Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Inhibition of apoptosis in serum starved porcine embryonic fibroblasts

In nuclear transplantation, serum starvation is a general method to synchronize donor cells at the quiescent stage (G(0)) of the cell cycle. However, serum starvation during culture of mammalian cells may induce cell death, especially through apoptosis, thus contributing to the low efficiency of nuclear transplantation. This study was performed to characterize apoptosis during serum starvation and to determine the effects of apoptosis inhibitors such as a protease inhibitor [alpha(2)-macroglobulin (MAC)] and antioxidants [N-acetylcysteine (NAC), glutathione (GSH)] on serum starved porcine embryonic fibroblasts (PEF). PEF, collected from day 25-30 porcine fetuses, were cultured for 5 days in media containing 0.5% FBS to induce quiescence. Serum starved PEF showed typical morphology of apoptotic cells and stained for DNA fragmentation by TUNEL assay (26.7%). All apoptosis inhibitors tested in this study significantly (P < 0.05) reduced apoptosis of serum starved PEF, with antioxidants having better results (MAC: 7.4% vs. NAC: 1.0%, and GSH: 0.8%). Equally and importantly, the treatment with apoptosis inhibitors did not change the proportion of G(0)/G(1) stage cells. Therefore, the addition of MAC and antioxidants during serum starvation of PEF reduces apoptosis of quiescent fibroblasts and may contribute to increasing the efficiency of nuclear transplantation by improving the quality of donor nuclei.     

Production of calves from G1 fibroblasts.

Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a "shake-off" procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the "shake-off" treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from "shake-off" cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.     

Induction of apoptosis in fibroblasts by c-myc protein.

Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.     

Bcl-2 is a key factor for cardiac fibroblast resistance to programmed cell death

Cardiac fibroblasts play an essential role in the physiology of the heart. These produce extracellular matrix proteins and synthesize angiogenic and cardioprotective factors. Although fibroblasts of cardiac origin are known to be resistant to apoptosis and to remain metabolically active in situations compromising cell survival, the underlying mechanisms are unknown. Here, we report that cardiac fibroblasts were more resistant than dermal or pulmonary fibroblasts to mitochondria-dependent cell death. Cytochrome c release was blocked in cardiac fibroblasts but not in dermal fibroblasts treated with staurosporine, etoposide, serum deprivation, or simulated ischemia, precluding caspase-3 activation and DNA fragmentation. Resistance to apoptosis of cardiac fibroblasts correlated with the expression of the anti-apoptotic protein Bcl-2, whereas skin and lung fibroblasts did not express detectable levels of this protein. Bcl-x(L,) Bax, and Bak were expressed at similar levels in cardiac, dermal, and lung fibroblasts. In addition, the death of cardiac fibroblasts during hypoxia was not associated with the cleavage of Bid but rather with Bcl-2 disappearance, suggesting the requirement of the mitochondrial apoptotic machinery to execute death receptor-induced programmed cell death. Knockdown of bcl-2 expression by siRNA in cardiac fibroblasts increased their apoptotic response to staurosporine, serum, and glucose deprivation and to simulated ischemia. Moreover, dermal fibroblasts overexpressing Bcl-2 achieved a similar level of resistance to these stimuli as cardiac fibroblasts. Thus, our data demonstrate that Bcl-2 is an important effector of heart fibroblast resistance to apoptosis and highlight a probable mechanism for promoting survival advantage in fibroblasts of cardiac origin.     

Combined experimental and computational analysis of DNA damage signaling reveals context‐dependent roles for Erk in apoptosis and G1/S arrest after genotoxic stress

Following DNA damage, cells display complex multi‐pathway signaling dynamics that connect cell‐cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell‐cycle re‐entry and proliferation, permanent cell‐cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time‐resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin‐induced DNA damage in the presence or absence of TNFα co‐treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell‐cycle regulatory responses. Two relational modeling approaches were then used to identify network‐level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term ‘time‐interval stepwise regression.’ Taken together, the results from these analysis methods revealed complex, cytokine‐modulated inter‐relationships among multiple signaling pathways following DNA damage, and identified an unexpected context‐dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug.     

Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.     

Apoptosis Dependent Decrease of the Intramembrane Ion Traffic in Cultured Mouse Fibroblasts Shown by Conductivity Dispersion

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.     

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes

The aim of the present study was to determine whether porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (SCNT) in pigs. Primary culture of porcine preadipocytes was established by de-differentiating mature fat cells taken from an adult pig. The cell cycle of the preadipocytes could be synchronized by serum starvation for 1 day, with a higher efficiency than control fetal fibroblasts. Incidence of premature chromosome condensation following nuclear transfer (NT) of preadipocytes was as high as that observed after NT with fetal fibroblasts. In vitro developmental rate of the NT embryos reconstructed with preadipocyte was equivalent to that of the fetal fibroblast derived embryos. Transfer of 732 NT embryos with preadipocytes to five recipients gave rise to five cloned piglets. These data demonstrate that preadipocyites collected from an adult pig are promising nuclear donor cells for pig cloning.     

