Induction of programmed death/apoptosis androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal

Shionogi Carcinoma 115 (SC 115) cells are a cloned cell line derived from androgen-dependent mouse mammary tumor. They can grow in serum-free culture if a physiological level of androgen is present in the medium, but can not proliferate in culture without testosterone. In the present study, the mechanism of cell death in SC 115 cells after androgen withdrawal was examined. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, androgen withdrawal induces programmed cell death (apoptosis) of SC 115 cells in serum-free culture. Northern blot analysis was used to identify a series of genes whose expression per cell is enhanced during the recruitment of cells from a nonproliferative (i.e. G0) state into G1 (i.e.,cyclins D1 and C), from G1 into the S phase of the cell cycle (i.e., cdk2), and during the programmed cell death pathway (i.e. testosterone repressed prostatic message-2 (TRPM-2), transforming growth factor-beta1 (TGF-beta1) and glucose regulated 78 kilodalton protein (GRP-78). Expression of TRPM-2, TGF-beta1, GRP-78, and calmodulin genes increases, but that of cyclins C and D1, and cdk2 genes decreases during programmed cell death of SC 115 cells. These results demonstrate that androgen-dependent SC 115 cells undergo programmed cell death induced by androgen withdrawal, and that this death does not require proliferation or progression into G1 of the proliferative cell cycle. SC 115 cells should be a good model for investigating programmed death of hormone-dependent cancer.     

Regulation of germ cell death in mammalian gonads

A large number of primordial germ cells (PGCs), as well as spermatogonia, undergo programmed cell death or apoptosis in the physiological context. In this process, environmental, cytoplasmic and nuclear factors are involved. Bcl-2 and its related molecules are known as general regulators of cell death, and some are important for survival of PGCs and spermatogonia. Steel factor, a ligand for c-Kit, also supports growth and survival of these cells. In addition, bone morphogenetic protein (BMP)8B and Desert Hedgehog (Dhh), which are secreted proteins, and a nuclear factor, c-Myc, play a role in spermatocyte survival. This suggests that germ cell survival or death at each stage of differentiation is precisely controlled by specific signalling pathways which consist of a number of molecules.     

Characterization of furazolidone apical-related effects to human polarized intestinal cells

Neuron numbers in developing vertebrate organisms are regulated by the availability of growth factors which promote their survival. However, neuron survival may also be regulated by growth factors which promote rather than prevent cell death. This study examined the effects of bone morphogenetic proteins (BMPs) in inducing apoptosis of MAH cells, an immortalized sympathoadrenal progenitor cell line. Treatment of MAH cells with BMP2 or BMP4 killed the cells in a dose-dependent manner. By contrast, treatment with BMP7 or TGFbeta1 failed to affect survival, suggesting that induction of apoptosis is specific to the dpp subgroup of BMPs. Survival after treatment with BMP2 or BMP4 required addition of fibroblast growth factor (FGF) and nerve growth factor (NGF), indicating that BMP treatment made the neurons dependent upon an exogenous factor for survival. Several experimental observations suggested an apoptotic mechanism for BMP-induced death. After BMP2 treatment, the cells progressively shrank and became pyknotic. Further, there was prominent endonucleosomic cleavage of DNA (laddering) as well as TUNEL staining. Moreover, BMP-induced death was inhibited by the caspase inhibitor z-VAD and was partially prevented by the endonuclease inhibitor aurintricarboxylic acid. These observations suggest that neuron numbers may be regulated by factors which promote death and that exposure to such factors may be a signal for the development of dependence upon other growth factors for survival.     

FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development

The development of calvarial bones is tightly co-ordinated with the growth of the brain and needs harmonious interactions between different tissues within the calvarial sutures. Premature fusion of cranial sutures, known as craniosynostosis, presumably involves disturbance of these interactions. Mutations in the homeobox gene Msx2 as well as the FGF receptors cause human craniosynostosis syndromes. Our histological analysis of mouse calvarial development demonstrated morphological differences in the sagittal suture between embryonic and postnatal stages. In vitro culture of mouse calvaria showed that embryonic, but not postnatal, dura mater regulated suture patency. We next analysed by in situ hybridisation the expression of several genes, which are known to act in conserved signalling pathways, in the sagittal suture during embryonic (E15-E18) and postnatal stages (P1-P6). Msx1 and Msx2 were expressed in the sutural mesenchyme and the dura mater. FGFR2(BEK), as well as Bmp2 and Bmp4, were intensely expressed in the osteogenic fronts and Bmp4 also in the mesenchyme of the sagittal suture and in the dura mater. Fgf9 was expressed throughout the calvarial mesenchyme, the dura mater, the developing bones and the overlying skin, but Fgf4 was not detected in these tissues. Interestingly, Shh and Ptc started to be expressed in patched pattern along the osteogenic fronts at the end of embryonic development and, at this time, the expression of Bmp4 and sequentially those of Msx2 and Bmp2 were reduced, and they also acquired patched expression patterns. The expression of Msx2 in the dura mater disappeared after birth. FGF and BMP signalling pathways were further examined in vitro, in E15 mouse calvarial explants. Interestingly, beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts, but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume both when placed on the osteogenic fronts and on the mid-sutural area, but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. We suggest that the local application of FGF on the osteogenic fronts accelerating suture closure in vitro, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors. Taken together, our data suggest that conserved signalling pathways regulate tissue interactions during suture morphogenesis and intramembranous bone formation of the calvaria and that morphogenesis of mouse sagittal suture is controlled by different molecular mechanisms during the embryonic and postnatal stages. Signals from the dura mater may regulate the maintenance of sutural patency prenatally, whereas signals in the osteogenic fronts dominate after birth.     

BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo

At E4.0 the inner cell mass of the mouse blastocyst consists of a core of embryonic ectoderm cells surrounded by an outer layer of primitive (extraembryonic) endoderm, which subsequently gives rise to both visceral endoderm and parietal endoderm. Shortly after blastocyst implantation, the solid mass of ectoderm cells is converted by a process known as cavitation into a pseudostratified columnar epithelium surrounding a central cavity. We have previously used two cell lines, which form embryoid bodies that do (PSA1) or do not (S2) cavitate, as an in vitro model system for studying the mechanism of cavitation in the early embryo. We provided evidence that cavitation is the result of both programmed cell death and selective cell survival, and that the process depends on signals from visceral endoderm (Coucouvanis, E. and Martin, G. R. (1995) Cell 83, 279-287). Here we show that Bmp2 and Bmp4 are expressed in PSA1 embryoid bodies and embryos at the stages when visceral endoderm differentiation and cavitation are occurring, and that blocking BMP signaling via expression of a transgene encoding a dominant negative mutant form of BMP receptor IB inhibits expression of the visceral endoderm marker, Hnf4, and prevents cavitation in PSA1 embryoid bodies. Furthermore, we show that addition of BMP protein to cultures of S2 embryoid bodies induces expression of Hnf4 and other visceral endoderm markers and also cavitation. Taken together, these data indicate that BMP signaling is both capable of promoting, and required for differentiation of, visceral endoderm and cavitation of embryoid bodies. Based on these and other data, we propose a model for the role of BMP signaling during peri-implantation stages of mouse embryo development.     

Induction of Bax-alpha precedes apoptosis in a human B lymphoma cell line: potential role of the bcl-2 gene family in surface IgM-mediated apoptosis

The members of the bcl-2 gene family are major regulators of programmed cell death, but their role in sIg-triggered apoptosis remains unclear. Using sensitive and resistant variants of the human B cell line BL-41, we studied the expression of the bcl-2 gene family during surface IgM-mediated apoptosis. We found constitutive Bcl-2 and Bcl-x expression, which remained unaltered after sIg cross-linking, in both resistant and sensitive cells. This and other experiments suggest that constitutive expression of Bcl-2 or Bcl-x alone is not sufficient to protect from activation-induced cell death in B cells. We therefore investigated Bax-alpha, the death-promoting splice variant of Bax, and found strong induction of both mRNA and protein upon sIg stimulation in sensitive cells. However, resistant subclones showed only weak expression, which was not inducible by sIg cross-linking. We provide evidence that up-regulation of Bax-alpha and the resulting imbalance of Bcl-2/Bax might be a major regulator of sIg-mediated apoptosis. Additionally, we found strong constitutive expression of Bcl-xs, the death promoting variant of Bcl-x, in sensitive cells, whereas resistant cells showed only weak Bcl-xs expression. Thus, we observed a much stronger expression of the death-promoting proteins Bax-alpha (inducible) and Bcl-xs (constitutive) in sensitive cells than in resistant cells. We therefore propose a potential role of the novel bcl-2 gene family members bcl-x and bax in surface IgM-triggered apoptosis.     

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2-lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.     

