Phospholipase C and D in the commitment to survival or death in the early lag phase of tetrahymena cultures

We made three kinds of experiments in order to elucidate aspects of physiological mechanisms involved in a series of specific events leading to either cell death or survival in the lag phase of culture growth. We studied the fate of newly inoculated Tetrahymena cells in small droplets at 'high' (more than 1000 cells ml(-1)) and 'low' cell densities (less than 600 cells ml(-1)) in a nutrionally complete, synthetic nutrient medium. Confirming previous results we found that the cells in high-density cultures multiplied to final densities around 500,000 cells ml(-1) and that cells in low-density cultures died before multiplying. The appearance of the cells was recorded with a video camera at 20 frames per second for 6 h or until they died. The results indicated that the death process took place within milliseconds. We also studied the effects of U 73122, an inhibitor of the phosphatidylinositol-specific phospholipase C, on cell survival at low densities. At low inhibitor concentrations low-density cells were rescued from dying. At high inhibitor concentrations all cells died, and phosphatidylinositol - but not phosphatidylserine and phosphatidylcholine - saved them. The results indicate that the paths leading to either cell death or to cell proliferation separate within the first few minutes after subcultivation into a new medium, since the first cells in each culture died within 4-30 min after inoculation. Our results also indicate that some PLC activity was required for stimulation of phospholipase D, and that cell death during the early lag phase is caused by a shortage in phosphatidylinositol before the phospholipase D activity is upregulated. These experiments are shedding light on the lethal consequences of a cellular depletion of the important signalling compound phosphatidylinositol in an in vivo system, and may help to elucidate mechanisms behind the century-old fact that eukaryote cells die when inoculated at too low a cell density to survive.     

EDR is a stress-related survival factor from stroma and other tissues acting on early haematopoietic progenitors (E-Mix)

The erythroid differentiation regulator (EDR) is a highly conserved autocrine factor produced in many tissues. Its haemoglobin synthesis-inducing activity for human and murine erythroleukaemia cell lines had been detected in WEHI-3 conditioned medium. EDR functions were analysed in detail. It is released from cells immediately in response to various stressful conditions and enhances cell survival particularly at a lower concentration range and low cell density. At high cell density and high EDR concentration the opposite effect of an increase in cell death was observed. Its essential function within a tissue is considered to be the maintenance of growth homeostasis. Cells kept in culture for weeks show a decreasing responsiveness to EDR supply. This was also noted in freshly cloned EDR-responsive mouse erythroleukaemia cells pointing to a molecular adaptation process. Human haematopoietic progenitors were amplified 7-fold by EDR when kept at low cytokine levels. At saturating levels progenitors giving rise to at least two lineages in semisolid medium (E-mix) respond to EDR with an average 1.87-fold increase in colony numbers and a bell-shaped dose-response curve. Of the more mature BFU-E compartment a response was observed particularly in cases with low colony numbers. Given the release from irradiated stromal cells and the ability to partly substitute for stromal cells in the Burkitt's lymphoma cell line BL-70, EDR functions as a stromal survival factor for stroma-responsive cells.     

Cell Density-Dependent Death Mode Switch of Cultured Cortical Neurons Under Serum-Free Starvation Stress

1. Cell death mode switch of cortical neurons from E17 rats was studied. Cells rapidly died under the serum-free condition. The time-course of cell death was markedly delayed by increasing cell density for primary culture in the trypan blue exclusion, LDH release, and MTT assays.2. By analyzing cell death by the use of double staining using PI/TUNEL and PI/Annexin V combinations, the mode in the low density culture was found to be necrosis, while that in the high density culture was apoptosis.3. The intracellular ATP level after the start of serum-free culture rapidly decline to 25% of 0-time level in the low density culture, but it was 60% in the high density culture. Both oligomycin and zVAD-fmk markedly decreased ATP levels and the population of TUNEL-positive neurons, while 3-aminobenzamide slightly increased these indices.4. Thus, it is strongly suggested that the cell death mode switch from necrosis to apoptosis is closely related to intracellular ATP levels, and some conditioned medium factors observed in the high density culture may affect both ATP level and cell death mode switch.     

Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts

Cell survival was significantly decreased in primary cultured rat calvarial osteoblasts in vitro at Day 0, 1, and 3 by replacement of the standard culture medium (alpha-modified minimum essential medium; alpha-MEM) with Dulbecco's modified eagle's medium (DMEM). Decreased cell survival was also observed following medium replacement in cultures of murine calvaria-derived osteoblastic cell line MC3T3-E1. Staining with Hoechst33342 revealed apoptotic cells with fragmented or condensed nuclei, while a fraction of the cell culture was stained with propidum iodide, indicating necrosis. Marked increases in DNA binding of both activator protein-1 and nuclear factor-kappaB were found in nuclear extracts of cells following medium replacement. The addition of either pyruvate or cysteine at each concentration found in alpha-MEM almost entirely prevented cell death associated with medium replacement at Day 3. These results suggest that pyruvate and cysteine may be essential factors for cell growth and survival in osteoblast cultures at the proliferative phase.     

