Correlation between steady-state cell concentration and cell death of hybridoma cultures in chemostat

In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (k(d)) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of k(d) vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The k(d) value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h(-1). The k(d) for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. (c) 1995 John Wiley & Sons, Inc.     

Fusicoccin induces in plant cells a programmed cell death showing apoptotic features

Summary. Programmed cell death plays a pivotal role in several developmental processes of plants and it is involved in the response to environmental stresses and in the defense mechanisms against pathogen attack. It has not yet been defined which part of the death signalling mechanism and which molecules involved in programmed cell death are common to animals and plants. In this paper we show that fusicoccin, a well-known phytotoxin, induces a strong acceleration in the appearance of Evans Blue-stainable (dead) cells in sycamore (Acer pseudoplatanus L.) cultures. This fusicoccin-induced cell death shows aspects common to the form of animal programmed cell death termed apoptosis: i.e., cell shrinkage, changes in nucleus morphology, increase in DNA fragmentation detectable by a specific immunological reaction, and presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Since fusicoccin has a well-identified molecular target, the plasma membrane H⁺-ATPase, and thoroughly investigated physiological effects, this toxin appears to be a useful tool to study the transduction of death signals leading to programmed cell death in plants.Correspondence and reprints: Dipartimento di Biotecnologie e Bioscienze, Universitä degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.     

在自然衰老和诱导条件下棉花悬浮细胞程序性死亡的发生

Cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities on the 17th day, and on the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmark of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors which included heatshock (42+/-3 degrees C for 8 hours), 10 micromol/L camptothecin, 20 micromol/L fumonisin B1 and 50 mmol/L cycloheximide. The cotton suspension cells which grew in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT differred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IBA and 0.1 mg/L KT, and they responded differentially to the heatshock, 10 micromol/L camptothecin and 20 micromol/L fumonisin B1, while the same to 50 mmol/L cycloheximide.     

Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta)

Two major pathways of programmed cell death (PCD)--the apoptotic and the autophagic cell death--were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin-proteasome system.     

Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death

Cell death can occur by two basically different processes. The original term, necrosis, is now reserved for the generally destructive series of events which include the release of lysosomal enzymes and loss of cell membrane integrity. In contrast, mild treatment with cell damaging agents, or withdrawal of growth factors, may result in a characteristic form of degradation of cellular DNA which is associated with cell death that has morphology known as apoptosis. In this study human leukemia cells were exposed to agents or conditions previously reported to cause necrosis or apoptosis, monitored by detection of DNA “ladders,” and the integrity of cellular DNA was determined on Southern blots. Nuclear DNA was distinguished from mitochondrial DNA by use of probes specific for nuclear genes or for mitochondrial DNA. When HL60, K562, MOLT4, or U937 cells were exposed to conditions which resulted in necrosis, mitochondrial DNA was damaged at approximately the same rate as nuclear DNA, but in apoptosis mtDNA was not degraded. Thus, the ratio of the relative (to untreated cells) abundance of mitochondrial DNA measured by a probe for 16S mitochondrial ribosomal RNA on Southern blots, to the relative abundance of DNA of any nuclear gene, was 1 or less in necrosis, but rose to values greater than 2 in apoptosis. It is concluded that the comparison of the degree of fragmentation of mitochondrial and nuclear DNA provides a quantitative way of distinguishing necrosis from apoptosis.     

Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures

Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical heterogeneity in cell death: combined synchrotron IR and fluorescence microscopy studies of single apoptotic and necrotic cells

The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures

Association of the availability of nutrients with the phenomenon of programmed cell death-apoptosis-was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. (c) 1995 John Wiley & Sons, Inc.     

The contribution of cell death to medium-released fractions of cell cultures

Summary Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [³H]thymidine and by subsequently measuring the release of label into complete culture medium or serum-and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [³H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the³H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account. This research benefited from use of the Cell Culture Facility supported by National Cancer Institute Grant CA 14733. This research was supported by a Muscular Dystrophy Association Grant to Dr. Heinz Herrmann and by American Cancer Society Grant RDP 8 and was submitted in partial fulfillmant of a Ph.D. degree at the University of Connecticut (T. C. Doetschman).     

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。     

Interplay between autophagy and programmed cell death in mammalian neural stem cells

Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390]     

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。     

Kinetic characterization of baculovirus-induced cell death in insect cell cultures

The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (t(d)) and a first-order death phase characterized by a half-life (t(1/2)). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection (c) 1993 John Wiley & Sons, Inc.