S-type Cells Research Articles

Activation of sonic hedgehog signaling pathway in S-type neuroblastoma cell lines

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

Abstract B76: MicroRNAs regulate cell phenotype and N-myc expression in human neuroblastoma cells

Abstract Identification of miRNAs that regulate neuroblast growth and differentiation could be important in understanding the onset and progression of the childhood cancer neuroblastoma, and in developing new therapeutics to fight this often fatal disease. Neuroblastoma tumors and derived cell lines are heterogeneous, with three basic phenotypes - neuroblastic (N), substrate-adherent (S), and cancer stem cells (I). These types differ significantly in their morphology, biochemistry and tumorigenic potential. Five miRNAs differentially expressed with respect to cell phenotype were identified from a miRNA microarray, validated by RT-PCR, and quantified by real-time RT-PCR taqman assays in 14 different cell lines (6 N-, 5 I- and 3 S-types). Out of these miRNAs, three are highly expressed in non-tumorigenic S-type cell lines compared to I- and N-type cell lines: miR-21 (10- fold, p<0.05), miR-221 (18-fold, p<0.05) and miR-335 (136-fold, p<0.05). Moreover, miRNA expression increased significantly with BrdU-induced I- to S-type differentiation of BE(2)-C. By contrast, miR-124a levels are 10-fold higher in N-type cells compared to both I- and S-type cells and are elevated 2-fold (p<0.05) following retinoic acid-induced neuronal differentiation of I-type BE(2)-C. Transient transfection of miR-124a into I-cells induced a neuronal-like phenotype with increased neurite growth, supporting its active role in neuronal differentiation. Another neuronal-specific miRNA, miR-375, expressed in N- and I- but not in S-type cells, significantly decreased with induced differentiation from an I- to S-type phenotype. A second factor affecting survival of neuroblastoma patients is overexpression/amplification of the protooncogene N-myc. Thus, the microarray was sorted for miRNAs whose levels correlated directly or inversely with N-myc gene copy number. Candidate miRNAs were quantified by real-time RT-PCR taqman assays in the same 14 cell lines (8 N-myc amplified and 6 non-amplified). Three members of the miR-17-92 cluster (miR-17-5p, miR-18a and miR-20a) are highly expressed in N-myc-amplified cell lines compared to non-amplified cells (p<0.05). Moreover, siRNA-induced reduction of N-myc protein (2.5-fold) down-regulated expression levels of all three miRNAs by 70% (p<0.05), suggesting N-myc up-regulates the expression of the miR-17-92 cluster. A screen of potential targets of the miR-17-92 cluster members revealed, interestingly, that N-myc mRNA itself is a predicted target of miR-17-5p. Down-regulation of this miRNA (95%) by transient transfection with specific inhibitors increased N-myc mRNA 2-fold (p<0.05) and protein 1.5-fold (p<0.05), confirming and extending results of Cloonan et al (2008). In N-myc amplified cell lines, N-myc mRNA levels negatively correlate with levels of miR-17-5p. Together these data reveal a possible negative feedback loop between N-myc and miR-17-5p. In conclusion, miRNAs that are differentially expressed with respect to both phenotype and N-myc amplification status may significantly contribute to the progression and aggressiveness of neuroblastoma. Citation Information: Cancer Res 2009;69(23 Suppl):B76.

Bcl6 Is Expressed in Neuroblastoma: Tumor Cell Type-Specific Expression Predicts Outcome

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and childhood. Whereas most low-risk patients do well, children with high-risk tumors often fail intensive treatment. Identification of novel biomarkers is critical to improve prognostication, tailor therapy, and develop new therapeutic targets. Differential RNA-level expression between tumor cells with neuroblastic (N-type) and Schwannian stromal (S-type) phenotypes was used to identify genes of potential interest based on tumor cell type-specific regulation. Gene expression microarray analysis revealed marked differences between N-type and S-type cells in their levels of BCL6 messenger RNA, a transcriptional regulator overexpressed in a variety of hematopoietic malignancies. S-type cells express higher levels of Bcl6 RNA and protein than N-type, and protein levels are significantly limited by proteasome function. An NB tumor tissue microarray linked to clinicopathologic data was immunohistochemically stained to measure Bcl6 protein levels. Bcl6 was detected in both the neuroblastic and Schwannian stromal regions, as distinguished histologically, and correlated with outcome. We found that expression in neuroblastic regions differentiates outcomes, in that Bcl6 expression in neuroblastic regions is associated with increased time to relapse and increased overall survival compared with absent expression in neuroblastic regions, regardless of Schwannian stromal expression. Thus, our findings suggest that Bcl6 may be useful as a prognostic marker and might represent a potential therapeutic target for high-risk NB.

Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

BackgroundNeuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously.MethodsHere, neuroblastoma (NB) cell line subtypes were characterized according to embryonic peripheral nervous system development markers (GAP43, Phox2b, Sox10, c-kit, GD2, NF68, vimentin, S100β, calcyclin and ABCG2), morphological features, gene expression and differentiation potential. I-type cells were investigated as a bipotential (neuronal and glial) differentiation stage.ResultsPositive immunostaining of NCSC (GAP43, c-kit, NF68, vimentin and Phox2b) and undifferentiated cell (ABCG2) markers was observed in all NB subtypes. N- and I-type cells displayed cytoplasmic membrane GD2 staining, while nuclear calcyclin was restricted to S-type. N- and I-type cells showed similar phenotype and immunoreactivity pattern. Differential gene expression was associated with each cell subtype. N- and I-type cells displayed similar differentiation capacity towards neuronal and glial lineage fates. S-type cells, upon induction, did not show a neuronal-like phenotype, despite gene expression changes.ConclusionResults suggest that N- and I-type NB cell subtypes represent an immature bilineage stage, able to progress towards neuronal and glial fates upon induction of differentiation. S-type cells appear irreversibly committed to a glial lineage fate.

Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma

Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.

Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by Sedimentation Field Flow Fractionation

Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

Bmi-1 Regulates the Differentiation and Clonogenic Self-renewal of I-type Neuroblastoma Cells in a Concentration-dependent Manner

Human neuroblastoma I-type cells have been proposed as a population of malignant neural crest stem cells, based on their high tumorigenic potential, expression of stem cell markers, and ability to differentiate into cells of neural crest lineages, including neuroblastic (N-type) and Schwann/glial (S-type) cells. Here, we demonstrate at single cell levels that a subpopulation of I-type cells possess clonogenic self-renewal capacity that requires the Polycomb group family transcription repressor Bmi-1. We further show that Bmi-1 expression levels exert an instructive influence on lineage commitment by I-type cells. Spontaneous and induced differentiation of I-type cells into S-type cells is accompanied by a marked reduction in the level of Bmi-1 expression, and enforced down-regulation of BMI-1 facilitates spontaneous differentiation of I-type cells into S-type cells. By contrast, N-type neuroblastoma cell lines and differentiated N-type cells express higher levels of Bmi-1 relative to I-type cells, and overexpression of BMI-1 promotes the differentiation of I-type cells along the neuronal lineage. Thus, Bmi-1 acts in a concentration-dependent manner in the control of the delicate balance between the self-renewal and differentiation of neuroblastoma I-type cells. These observations suggest that graded activation of a master regulator within individual tumors could trigger divergent developmental programs responsible for both tumor growth and heterogeneity.

Differential sensitivity of chemoresistant neuroblastoma subtypes to MAPK-targeted treatment correlates with ERK, p53 expression, and signaling response to U0126

