Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.     

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

Ceramide triggers metacaspase-independent mitochondrial cell death in yeast

The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains

We have previously demonstrated that hexanoyl-D-erythro-sphingosine (C(6)-ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C zeta (PKC zeta) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKC zeta, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKC zeta was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C(6)-ceramide treatment results in an increased localization and phosphorylation of PKC zeta within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKC zeta with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKC zeta-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKC zeta phosphorylation of Akt3 at Ser(34) was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKC zeta and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.     

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.     

Increased Tyrosine Kinase Activity but Not Calcium Mobilization Is Required for Ceramide-Induced Apoptosis

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.     

High levels of exogenous C2-ceramide promote morphological and biochemical evidences of necrotic features in thyroid follicular cells

CD95 and ceramide are known to be involved in the apoptotic mechanism. The triggering of CD95 induces a cascade of metabolic events that progressively and dramatically modifies the cell shape by intense membrane blebbing, leading to apoptotic bodies production. Although the CD95 pathway has been abundantly described in normal thyrocytes, the effects of cell permeable synthetic ceramide at morphological and biochemical levels are not fully known. In the present study, we show that thyroid follicular cells (TFC) exposed to 20 microM of C(2)-ceramide for 4 h are characterized by morphological features of necrosis, such as electron-lucent cytoplasm, mitochondrial swelling, and loss of plasma membrane integrity without drastic morphological changes in the nuclei. By contrast, TFC treated with 2 microM of C(2)-ceramide for 4 h are able to accumulate GD3, activate caspases cascade, and induce apoptosis. Furthermore, we provide evidence that 20 microM of C(2)-ceramide determine the destruction of mitochondria and are not able to induce PARP cleavage and internucleosomal DNA fragmentation, suggesting that the apoptotic program is not activated during the death process and nuclear DNA is randomly cleaved as the consequence of cellular degeneration. Pretreatment with 30 microM of zVAD-fmk rescued TFC from 2 microM of C(2)-ceramide-induced apoptosis, whereas, 20 microM of C(2)-ceramide exposure induced necrotic features. Deltapsi(m) was obviously altered in cells treated with 20 microM of C(2)-ceramide for 4 h (75% +/- 3.5%) compared with the low percentage (12.5% +/- 0.4%) of cells with altered Deltapsi(m) exposed to 2 microM of C(2)-ceramide. Whereas, only 20% +/- 1.1% of cells treated with anti-CD95 for 1 h showed altered Deltapsi(m). Additionally, Bax and Bak, two pro-apoptotic members, seem to be not oligomerized in the mitochondrial membrane following ceramide exposure. These results imply that high levels of exogenous ceramide contribute to the necrotic process in TFC, and may provide key molecular basis to the understanding of thyroid signaling pathways that might promote the apoptotic mechanism in thyroid tumoral cells.     

Integration of glycosphingolipid metabolism and cell-fate decisions in cancer and stem cells: Review and Hypothesis

The metabolism of glycosphingolipids is strictly regulated during the mitotic cell cycle. Before the G1-to-S transition, the ceramide and glucosylceramide concentration is elevated. Ceramide induces apoptosis synergistically with the pro-apoptotic protein prostate apoptosis response 4 (PAR-4) that may be asymmetrically inherited during cell division. Only one daughter cell dies shortly after mitosis, a mechanism we suggested to regulate the number of neural stem cells during embryonic development. The progeny cells, however, may protect themselves by converting ceramide to glucosylceramide and other glycosphingolipids. In particular, complex gangliosides have been found to sustain cell survival and differentiation. The cell cycle may thus be a turning point for (glyco)sphingolipid metabolism and explain rapid changes of the sphingolipid composition in cells that undergo mitotic cell-fate decisions. In the proposed model termed "Shiva cycle", progression through the cell cycle, differentiation, or apoptosis may rely on a delicate balance of (glyco)sphingolipid second messengers that modulate the retinoblastoma-dependent G1-to-S transition or caspase-dependent G1-to-apoptosis program. Ceramide-induced cell cycle delay at G0/G1 is either followed by ceramide-induced apoptosis or by conversion of ceramide to glucosylceramide, a proposed key regulatory rheostat that rescues cells from re-entry into a life/death decision at G1-to-S. We propose a mechanistic model for sphingolpid-induced protein scaffolds ("slip") that regulate cell-fate decisions and will discuss the biological consequences and pharmacological potential of manipulating the (glyco)sphingolipid-dependent cell fate program in cancer and stem cells.     

Distinct apoptotic phenotypes induced by radiation and ceramide in both p53-wild-type and p53-mutated lymphoblastoid cells

The tumour suppressor gene p53 and the intracellular signalling molecule ceramide have both been shown to play crucial roles in the induction of apoptosis by ionising radiation. In this study we examined whether p53 and ceramide are involved in independent signal pathways, inducing different types of apoptosis. TK6 (p53^wt/wt) and WTK1 (p53^mut/mut) lymphoblastoid cells were treated with ionising radiation or N-acetyl-d-sphingosine (C₂-ceramide). Flow cytometry and fluorescence microscopy studies were performed to characterise the time kinetics and morphological features of induced apoptosis. Ceramide- and radiation-induced apoptotic cells display characteristic differences in morphology and DNA staining and ceramide-induced apoptosis is expressed much faster than radiation-induced apoptosis. Radiation-induced apoptosis is p53-dependent and ceramide-induced apoptosis is p53-independent. The p53 pathway and the ceramide pathway are two independent signal pathways leading to distinct types of apoptosis. Since p53 is very often dysfunctional in tumour cells, modifying the ceramide pathway is a promising strategy to increase tumour sensitivity to radiation and other anticancer agents. Received: 19 April 2001 / Accepted: 15 October 2001     