Overexpression of a Hu-bcl-2 transgene in Lurcher mutant mice delays Purkinje cell death

Cerebellar Purkinje cells in the heterozygous Lurcher mutant undergo cell autonomous degeneration beginning in the second week of postnatal development and becoming almost total around 30-45 days. The Lurcher mutation was recently identified as gain-of-function defect in the delta 2 glutamate receptor causing a constitutive current leak, suggesting that +/Lc Purkinje cells die by an excitotoxic mechanism. In previous studies we have shown that overexpression of bcl-2, a key regulator of cell death, in the heterozygous Lurcher mutant does not prevent +/Lc Purkinje cell death. To investigate further the mechanisms of +/Lc Purkinje cell death, we have crossed +/Lc mutants with a second line of Hu-bcl-2 transgenics (NSE73a) that shows an earlier onset of transgene expression and higher expression levels. Analysis of eight +/Lc-NSE73a mutants (4 at 2 months and 4 at 5-6 months) showed that Hu-bcl-2 overexpression delayed, but ultimately could not prevent +/Lc Purkinje cell death.     

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by a equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.     

IL-6 rescues resting mouse T cells from apoptosis

It has previously been demonstrated that mature mouse T cells live for many weeks in vivo. In contrast, explanted lymph node or splenic T cells undergo spontaneous death within days, suggesting that survival factors supplied in vivo are not present in normal tissue culture medium. We discovered that IL-6 can rescue resting T cells from apoptosis in vitro. We show that recombinant mouse IL-6 as well as IL-6 in endothelial cell supernatants are sufficient to rescue T cells from death in the absence of additional cytokines. We show that CD4+ T cells express Bcl-2 immediately following isolation from the mouse, but after 24 h in culture Bcl-2 is undetectable. If during this time period the T cells are incubated with rIL-6, Bcl-2 expression is not down-regulated. It is, therefore, possible that IL-6 rescue from death is mediated by maintenance or induction of Bcl-2 expression. Addition of rIL-6 does not by itself induce blastogenesis or proliferation, and therefore, this cytokine appears to be a true survival factor rather than a mitogenic factor for resting T cells. Together, these results support a potential role for IL-6 as one of the factors important for prolonging resting T cell survival in vivo.     

MyD genes in negative growth control

Two interrelated cellular processes are invoked simultaneously upon induction of differentiation, the regulated progression of cells through successive stages of cell differentiation and growth inhibition which ultimately leads to growth arrest. In tissues with rapid cell turnover terminally differentiated cells undergo programmed cell death. Terminal differentiation, thus, represents one form of negative growth control. It was surmised that the molecular engine which drives the differentiation process forward requires induction of positive regulators of terminal cell differentiation, to be found among differentiation primary response genes, as well as suppression of negative regulators, which correspond to genes which control cellular growth. This line of thought has prompted the isolation of myeloid differentiation primary response (MyD) genes activated in the absence of de novo protein synthesis, upon IL-6 induced terminal differentiation of murine M1 myeloblastic leukemia cells, where the cells growth arrest and ultimately undergo programmed cell death. As delineated in this review many of the genes identified as MyD genes, including both known genes [IRF-1, (AP-1)Fos/Jun.EGR-1] and novel ones (MyD88, MyD116, MyD118), turned out to play a role in negative growth control, including growth suppression and apoptosis, in many cell types, of both hematopoietic and non hematopoietic origins.     

Formation of Rathke's pouch requires dual induction from the diencephalon

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.     

The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.     

v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells

Interleukin-3 (IL-3) is required for the proliferation, survival and differentiation of myeloid progenitors. In the absence of IL-3, murine myeloid 32D.3 cells accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death, or apoptosis. Here we demonstrate that enforced expression of the v-raf oncogene suppresses apoptosis of myeloid 32D.3 cells following the withdrawal of IL-3. Surprisingly, steady state levels of Bcl-2, an oncogene known to suppress apoptosis, were not dependent upon IL-3 in 32D.3 cells and its levels were not augmented in v-raf clones. This suggests that ability of v-raf to suppress apoptosis in the absence of ligand is either Bcl-2 independent or that v-raf kinase promotes Bcl-2 function. v-raf also promoted growth of these cells in the presence of IL-3. v-raf clones proliferated at an increased rate due to a shortened G1 phase and had decreased requirements for IL-3 for growth. Therefore, transformation of myeloid cells by v-raf involves signaling pathways which promote both cell cycle progression and cell survival.