xL, Mcl-1, and A1. A Gly-159-->Ala substitution in BH1 of Bcl-xL disrupted its heterodimerization with Bax and abrogated its inhibition of apoptosis in mammalian cells. This suggests that the susceptibility to apoptosis is determined by multiple competing dimerizations in which Bax may be a common partner.
Cells are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide. Apoptosis comprises an intrinsic cellular defence against tumorigenesis, which, when suppressed, may contribute to the development of malignancies. The bcl-2 oncogene, which is activated in follicular lymphomas, functions as a potent suppressor of apoptosis under diverse conditions. Here we describe the complementary DNA cloning and functional analysis of a new Bcl-2 homologue, Bak, which promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. This raises the possibility that Bak is directly involved in activating the cell death machinery.
A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response. The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation. The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins. We report here the identification of three cellular proteins that bind E1B 19K. One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer). This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref. 17) in yeast. In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.
Transgenic mice homozygously lacking in the bcl-2 gene were generated using homologous recombination in embryonal stem cells. The complete absence of Bcl-2 alpha and -beta proteins did not interfere with normal embryonic development. Abnormalities became evident after birth, although the severity varied among homozygous null mice, bcl-2-/- mice displayed pleiotropic abnormalities similar to those in the previously described bcl-2-/- mice, including growth retardation, smaller ears, short lives, polycystic kidney, atrophic thymus and spleen with accelerated apoptotic cell death of lymphocytes, and hair hypopigmentation in the second hair follicle cycle. Our bcl-2-/- mice also revealed novel defects in the small intestine, characterized by retarded development, accelerated exfoliation of epithelial cells, and very few mitotic progenitor cells.
The bax gene promoter region contains four motifs with homology to consensus p53-binding sites. In cotransfection assays using p53-deficient tumor cell lines, wild-type but not mutant p53 expression plasmids transactivated a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase. In addition, wild-type p53 transactivated reporter gene constructs containing a heterologous minimal promoter and a 39-bp region from the bax gene promoter in which the p53-binding site consensus sequences reside. Introduction of mutations into the consensus p53-binding site sequences abolished p53 responsiveness of reporter gene plasmids. Wild-type but not mutant p53 protein bound to oligonucleotides corresponding to this region of the bax promoter, based on gel retardation assays. Taken together, the results suggest that bax is a p53 primary-response gene, presumably involved in a p53-regulated pathway for induction of apoptosis.
To extend the mammalian cell death pathway, we screened for further Bcl-2 interacting proteins. Both yeast two-hybrid screening and lambda expression cloning identified a novel interacting protein, Bad, whose homology to Bcl-2 is limited to the BH1 and BH2 domains. Bad selectively dimerized with Bcl-xL as well as Bcl-2, but not with Bax, Bcl-xs, Mcl-1, A1, or itself. Bad binds more strongly to Bcl-xL than Bcl-2 in mammalian cells, and it reversed the death repressor activity of Bcl-xL, but not that of Bcl-2. When Bad dimerized with Bcl-xL, Bax was displaced and apoptosis was restored. When approximately half of Bax was heterodimerized, death was inhibited. The susceptibility of a cell to a death signal is determined by these competing dimerizations in which levels of Bad influence the effectiveness of Bcl-2 versus Bcl-xL in repressing death.
bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x.
The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature
neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic.
Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished.
The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability
of immature cells during the development of the nervous and hematopoietic systems.
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although
much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death
occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo
apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival
contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative
disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold
may have the potential to change the natural progression of some of these diseases.
In vivo thymocyte maturation models were used to investigate the differentiation role of Bcl-2. In alpha/beta T cell receptor (TCR) class II-restricted transgenic mice, Bcl-2 was upregulated at the CD4+ CD8+ stage during positive selection. The lckpr-bcl2 transgene was bred onto MHC classes I-I- and II-I-, MHC-I-, and alpha/beta TCR backgrounds to determine whether Bcl-2 promoted thymocyte maturation in the absence of coreceptor-MHC interaction. Bcl-2 rescued CD8+ thymocytes in class I-I- and alpha/beta TCR in mice; however, they were not exported to the periphery. Bcl-2 had no effect on CD4 lineage maturation in class II-I- mice. No single-positive thymocytes accumulate in MHC-I- mice despite overexpressed Bcl-2. Thus, Bcl-2 enables selection of certain TCRs on class II molecules and their differentiation along the CD8 pathway; however, Bcl-2 did not substitute for positive selection. In RAG-1-I- mice, Bcl-2 promoted differentiation to the CD4+ CD8+ stage. Bcl-2 can promote thymocyte maturation at several control points.