Hepatocyte growth factor protects gastric epithelial cells against ceramide-induced apoptosis through induction of cyclooxygenase-2

Forced overexpression of cyclooxygenase-2 (COX-2) in intestinal cells has been shown to be associated with resistance to apoptosis. However, the role of physiologically-induced COX-2 in the regulation of apoptosis remains unclear. In the present study, we examined whether hepatocyte growth factor (HGF)-induced COX-2 affects ceramide-induced apoptosis in RGM-1 gastric epithelial cells. An externally applied cell permeable ceramide analogue, C2-ceramide, caused RGM-1 cell death in a dose-dependent manner, whereas an inactive ceramide analogue, C2-dihydroceramide, did not. TdT-mediated dUTP nick end labeling (TUNEL) assay showed that the C2-ceramide-induced cell death was apoptosis. Application of HGF rapidly induced the expression of COX-2, and HGF prevented the apoptotic cell death induced by C2-ceramide. However, the anti-apoptotic action of HGF was antagonized by coapplication of NS-398, a selective inhibitor of COX-2. Thus, these results indicate that COX-2 is involved in the survival signaling from HGF in gastric epithelial cells, and suggest a role for physiologically-induced COX-2 in the protection of the cells from apoptosis.     

DAP kinase activity is critical for C(2)-ceramide-induced apoptosis in PC12 cells.

Exposure of PC12 cells to C(2)-ceramide results in dose- dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-gamma-induced apoptosis of HeLa cells, in the C(2)-ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 microm C(2)-ceramide, both the total (assayed in the presence of Ca(2+)/calmodulin) and Ca(2+)/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 microm C(2)-ceramide, the Ca(2+)/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C(2)-ceramide concentration. C(8)-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C(2)-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C(2)- and C(8)-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C(2)-, and C(8)-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.     

Ceramide Induces Non-Apoptotic Cell Death in Human Glioma Cells

Ceramide causes either apoptosis or non-apoptotic cell death depending on model system and experimental conditions. The present study was undertaken to examine the effect of ceramide on cell viability and its molecular events leading to cell death in A172 human glioma cells. Ceramide induced cell death in a dose-dependent manner and the cell death was dependent on generation of reactive oxygen species and lipid peroxidation. TUNEL assay, Hoechst 33258 staining, and flow cytometric analysis did not show typical apoptotic morphological features. Ceramide caused phosphorylation of extracellular signal-regulated kinase (ERK) and p38, but the cell death was not affected by inhibitors of MAPK subfamilies. Ceramide caused ATP depletion without loss of mitochondrial membrane potential. Ceramide did not induce caspase activation and ceramide-induced cell death was also not altered by inhibitors of caspase activation. Transfection of dominant inhibitory mutant of IκBα (S32A/36A) and pretreatment of pyrrolidinedithiocarbamate, an inhibitor of NF-κB, enhanced ceramide-induced cell death. These results indicate that ceramide causes non-apoptotic, caspase-independent cell death by inducing reactive oxygen species generation in A172 human glioma cells. NF-κB is involved in the regulation of ceramide-induced cell death in human glioma cells.     

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.     

Activation of Cell Cycle Regulatory Proteins in the Apoptosis of Terminally Differentiated Oligodendrocytes

There is increasing evidence that proteins normally involved in the cell cycle play a role in the regulation of neuronal apoptotic death following various insults. However, it is not clear if the same mechanisms regulate cell death of oligodendrocytes as well. In this study, we investigated the mechanism of ceramide-induced apoptosis in primary rat oligodendrocytes. We show that ceramide treatment initiates a cascade of biochemical events involving cell cycle regulatory proteins. Although at the time of induction of cell death the oligodendrocytes are postmitotic, activation of c-myc and translocation of Cdc25A into the nucleus can be demonstrated. Of particular interest are the findings of the up-regulation of PCNA and down-regulation of p21WAF1/CIP1 protein, an inhibitor of cell-cycle progression. The current results show that activation of regulatory cell-cycle proteins at the oligodendrocytes G1-S checkpoint may constitute a crucial step of the death pathway of oligodendrocytes.     

Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation

BACKGROUND: Leukaemia cells differ from their normal counterparts in that their ability to properly regulate survival, proliferation, differentiation, and apoptosis is aberrant. Understanding the molecular mechanisms controlling cell proliferation and developing therapeutic strategies to correct nonfunctional regulatory mechanisms are emerging areas of medical research. Ceramide, a metabolite of membrane sphingomyelin hydrolysis, has recently emerged as a key regulator of cellular proliferation, differentiation, and apoptosis in leukaemia cells. METHODS: Leukaemia cell lines were treated with a biologically active analogue of ceramide, C(2)-ceramide. Cell cycle status was assessed flow cytometrically using propidium iodide. Induction of apoptosis was confirmed by annexin V staining of externalised phosphatidylserine and retinoblastoma activation was determined by Western blotting. RESULTS: C(2)-ceramide induced activation of retinoblastoma tumour suppressor protein, G(0)/G(1) cell cycle arrest, or apoptosis in leukaemia cell lines. In addition, these effects differed depending upon cell type, thus confirming the pleiotropic nature of the ceramide signalling pathway. Most cells studied responded to exogenous C(2)-ceramide by entering growth arrest, evidently resulting from activation of retinoblastoma protein, and by displaying some degree of apoptosis. CONCLUSIONS: Taken together, these findings suggest that signalling via ceramide has novel therapeutic applications for treatment of leukaemia.