Apoptosis,Pyroptosis, and Necroptosis—Oh My! The Many Ways a Cell Can Die

Cell death is an essential process in all living organisms and occurs through different mechanisms. The three main types of programmed cell death are apoptosis, pyroptosis, and necroptosis, and each of these pathways employs complex molecular and cellular mechanisms. Although there are mechanisms and outcomes specific to each pathway, they share common components and features. In this review, we discuss recent discoveries in these three best understood modes of cell death, highlighting their singularities, and examining the intriguing notion that common players shape different individual pathways in this highly interconnected and coordinated cell death system. Understanding the similarities and differences of these cell death processes is crucial to enable targeted strategies to manipulate these pathways for therapeutic benefit.     

Basic fibroblast growth factor-induced cell death is effected through sustained activation of p38MAPK and up-regulation of the death receptor p75NTR

Basic fibroblast growth factor (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro. In this study we demonstrate that this is dependent on the rapid and sustained activation of p38(MAPK), in contrast to the transient activation of p38(MAPK) associated with bFGF-induced cell proliferation. Stem cell factor-induced survival of TC-32 cells was also associated with transient activation of p38(MAPK). Inhibition of p38(MAPK) by SB202190 and p38(MAPK) small interfering RNA reduces bFGF-induced death in TC-32 cells, consistent with the hypothesis that activation of p38(MAPK) is essential for induction of death by bFGF. This appears to be dependent on sustained activation of p38(MAPK), demonstrated by inhibition of bFGF-induced cell death following addition of SB202190 to TC-32 cells 5 min after exposure to bFGF (20 ng/ml) and activation of p38(MAPK). Prolonged activation of p38(MAPK) is accompanied by a rapid and sustained phosphorylation of Ras and ERK; inhibition of ERK phosphorylation using the MEK-1 inhibitor PD98059 rescued approximately 30% of cells from bFGF-induced death suggesting ERK plays a secondary role in the induction of death. This hypothesis is supported by observations in the A673 cell line; bFGF induced sustained activation of ERK and transient activation of p38(MAPK), which was not associated with cell death. These data demonstrate that sustained activation of p38(MAPK) is essential for activation of the death cascade following exposure of Ewing's sarcoma family of tumors cells to bFGF and provide evidence that activation of p38(MAPK) results in an up-regulation of the death receptor p75(NTR).     

To close or not to close: Plasmodesmata in defense

Cell death is a biological process that occurs during differentiation and maturation of certain cell types, during senescence, or as part of a defense mechanism against microbial pathogens. Intercellular coordination is thought to be necessary to restrict the spread of death signals, although little is known about how cell death is controlled at the tissue level. The recent characterization of a plasmodesmal protein, PDLP5, has revealed an important role for plasmodesmal control during salicylic acid-mediated cell death responses. Here, we discuss molecular factors that are potentially involved in PDLP5 expression, and explore possible signaling networks that PDLP5 interacts with during basal defense responses.     

Pathogen-induced programmed cell death in plants,a possible defense mechanism

As much as the definition of life may be controversial, the definition of death also may prove problematic. In recent years it became apparent that the death of a living cell may follow more than one possible scenario: it may result from an externally applied physical injury (an accidental death), or it may be the outcome of activating an internal pathway for cell suicide (a programmed death). That cells can participate in their own execution may indicate that certain types of cell deaths that were previously considered to be caused by foreign agents such as pathogens or drugs may actually result from the activation of a programmed cell death pathway that is normally latent in cells. Here, we describe the activation of such a cell suicide pathway in plant cells upon the recognition of an invading pathogen. We discuss the possible use of this pathway as a defense mechanism against infection and the possibility that in many ways the use of this type of cell death in plants is functionally analogous to that used by mammalian cells in response to infection by pathogens. Dev. Genet. 21:279–289, 1997. © 1997 Wiley-Liss, Inc.     

Cell cycle re-entry sensitizes podocytes to injury induced death

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.     

Development of a multipotent clonal human periodontal ligament cell line

Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor’s Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor’s Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Analysis of endothelial cell locomotion: Differential effects of motility and contact inhibition

Video microscopy and digital time-lapse recording were used to monitor locomotion and proliferation of bovine pulmonary artery endothelial (BPAE) cells cultured with varying concentrations of basic fibroblast growth factor (bFGF). Cell trajectories were reconstructed using a generalized nearest-neighbor algorithm and analyzed to determine how cell motility is affected by cell-cell collisions, cell divisions, and increasing cell density. The temporal evolution patterns of the average speed of locomotion for all cells in a culture were computed and the effects of varying bFGF concentrations were analyzed. Intermediate concentrations of bFGF (30 and 50 ng/mL) significantly increased the speed of locomotion above the levels we observed with 0 and 100 ng/mL concentrations of bFGF. Increases in cell density due to proliferation were immediately accompanied by a decrease in the average speed of locomotion of the cell population. Finally, the effect of bFGF concentration on the overall cell proliferation rates was assessed. With the addition of 30 or 50 ng/mL of bFGF to the culture media, the observed cell proliferation rates increased significantly. The proliferation rates decreased when the bFGF concentration increased to 100 ng/mL. These results show that bFGF concentrations that increase the motility of BPAE cells also increase the observed cell proliferation rates. (c) 1994 John Wiley & Sons, Inc.     