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Cell death is a common feature of neural development in all vertebrates. The bcl-2 proto-oncogene has been shown to protect a variety of cell types from programmed cell death. We have examined the distribution of bcl-2 protein in the developing and adult nervous systems. bcl-2 protein is widespread during embryonic development. Proliferating neuroepithelial cells of ventricular zones as well as the postmitotic cells of the cortical plate, cerebellum, hippocampus and spinal cord express bcl-2. Postnatally, bcl-2 is principally retained in the granule cells of the cerebellum and dentate gyrus of the hippocampus. bcl-2 expression in the CNS declines with aging. In the peripheral nervous system, neurons and supporting cells of sympathetic and sensory ganglia retain substantial bcl-2 protein throughout life. The widespread expression of bcl-2 in CNS and PNS neurons during embryonic development and its selective retention in the adult PNS is consistent with a role for bcl-2 in regulating neuronal survival. In addition, the expression of bcl-2 in some neuronal populations beyond the recognized period of cell death is suggestive of a role for bcl-2 beyond simply protecting neurons from developmental cell death.
Bcl-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths. An emerging family of Bcl-2-related proteins share two highly conserved regions referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-2. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitution of Gly 145 in BH1 domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in interleukin-3 deprivation, gamma-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homodimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.
The p53 tumor suppressor gene product can induce apoptotic cell death through an unknown mechanism. Here we demonstrate that a temperature-sensitive p53 induces temperature-dependent decreases in the expression of the apoptosis-suppressing gene bcl-2 in the murine leukemia cell M1, while simultaneously stimulating increases in the expression of bax, a gene which encodes a dominant-inhibitor of the Bcl-2 protein. Mice deficient in p53 exhibit increases in Bcl-2 and decreases in Bax protein levels in several tissues as determined by immunohistochemical and immunoblot methods. The findings suggest a potential mechanism by which p53 regulates apoptosis, as well as responses to radiation and chemotherapeutic drugs in cancer.
A rat IgM monoclonal antibody has been developed which recognized a mouse germ cell nuclear antigen (GCNA1). GCNA1 is present in prospermatogonia (gonocytes) in males and in oogonia and oocytes of females within the gonadal ridge from Embryonic Day 11.5 onward, but rarely in primordial germ cells prior to their arrival at the gonadal ridge. Immunolocalization demonstrates that GCNA1 is abundant in nuclei of spermatogonia and early spermatocytes, but decreases during subsequent spermatocyte and round spermatid development, and is not detected beyond step 10 elongating spermatids. The antigen is approximately 80-110 kDa on immunoblots of isolated pachytene spermatocytes and round spermatids. However, GCNA1 appears to be absent from sperm in the epididymis and vas deferens, Sertoli cells, TM3 cells (Leydig-like) and TM4 cells (Sertoli-like), lung, liver, kidney, spleen, heart, skin, brain, epididymis, and ovary. GCNA1 is present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in oocytes of neonatal females until Postpartum Day 12. GCNA1 is first lost from oocytes in the medulla of the ovary as they arrest at the dictyate stage and gain a layer of granulosa cells. In addition, antigen is present in moderate amounts in F9 embryonal carcinoma cells and SCC-PSA1 pluripotent terato-carcinoma cells. Thus, GCNA1 serves as a common marker of the germ cell lineage in male and female mice after primordial germ cells arrive in the gonadal ridge until they reach the diplotene/dictyate stage of the first meiotic division.
A cascade of new research findings is giving researchers insights into the genes that control programmed cell death. The genes that carry out the suicide program are coming into the light. One particularly hot area of the cell-death field concerns several genes that seem to direct and execute the death program inside cells.
Bcl-2 protein is able to repress a number of apoptotic death programs. To investigate the mechanism of Bcl-2's effect, we examined whether Bcl-2 interacted with other proteins. We identified an associated 21 kd protein partner, Bax, that has extensive amino acid homology with Bcl-2, focused within highly conserved domains I and II. Bax is encoded by six exons and demonstrates a complex pattern of alternative RNA splicing that predicts a 21 kd membrane (alpha) and two forms of cytosolic protein (beta and gamma). Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death induced by cytokine deprivation in an IL-3-dependent cell line. Overexpressed Bax also counters the death repressor activity of Bcl-2. These data suggest a model in which the ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus.
