Expression of p53 protein isoforms predicts survival in patients with multiple myeloma.

Loss and/or mutation of the TP53 gene are associated with short survival in multiple myeloma, but the p53 landscape goes far beyond. At least 12 p53 protein isoforms have been identified as a result of a combination of alternative splicing, alternative promoters and/or alternative transcription site starts, which are grouped as α, ß, γ, from transactivation domain (TA), long, and short isoforms. Nowadays, there are no studies evaluating the expression of p53 isoforms and its clinical relevance in multiple myeloma (MM). We used capillary nanoimmunoassay to quantify the expression of p53 protein isoforms in CD138-purified samples from 156 patients with newly diagnosed MM who were treated as part of the PETHEMA/GEM2012 clinical trial and investigated their prognostic impact. Quantitative real-time polymerase chain reaction was used to corroborate the results at RNA levels. Low and high levels of expression of short and TAp53ß/γ isoforms, respectively, were associated with adverse prognosis in MM patients. Multivariate Cox models identified high levels of TAp53ß/γ (hazard ratio [HR], 4.49; p < .001) and high-risk cytogenetics (HR, 2.69; p < .001) as independent prognostic factors associated with shorter time to progression. The current cytogenetic-risk classification was notably improved when expression levels of p53 protein isoforms were incorporated, whereby high-risk MM expressing high levels of short isoforms had significantly longer survival than high-risk patients with low levels of these isoforms. This is the first study that demonstrates the prognostic value of p53 isoforms in MM patients, providing new insights on the role of p53 protein dysregulation in MM biology.

Insights into epigenetic regulation of microRNA-155 expression in multiple myeloma.

CONTEXT: MiR-155 plays a critical role in the development of B-cell malignancies. Previous studies have shown a deregulation of miR-155 in specific cytogenetic subtypes of multiple myeloma (MM). However, the mechanisms that regulate miR-155 expression in MM are not fully understood. OBJECTIVE: In the present study, we explored the regulation of miRNA-155 in MM by DNA methylation mechanisms and the impact of miR-155 expression in survival of MM patients. METHOD: Primary samples were obtained from 95 patients with newly diagnosed myeloma. Methylation was analyzed by Methylation Specific PCR, sequencing of bisulfite treated DNA and luciferase assay. RESULTS: qRT-PCR analysis revealed that miR-155 was differentially expressed in MM and its upregulation was associated with longer survival. DNA methylation of CpG island present in the first exon of miR-155 host gene was associated with its low expression in MM cell lines and patient samples. Our results showed for the first time that in vitro methylation of part of the promoter and first exon abrogated the miR-155 expression. We further showed that miR-155 expression in MM cell lines was increased by demethylating 5-aza-dC treatment and decreased by RNA-directed DNA methylation. Additionally, we found that LPS "immunological challenge" was insufficient to induce miR-155 expression in MM cell lines with methylated DNA around transcription start site (TSS). CONCLUSION: This study provides evidence that DNA methylation contributes to miR-155 expression in myeloma cells. Interestingly, the survival data showed an association between miR-155 expression and outcome of MM.

Molecular Mechanisms of p53 Deregulation in Cancer: An Overview in Multiple Myeloma.

The p53 pathway is inactivated in the majority of human cancers. Although this perturbation frequently occurs through the mutation or deletion of p53 itself, there are other mechanisms that can attenuate the pathway and contribute to tumorigenesis. For example, overexpression of important p53 negative regulators, such as murine double minute 2 (MDM2) or murine double minute 4 (MDM4), epigenetic deregulation, or even alterations in TP53 mRNA splicing. In this work, we will review the different mechanisms of p53 pathway inhibition in cancer with special focus on multiple myeloma (MM), the second most common hematological malignancy, with low incidence of p53 mutations/deletions but growing evidence of indirect p53 pathway deregulation. Translational implications for MM and cancer prognosis and treatment are also reviewed.