p53与细胞死亡

p53是一种重要的肿瘤抑制因子,是迄今发现与人类肿瘤相关性最高的分子之一。超过50%的人类肿瘤含有p53基因突变。因此,p53是肿瘤治疗中的重要分子靶点。p53依赖的细胞凋亡是其抑制肿瘤的重要机制之一。然而,最近研究发现,p53不仅参与细胞凋亡,还与程序性细胞坏死、细胞自噬以及铁诱导的细胞死亡等细胞死亡途径相关。促使肿瘤细胞死亡是肿瘤治疗的重要目标。因此,进一步了解p53与细胞死亡之间的关系,将有助于探索以p53为靶点的肿瘤治疗和p53相关肿瘤细胞耐药机制。     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Programming of cell death during xylogenesis

Death of tracheary elements which compose vessels and tracheids is a typical example of programmed cell death in plants. Anin vitro system usingZinnia mesophyll cells which differentiate directly into tracheary elements has provided various types of data on the cell death process. In this paper, we will summarize recent results obtained using theZinnia system and discuss the programming of cell death during tracheary element differentiation. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.     

Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium

Although the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell-associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin-releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell-associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell-associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.     

Divergence in Signaling Pathways Involved in Promotion of Cell Viability Mediated by bFGF,NGF, and EGF in PC12 Cells

We employed a series of inhibitors of intracellular cascade to disclose the precise molecular mechanisms by which basic fibroblast growth factor (bFGF) promotes viability of PC12 cells and compared with nerve growth factor (NGF) and epidermal growth factor (EGF). The MEK 1 and 2 inhibitors, U0126 and PD98059, significantly suppressed cell viability mediated by bFGF in a dose-dependent manner, and to a greater extent compared with EGF and NGF. The degree of MEK dependency for growth factor-mediated cell viability was estimated to be in the order of bFGF, EGF, and NGF. Rapamycin strongly inhibited the effect of NGF on cell viability, compared with bFGF and EGF. The mechanisms of action of NGF-mediated cell viability may depend largely on p70 S6 kinase-related signal transduction pathways comparing to bFGF and EGF. The present findings suggest that different signal transduction systems may be involved in the molecular mechanisms by which bFGF, NGF, and EGF mediate cell viability.     

Density-dependent differentiation in nontransformed human retinal progenitor cells in response to basic fibroblast growth factor- and transforming growth factor-alpha

Multipotential retinal precursors give rise to all cell types seen in multilayered retina. The generation of differentiation and diversity of neuronal cell types is determined by both extrinsic regulatory signals and endogenous genetic programs. We have previously reported that cell commitment in human retinal precursor cells (SV-40T) can be modified in response to exogenous growth factors, basic fibroblast growth factor, and transforming growth factor alpha (bFGF and TGFalpha). We report in this study that nontransformed human retinal precursors differentiate into photoreceptors by a cell density-dependent mechanism, and the effects were potentiated by bFGF and TGFalpha alone or in combination. A larger proportion of multipotential precursors plated at a density of 1 x 10(4) cells/cm(2) differentiated into neurons (photoreceptors) compared to cells plated at 3-5 x 10(4)/cm(2) and 1 x 10(5) cells/cm(2) under serum-free conditions and the effects were amplified seven- to eightfold in response to growth factors. Basic fibroblast growth factor (bFGF) and TGFalpha can induce 90% of the cells to assume a photoreceptor phenotype at a lower cell density, compared to only 30 and 25% of the cells acquiring a photoreceptor phenotype at intermediate and higher cell densities. Furthermore, at a lower cell density, 60-70% of the cells incorporate Bromodeoxyuridine (Brdu), suggesting that cells in a cell cycle may make a commitment to a specific fate in response to neurotrophins. Neurons with a photoreceptor phenotype were positive for three different sets of antibodies for rods/cones. Cells also exhibited upregulation of other proteins such as a D4 receptor protein expressed in photoreceptors, protein kinase Calpha (PKCalpha) expressed in rod bipolars and blue cones, and some other neuronal cell types. This was also confirmed by Western blot analysis. Newly derived photoreceptors survive for a few days before significant cell death ensues under serum-free conditions. To summarize, differentiation in precursors is density dependent, and growth factors amplify the effects.     

Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen whose actions are mediated by binding to specific cell surface receptors on a variety of cell types. However, the amino acid sequence of bFGF does not contain a classical signal peptide sequence and the extent to which cellular stores of this mitogen are released is still a matter of some controversy. In the present study we examined the release of immunoreactive bFGF into serum-free conditioned medium of bovine corneal endothelial cells (BCE) and a human astrocytoma cell line, U87-MG. Western blotting analysis of BCE conditioned medium using N-terminal specific anti-bFGF serum revealed a single immunoreactive band of 32 kilodaltons, which was reduced to 18 kilodaltons in the presence of 8 M urea. Using a sensitive two-site immunoradiometric assay we were able to quantify the release of immunoreactive bFGF into the culture medium by BCE cells and by the human astrocytoma cell line U87-MG. In each case the release of bFGF was cell density dependent, but under all conditions the level of bFGF released was significantly greater in the transformed astrocytoma line, ranging from 15- to 50-fold higher than in the BCE cultures under various conditions. At 30% confluence the concentration of immunoreactive bFGF in the medium was maintained at a constant level for up to 24 h. However, the level of immunoreactive bFGF declined rapidly in confluent cells.(ABSTRACT TRUNCATED AT 250 WORDS)