We report the isolation of bcl-x, a bcl-2-related gene that can function as a bcl-2-independent regulator of programmed cell death (apoptosis). Alternative splicing results in two distinct bcl-x mRNAs. The protein product of the larger mRNA, bcl-xL, is similar in size and predicted structure to Bcl-2. When stably transfected into an IL-3-dependent cell line, bcl-xL inhibits cell death upon growth factor withdrawal at least as well as bcl-2. Surprisingly, the second mRNA species, bcl-xS, encodes a protein that inhibits the ability of bcl-2 to enhance the survival of growth factor-deprived cells. In vivo, bcl-xS mRNA is expressed at high levels in cells that undergo a high rate of turnover, such as developing lymphocytes. In contrast, bcl-xL is found in tissues containing long-lived postmitotic cells, such as adult brain. Together these data suggest that bcl-x plays an important role in both positive and negative regulation of programmed cell death.
The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous
inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature
T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast,
stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus,
and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune
system after birth.
bcl-2-/-mice complete embryonic development, but display growth retardation and early mortality postnatally. Hematopoiesis including lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution. Thymocytes require an apoptotic signal to manifest accelerated cell death. Renal failure results from severe polycystic kidney disease characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium. bcl-2-/-mice turn gray with the second hair follicle cycle, implicating a defect in redox-regulated melanin synthesis. The abnormalities in these loss of function mice argue that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway.
The p53 tumour suppressor gene is the most widely mutated gene in human tumorigenesis. p53 encodes a transcriptional activator whose targets may include genes that regulate genomic stability, the cellular response to DNA damage, and cell-cycle progression. Introduction of wild-type p53 into cell lines that have lost endogenous p53 function can cause growth arrest or induce a process of cell death known as apoptosis. During normal development, self-reactive thymocytes undergo negative selection by apoptosis, which can also be induced in immature thymocytes by other stimuli, including exposure to glucocorticoids and ionizing radiation. Although normal negative selection involves signalling through the T-cell receptor, the induction of apoptosis by other stimuli is poorly understood. We have investigated the requirement for p53 during apoptosis in mouse thymocytes. We report here that immature thymocytes lacking p53 die normally when exposed to compounds that may mimic T-cell receptor engagement and to glucocorticoids but are resistant to the lethal effects of ionizing radiation. These results demonstrate that p53 is required for radiation-induced cell death in the thymus but is not necessary for all forms of apoptosis.
BCL-2-IMMUNOGLOBULIN TRANSGENIC MICE DEMONSTRATE EXTENDED B-CELL SURVIVAL AND FOLLICULAR LYMPHOPROLIFERATION
- J Mcdonnell T
- MCDONNELL T.J.
IMMUNOHISTOCHEMICAL ANALYSIS OF MCL-1 AND BCL-2 PROTEINS IN NORMAL AND NEOPLASTIC LYMPH-NODES
- Immunohistochemical Krajewski S
- Analysis
- Mcl
- KRAJEWSKI S
BCL-2 IS UP-REGULATED AT THE CD4(+)CD8(+) STAGE DURING POSITIVE SELECTION AND PROMOTES THYMOCYTE DIFFERENTIATION AT SEVERAL CONTROL POINTS
- LINETTE G.P.
MASSIVE CELL-DEATH OF IMMATURE HEMATOPOIETIC-CELLS AND NEURONS IN BCL-X-DEFICIENT MICE
- Massive Cell-Death Of Immature Hematopoietic-Cells And Neurons In Bcl-X-Deficient Motoyama N
- Mice
- MOTOYAMA N
EXPRESSION OF MEMBERS OF THE BCL-2 GENE FAMILY IN THE IMMATURE RAT OVARY - EQUINE CHORIONIC GONADOTROPIN-MEDIATED INHIBITION OF GRANULOSA-CELL APOPTOSIS IS ASSOCIATED WITH DECREASED BAX AND CONSTITUTIVE BCL-2 AND BCL-X(LONG) MESSENGER-RIBONUCLEIC-ACID LEVELS
- TILLY J.L.
BCL2 PROTEIN IS TOPOGRAPHICALLY RESTRICTED IN TISSUES CHARACTERIZED BY APOPTOTIC CELL-DEATH
- HOCKENBERY D.M.
IMMEDIATE-EARLY UP-REGULATION OF BAX EXPRESSION BY P53 BUT NOT TGF-BETA-1 - A PARADIGM FOR DISTINCT APOPTOTIC PATHWAYS
- Immediate-Early Up-Regulation Of Selvakumaran M
- Bax
- By
- But
- Tgf-Beta
- SELVAKUMARAN M