[Nucelotide receptors in cancer cells]. / Receptory nukleotydowe w komórkach nowotworowych.

Despite a great scientific effort, the causes, development as well as progression of many tumors still remains unknown. Since that, it is important to investigate possible mechanisms which may lead to better understanding of cancer biology. Nucleotide and adenosine receptors may be consider as a promising direction of anti-tumor scientific research. Nucleotide receptors P2 and adenosine receptors P1 forms a great family of proteins whose activity has been proved to be involved in many cellular processes vital for tumorogenesis. This review gives basic insights into the mechanisms of proproliferative as well as antiproliferative action of nucleotide receptors in tumor cells.

Factors determining the sensitivity to proteasome inhibitors of multiple myeloma cells.

Multiple myeloma is an incurable cancer that originates from antibody-producing plasma cells. It is characterized by an intrinsic ability to produce large amounts of immunoglobulin-like proteins. The high rate of synthesis makes myeloma cells dependent on protein processing mechanisms related to the proteasome. This dependence made proteasome inhibitors such as bortezomib and carfilzomib one of the most important classes of drugs used in multiple myeloma treatment. Inhibition of the proteasome is associated with alteration of a number of important biological processes leading, in consequence, to inhibition of angiogenesis. The effect of drugs in this group and the degree of patient response to the treatment used is itself an extremely complex process that depends on many factors. At cellular level the change in sensitivity to proteasome inhibitors may be related to differences in the expression level of proteasome subunits, the degree of proteasome loading, metabolic adaptation, transcriptional or epigenetic factors. These are just some of the possibilities that may influence differences in response to proteasome inhibitors. This review describes the main cellular factors that determine the degree of response to proteasome inhibitor drugs, as well as information on the key role of the proteasome and the performance characteristics of the inhibitors that are the mainstay of multiple myeloma treatment.

The protective effect of silver nanoparticles' on epithelial cornea cells against ultraviolet is accompanied by changes in calcium homeostasis and a decrease of the P2X7 and P2Y2 receptors.

PURPOSE: The aim of the study was to evaluate the effect of silver nanoparticles hydrocolloids (AgNPs) on human corneal epithelial cells. Epithelial cells form the outermost and the most vulnerable to environmental stimuli layer of the cornea in the eye. Mechanical stress, UV radiation, and pathogens such as bacteria, viruses, and parasites challenge the fragile homeostasis of the eye. To help combat stress, infection, and inflammation wide portfolio of interventions is available. One of the oldest treatments is colloidal silver. Silver nanoparticle suspension in water is known for its anti-bacterial anti-viral and antiprotozoal action. However, AgNPs interact also with host cells, and the character of the interplay between corneal cells and silver seeks investigation. METHODS: The human epithelial corneal cell line (HCE-2) was cultured in vitro, treated with AgNPs, and subjected to UV. The cell's viability, migration, calcium concentration, and expression/protein level of selected proteins were investigated by appropriate methods including cytotoxicity tests, "wound healing" assay, Fluo8/Fura2 AM staining, qRT-PCR, and western blot. RESULTS: Incubation of human corneal cells (HCE-2) with AgNP did not affect cells viability but limited cells migration and resulted in altered calcium homeostasis, decreased the presence of ATP-activated P2X7, P2Y2 receptors, and enhanced the expression of PACAP. Furthermore, AgNPs pretreatment helped restrain some of the deleterious effects of UV irradiation. Interestingly, AgNPs had no impact on the protein level of ACE2, which is important in light of potential SARS-CoV-2 entrance through the cornea. CONCLUSIONS: Silver nanoparticles are safe for corneal epithelial cells in vitro.

Restoration of microRNA-214 expression reduces growth of myeloma cells through positive regulation of P53 and inhibition of DNA replication.