Sulbutiamine Counteracts Trophic Factor Deprivation Induced Apoptotic Cell Death in Transformed Retinal Ganglion Cells

Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B₁ and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture.     

TNF triggers mitogenic signals in NIH 3T3 cells but induces apoptosis when the cell cycle is blocked

Tumor necrosis factor (TNF) is known to be a mediator of a variety of cellular responses including apoptotic death or proliferation depending on the target cell and the environmental conditions. We show here that TNF triggers both growth and death signals in NIH 3T3 murine fibroblasts. In cells arrested in G(0) by serum deprivation, TNF drives approximately 50% of them to enter the cell cycle, but kills the cells that remain quiescent. The presence of serum prevents toxic effects of TNF, suggesting that TNF can cooperate to drive cells through the cell cycle, but is unable to do so by itself and alternatively it triggers death signals in cells unable to proliferate. Interestingly, TNF induces a similar toxic effect in cells forced to stay at the G(1)/S border, S or M phases. We have explored the TNF apoptotic pathway in arrested cells. This mechanism is not due to the loss of the anti-apoptotic capacity of NFkappaB and is mediated by mitochondria since Bcl-2 overexpression partially inhibits cell death. There are, however, interesting differences in the kinetics of mitochondrial events which indicate that this form of sensitization to TNF leads to an apoptotic mechanism different from that observed after sensitization by RNA synthesis inhibition.     

Analysis of aclarubicin-induced cell death in human fibroblasts

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Anti-apoptotic and antiproliferative effect of bcl-2 gene transferred to E1A+cHa-ras-transformed cells

Transformed rat embryo fibroblasts E1A + cHa-ras known to possess high proapoptotic sensitivity and not to be arrested after DNA damage or upon serum starvation, were transfected with bcl-2 gene using calcium-phosphate precipitation method. Triple transformants E1A + cHa-ras + bcl-2 appeared to be protected from damage- and serum depletion-induced apoptosis and to restore cell cycle checkpoint control. Using the method of flow cytometry we have shown that these transformants are arrested in different phases of cell cycle in response to irradiation, adriamycin treatment and serum deprivation. Overexpression of bcl-2 in E1A + cHa-ras-transformed cells entirely suppresses adriamycin-induced apoptosis and significantly reduces the level of apoptosis triggered by irradiation and growth factor withdrawal, as we have revealed by the test of clonogenic survival and electrophoretic analysis of oligonucleosomal DNA fragmentation. Our results have demonstrated, for the first time, that the oncogenic Ras co-immunoprecipitates with transfected Bcl-2 in E1A + cHa-ras + bcl-2 transformed cells after irradiation but not after adriamycin treatment. Bcl-2-Ras complexes were also observed in transformants E1A + cHa-ras + bcl-2 after serum starvation. Taken together, these data suggest that Bcl-2 and Ras interaction might play a crucial role in the cell cycle checkpoints restoration and apoptotic events regulation in transformants E1A + cHa-ras + bcl-2 exposed to DNA-damaging factors or growth factor-deprived.     

Block of a mitochondrial-mediated apoptotic pathway in Tax-expressing murine fibroblasts

Although the viral transactivator Tax has been established as an essential effector of HTLV-I-mediated oncogenesis, its exact role(s) in the pathogenesis of HTLV-I-associated diseases, which include both a neurodegenerative pathology and leukemia/lymphoma, remains to be clarified. It was recently advanced that dysregulation of the apoptotic process can lead to pathophysiological changes which result in either degenerative diseases or cancer. As the apoptotic potential of Tax is still debated, we addressed this question by testing the susceptibility of Tax(+) and Tax(-) murine fibroblasts to apoptosis under conditions of growth factor withdrawal or treatment with TNFalpha, which trigger apoptosis through different pathways, i.e., mitochondrial and receptor-mediated pathways, respectively. Results showed that Tax-expressing cells are protected from apoptotic death induced by serum deprivation but are sensitive to TNFalpha-mediated apoptosis, suggesting that Tax expression has different effects on cell death, depending on the apoptotic stimulus used. Analysis of the mechanism(s) involved in the resistance to serum depletion-induced apoptosis indicated that Tax(+) cells do not undergo release of cytochrome c from the mitochondrial intermembrane space or redistribution of Bax from the cytosol to mitochondria, two phenomena critical to the mitochondrial apoptotic pathway.     

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.