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model

Activated microglia release inflammatory mediators that display either beneficial or harmful effects on neuronal survival and signaling. In the present study we demonstrate that exposure to lipopolysaccharide leads to an increase in the lysosomal cysteine proteases, cathepsin B, K, S, and X, in culture supernatants of the microglia cell line BV-2. In addition, we observed an up-regulation of cathepsins in the cytoplasmic fraction in response to stimulation with lipopolysaccharide. Conditioned medium from these cells was toxic to the neuroblastoma cell line Neuro2a. Experiments with membrane-permeable and membrane-impermeable cysteine protease inhibitors suggested that blocking extracellular cathepsins had no effect on microglia-mediated neuron death in this medium transfer model. However, intracellular cathepsins seem to trigger the release of neurotoxic factors. In lipopolysaccharide-stimulated BV-2 cells, inhibition of intracellular cathepsins significantly diminished microglial activation characterized by reduced expression of different proinflammatory cytokines, thereby reducing the neurotoxic effects of the medium. This hitherto unknown intracellular effect of cysteine proteases in activated microglia might connect chronic neuroinflammation with neurodegeneration.     

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.     

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24°C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38°C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38°C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.     

Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.     

Low-Density Induced Apoptosis of Cortical Neurons Is Inhibited by Serum Factors

1. We investigated the survival of neurons under serum-free conditions without any exogenous signal molecules, using primary cultures of rat cerebral cortex.2. Survival activity, measured with Alamar Blue, showed a cell density dependency under serum-free conditions.3. The addition of fetal bovine serum suppressed the apoptotic cell death accompanied by DNA-laddering and fragmentation specific in low-density cultures, resulting in the disappearance of the cell density dependency of survival.4. These findings suggest that serum factors may substitute for endogenous survival factors from cortical neurons in high-density cultures.     

Insulin-like growth factor-mediated muscle cell survival: central roles for Akt and cyclin-dependent kinase inhibitor p21

Polypeptide growth factors activate specific transmembrane receptors, leading to the induction of multiple intracellular signal transduction pathways which control cell function and fate. Recent studies have shown that growth factors promote cell survival by stimulating the serine-threonine protein kinase Akt, which appears to function primarily as an antiapoptotic agent by inactivating death-promoting molecules. We previously established C2 muscle cell lines lacking endogenous expression of insulin-like growth factor II (IGF-II). These cells underwent apoptotic death in low-serum differentiation medium but could be maintained as viable myoblasts by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we show that IGF-I promotes muscle cell survival through Akt-mediated induction of the cyclin-dependent kinase inhibitor p21. Treatment of myoblasts with IGF-I or transfection with an inducible Akt maintained muscle cell survival and enhanced production of p21, and ectopic expression of p21 was able to sustain viability in the absence of growth factors. Blocking of p21 protein accumulation through a specific p21 antisense cDNA prevented survival regulated by IGF-I or Akt but did not block muscle cell viability mediated by PDGF-BB. Our results define Akt as an intermediate and p21 as a critical effector of an IGF-controlled myoblast survival pathway that is active during early myogenic differentiation and show that growth factors are able to maintain cell viability by inducing expression of pro-survival molecules.     

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.Correspondence to: M.G. Höfle     

Control of lens epithelial cell survival

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Dual control of muscle cell survival by distinct growth factor-regulated signaling pathways

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.     

Serum-free,chemically defined medium to evaluate the direct effects of growth factors and inhibitors on proliferation and function of neonatal rat cardiac muscle cells in culture

Summary Neonatal rat cardiac myocytes were isolated and cultured to evaluate the effects of growth factors and inhibitors on proliferation, survival, and functions in a serum-free medium. Insulin and transferrin in MCDB 107 nutrient medium elicited DNA and protein synthesis in cells on a fibronectin-coated culture surface in serum-free medium. Insulin was most effective on both DNA and protein synthesis in serum-free culture conditions. The serum-free, hormone-supplemented medium eliminated the contamination of noncardiac myocytes and supported the long-term survival (over 18 d) of cardiac myocytes. Dexamethasone was required to induce optimal contractility with or without insulin and transferrin. Serum contained both negative and positive effectors of DNA and protein synthesis of the cardiac myocytes. Concentrations of serum (above 5%) inhibited DNA and protein synthesis. Low density lipoprotein (LDL) accounted in part for the inhibitory activity. The serum-free culture system provides a useful model to elucidate the role of hormones, growth factors, and drugs in heart cell regeneration and function.     

Acute lethality in irradiated flour beetles: Evidence against a role of bacterial infection

The time course of mortality and the mean survival time of flour beetles dying after X-irradiation varied slightly in different strains and species. Within a given strain, however, both minimum and mean survival time were independent of dose over a wide range of exposures and independent of percent mortality. These findings suggest the operation of a single mode of death in irradiated beetles, in contrast to the “hierarchy of modes of death” recognized in irradiated mammals. Invasion of blood and tissues by enteric bacteria which gain entrance through the damaged intestinal lining, and subsequent spread of such infection, have been implicated in the lethal syndrome of irradiated mammals. The present investigation indicates that bacterial invasion does not contribute to the death of irradiated beetles. This conclusion is based on the lack of influence on survival of (1) administration of neomycin at levels which eliminated virtually all bacteria from the insects, (2) crowding or isolation, or (3) maintenance of irradiated beetles on medium in which other beetles previously had been raised. In addition, analysis of the distribution of deaths among a large number of vials each containing a small number of beetles disclosed no deviation from expectation based on random distribution; thus it seems unlikely that there is any “spread” of contagion within vials.     

Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.