MicroRNA have been demonstrated to be deregulated in multiple myeloma. We have previously reported that miR-214 is down-regulated in multiple myeloma compared to in normal plasma cells. The functional role of miR-214 in myeloma pathogenesis was explored by transfecting myeloma cell lines with synthetic microRNA followed by gene expression profiling. Putative miR-214 targets were validated by luciferase reporter assay. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this microRNA, gene expression profiling of the H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. miR-214 directly down-regulated the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-untranslated regions. In addition, gankyrin inhibition induced an increase of P53 mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, MiR-214 functions as a tumor suppressor in myeloma by positive regulation of p53 and inhibition of DNA replication.

A Comprehensive Assessment of the Biocompatibility and Safety of Diamond Nanoparticles on Reconstructed Human Epidermis.

Diamond nanoparticles, also known as nanodiamonds (NDs), exhibit remarkable, awe-inspiring properties that make them suitable for various applications in the field of skin care products. However, a comprehensive assessment of their compatibility with human skin, according to the irritation criteria established by the Organization for Economic Cooperation and Development (OECD), has not yet been conducted. The purpose of this study was to evaluate if diamond nanoparticles at a concentration of 25 µg/mL, incubated with reconstituted human epidermis (EpiDermTM) for 18 h, conform to the OECD TG439 standard used to classify chemical irritants. For this purpose, a cell viability test (MTT assay), histological assessment, and analysis of pro-inflammatory cytokine expression were performed. The results indicated that NDs had no toxic effect at the tested concentration. They also did not adversely affect tissue structure and did not lead to a simultaneous increase in protein and mRNA expression of the analyzed cytokines. These results confirm the safety and biocompatibility of NDs for application in skincare products, thereby creating a wide range of possibilities to exert an impact on the advancement of contemporary cosmetology in the future.

Do Small Molecules Activate the TrkB Receptor in the Same Manner as BDNF? Limitations of Published TrkB Low Molecular Agonists and Screening for Novel TrkB Orthosteric Agonists.

TrkB is a tyrosine kinase receptor that is activated upon binding to brain-derived neurotrophic factor (BDNF). To date, the search for low-molecular-weight molecules mimicking BDNF's action has been unsuccessful. Several molecules exerting antidepressive effects in vivo, such as 7,8-DHF, have been suggested to be TrkB agonists. However, more recent publications question this hypothesis. In this study, we developed a set of experimental procedures including the evaluation of direct interactions, dimerization, downstream signaling, and cytoprotection in parallel with physicochemical and ADME methods to verify the pharmacology of 7,8-DHF and other potential reference compounds, and perform screening for novel TrkB agonists. 7,8 DHF bound to TrkB with Kd = 1.3 µM; however, we were not able to observe any other activity against the TrkB receptor in SN56 T48 and differentiated SH-SY5Y cell lines. Moreover, the pharmacokinetic and pharmacodynamic effects of 7,8-DHF at doses of 1 and 50 mg/kg were examined in mice after i.v and oral administration, respectively. The poor pharmacokinetic properties and lack of observed activation of TrkB-dependent signaling in the brain confirmed that 7,8-DHF is not a relevant tool for studying TrkB activation in vivo. The binding profile for 133 molecular targets revealed a significant lack of selectivity of 7,8-DHF, suggesting a distinct functional profile independent of interaction with TrkB. Additionally, a compound library was screened in search of novel low-molecular-weight orthosteric TrkB agonists; however, we were not able to identify reliable drug candidates. Our results suggest that published reference compounds including 7,8-DHF do not activate TrkB, consistent with canonical dogma, which indicates that the reported pharmacological activity of these compounds should be interpreted carefully in a broad functional context.

Zebularine-induced myeloma cell death is accompanied by decreased c-Myc expression.

PURPOSE: Epigenetic therapies have proven to be clinically effective in several hematological malignancies. Here, we aimed to evaluate the effect of a second-generation DNA demethylation agent, zebularine, on multiple myeloma (MM). METHODS: Western blot, ELISA, qRT-PCR, proliferation assays and cell transfection were used to investigate the mechanism of action of zebularine in MM. RESULTS: We found that zebularine induced apoptosis and DNA demethylation in most of the MM cell lines tested. Its cytotoxic effect was associated with a time-dependent decrease in the level of c-Myc protein. Moreover, zebularine induced H2AX phosphorylation, a surrogate marker of DNA damage, in five out of eight MM cell lines tested. CONCLUSIONS: Our study revealed novel effects of zebularine on MM that may have potential implications for DNA methylation-based therapies.

Quantitative expression of Ikaros, IRF4, and PSMD10 proteins predicts survival in VRD-treated patients with multiple myeloma.

The search for biomarkers based on the mechanism of drug action has not been thoroughly addressed in the therapeutic approaches to multiple myeloma (MM), mainly because of the difficulty in analyzing proteins obtained from purified plasma cells. Here, we investigated the prognostic impact of the expression of 12 proteins involved in the mechanism of action of bortezomib, lenalidomide, and dexamethasone (VRD), quantified by capillary nanoimmunoassay, in CD138-purified samples from 174 patients with newly diagnosed MM treated according to the PETHEMA/GEM2012 study. A high level of expression of 3 out of 5 proteasome components tested (PSMD1, PSMD4, and PSMD10) negatively influenced survival. The 5 analyzed proteins involved in lenalidomide's mode of action were associated with time to progression (TTP); low levels of cereblon and IRF4 protein and high levels of Ikaros, AGO2, and Aiolos were significantly associated with shorter TTP. Although the glucocorticoid receptor (GCR) level by itself had no significant impact on MM prognosis, a high XPO1 (exportin 1)/GCR ratio was associated with shorter TTP and progression-free survival (PFS). The multivariate Cox model identified high levels of PSMD10 (hazard ratio [HR] TTP, 3.49; P = .036; HR PFS, 5.33; P = .004) and Ikaros (HR TTP, 3.01, P = .014; HR PFS, 2.57; P = .028), and low levels of IRF4 protein expression (HR TTP, 0.33; P = .004; HR PFS, 0.35; P = .004) along with high-risk cytogenetics (HR TTP, 3.13; P < .001; HR PFS, 2.69; P = .002), as independently associated with shorter TTP and PFS. These results highlight the value of assessing proteins related to the mechanism of action of drugs used in MM for predicting treatment outcome.

Factors Regulating microRNA Expression and Function in Multiple Myeloma.

Intensive research has been undertaken during the last decade to identify the implication of microRNAs (miRNAs) in the pathogenesis of multiple myeloma (MM). The expression profiling of miRNAs in MM has provided relevant information, demonstrating different patterns of miRNA expression depending on the genetic abnormalities of MM and a key role of some miRNAs regulating critical genes associated with MM pathogenesis. However, the underlying causes of abnormal expression of miRNAs in myeloma cells remain mainly elusive. The final expression of the mature miRNAs is subject to multiple regulation mechanisms, such as copy number alterations, CpG methylation or transcription factors, together with impairment in miRNA biogenesis and differences in availability of the mRNA target sequence. In this review, we summarize the available knowledge about the factors involved in the regulation of miRNA expression and functionality in MM.

The P2Y14 receptor activity in glioma C6 cells.

In this study, we demonstrated the presence and the activity of the P2Y14 receptor in glioma C6 cells. We found that P2Y14 could exist in two forms, highly predominating glycosylated and non-glycosylated. Binding of UDP-glucose evoked two responses: calcium signal and adenylate cyclase inhibition, both pertussis toxin-sensitive. Separate glycosylation pattern and functional profile of these two receptor forms were observed in non-starved and serum-starved cells. During long-term serum deprivation (96 h), the level of glycosylated form strongly decreased, while non-glycosylated increased, what was correlated with the decrease of calcium signaling activity and stronger adenylate cyclase inhibition, suggesting that receptor N-glycosylation may modulate its functional activity.

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells.

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.

Mitochondrial localization of P2Y1, P2Y2 and P2Y12 receptors in rat astrocytes and glioma C6 cells.

We have previously shown that P2Y1, P2Y2 and P2Y12 nucleotide receptors are functionally expressed and active on the cell surface of rat glioma C6 cells. In the present study, we have immunocytochemically shown their sub-cellular colocalization with mitochondria in these cells. The same colocalization of above receptors has been found in rat astrocytes. Additionally, differences in intracellular distribution of examined receptors between both cell lines have been observed. This data indicates that P2Y1, P2Y2 and P2Y12 receptor proteins exist within mitochondria of astrocytes and C6 cells, although their role in these sub-cellular structures remains unclear.

DEPTOR maintains plasma cell differentiation and favorably affects prognosis in multiple myeloma.

BACKGROUND: The B cell maturation process involves multiple steps, which are controlled by relevant pathways and transcription factors. The understanding of the final stages of plasma cell (PC) differentiation could provide new insights for therapeutic strategies in multiple myeloma (MM). Here, we explore the role of DEPTOR, an mTOR inhibitor, in the terminal differentiation of myeloma cells, and its potential impact on patient survival. METHODS: The expression level of DEPTOR in MM cell lines and B cell populations was measured by real-time RT-PCR, and/or Western blot analysis. DEPTOR protein level in MM patients was quantified by capillary electrophoresis immunoassay. RNA interference was used to downregulate DEPTOR in MM cell lines. RESULTS: DEPTOR knockdown in H929 and MM1S cell lines induced dedifferentiation of myeloma cells, as demonstrated by the upregulation of PAX5 and BCL6, the downregulation of IRF4, and a clear reduction in cell size and endoplasmic reticulum mass. This effect seemed to be independent of mTOR signaling, since mTOR substrates were not affected by DEPTOR knockdown. Additionally, the potential for DEPTOR to be deregulated in MM by particular miRNAs was investigated. The ectopic expression of miR-135b and miR-642a in myeloma cell lines substantially diminished DEPTOR protein levels, and caused dedifferentiation of myeloma cells. Interestingly, the level of expression of DEPTOR protein in myeloma patients was highly variable, the highest levels being associated with longer progression-free survival. CONCLUSIONS: Our results demonstrate for the first time that DEPTOR expression is required to maintain myeloma cell differentiation and that high level of its expression are associated with better outcome. Primary samples used in this study correspond to patients entered into GEM2010 trial (registered at www.clinicaltrials.gov as #NCT01237249, 4 November 2010).

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells.

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.

Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

Post-transcriptional Modifications Contribute to the Upregulation of Cyclin D2 in Multiple Myeloma.

PURPOSE: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3'UTR in CCND2 overexpression in multiple myeloma. EXPERIMENTAL DESIGN: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3'UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3' rapid amplification of cDNA ends (RACE)-PCR. RESULTS: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3'RACE experiments revealed that changes in CCND2 3'UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3'UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. CONCLUSIONS: Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.

Modulation of ERK1/2 activity is crucial for sphingosine-induced death of glioma C6 cells.

In this study the contribution of the ERK1/2 pathway to sphingosine-induced death and morphological changes of the actin cytoskeleton in glioma C6 cells was investigated. Surprisingly, the level of ERK1/2 phosphorylation does not change after incubation of cells with sphingosine. Despite this, sphingosine induces rounding and detachment of cells without formation of apoptotic bodies. To shed light on this process, a specific inhibitor of ERK1/2 phosphorylation, U0126, was used. Cells incubated simultaneously with sphingosine and U0126 not only detached, but also exhibited formation of apoptotic-like blebs. These data suggest that during sphingosine-induced glioma C6 cell death apoptotic blebbing is dependent on ERK1/2 signalling and occurs only when ERK1/2 activity is decreased or abolished.