Purpose Neuroblastoma tumors are comprised of neuroblastic (N), substrate-adherent (S), and intermediate (I) cells. Because cell growth and differentiation often involve p44/p42 mitogen-activated protein kinase (MAPK) pathway signaling, we explored MAPK signaling and growth response in three NB cell types after MAPK kinase (MEK) inhibition to evaluate the feasibility of MAPK-targeted treatment strategies. Methods Three human NB cell cultures, SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type), were treated in monolayer cultures with increasing concentrations of the MEK inhibitor U0126. MAPK pathway intermediates MEK and ERK, their activated (phosphorylated) forms p-MEK and p-ERK, and p53 expression were assessed by Western blot at 1 and 24 hours. At 72 hours, cell counts determined growth inhibition and DNA fragmentation ELISA assessed apoptosis. Results Among all three lines, total ERK and MEK expression were unaffected by U0126. However, constitutive total ERK and p53 expression were significantly greater in BE(2)-C (I-type) cells than in SH-SY5Y (N-type) and SK-N-AS (S-type). Active ERK (p-ERK) levels decreased in dose response to U0126 at 1 and 24 hours in all lines. Conversely, p-MEK levels increased with increasing U0126 concentrations at 1 hour in SH-SY5Y (N-type) and at 24 hours in all lines. BE(2)-C (I-type) cell counts decreased in concentration-dependent fashion with U0126, whereas SH-SY5Y (N-type) and SK-N-AS (S-type) showed a biphasic response with increased cell counts at 1 μmol/L U0126 and slightly decreased cell counts at 10 μmol/L U0126. Conclusion This study demonstrates that BE(2)-C (I-type) cells exhibit greater constitutive total ERK and p53 expression than SH-SY5Y (N-type) and SK-N-AS (S-type). Although all three lines exhibit p-ERK decreases with MEK inhibition, only BE(2)-C (I-type) cells significantly decrease their proliferation with U0126 treatment. Although MEK inhibition holds promise in targeting I-type NB cells, successfully treating this heterogeneous tumor may require combining agents against N- and S-type cells.

Disorganization of the Facial Nucleus after Nerve Lesioning and Regeneration in the Rat: Effects of Transplanting Candidate Reparative Cells to the Site of Injury

After the facial nerve has been damaged, functional recovery is impaired by synkinesis: the abnormal movement of part of the face during voluntary movement of another part. This is primarily because of aberrant regenerating axons that fail to reach the correct distal targets. We studied the effects of transplanting Schwann cells, mixed olfactory ensheathing cells, and S-type olfactory ensheathing cells on the histological and functional organization of the connections between the facial nucleus and distal muscles. In a control group of adult rats (n = 18), the proximal part of the extracranial facial nerve was completely severed and immediately repaired by suture. In an experimental group (n = 34), a bolus of cultured cells was applied to the cut ends before suture (Schwann cells, n = 10; olfactory ensheathing cells, n = 18; S-type cells, n = 6). A period of regeneration was permitted, after which the animals were assessed functionally by measurement of eye closure in response to a puff of air. The site of lesion and the somatotopic organization of the facial nucleus were assessed histologically, after application of different retrograde fluorescent tracers simultaneously to the buccal and temporal divisions of the facial nerve. The rate of recovery of eye closure was increased by the use of the cell transplants, but the disorganization of the facial nucleus and aberrant nerve branching were unchanged. Perhaps regenerating axons become misguided throughout the whole length of the nerve, not only at the site of the lesion, and strategies aimed at improving axon path-finding solely at the lesion site will therefore be inadequate.

Characteristics of Stem Cells from Human Neuroblastoma Cell Lines and in Tumors

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed 1-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.

Integrin Expression Regulates Neuroblastoma Attachment and Migration

Neuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that β1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of β1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of β1 integrin, whereas tumorigenic N-cells express little β1 integrin. Alterations in (3, integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that β1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of α5β1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease β1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of αvβ3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration.

Chemotherapy-induced Apoptosis of S-type Neuroblastoma Cells Requires Caspase-9 and Is Augmented by CD95/Fas Stimulation

Stromal or S-type tumor cells are a distinct lineage found in neuroblastoma tumors and have an important role in the biology of this disease. Anticancer agents induce apoptosis through death receptor- and mitochondria-initiated pathways. The object of this work was to determine the involvement of these pathways in the response to doxorubicin (Dox) and cisplatin (CDDP) in S-type neuroblastoma cells. Both drugs activated caspase-9 and caspase-3 but not caspase-8. Caspase-9-specific inhibition blocked S-type cell death induced by Dox. SH-EP1 cells transfected to express dominant negative mutant caspase-9, but not those expressing DN caspase-8, were resistant to Dox- and CDDP-induced apoptosis. The lack of caspase-8 involvement in chemotherapy-induced death was not the result of an intrinsic inability of these cells to activate this enzyme because when they were treated with tumor necrosis factor-related apoptosis-inducing ligand, caspase-8 was activated. We also found that both drugs up-regulated CD95/Fas expression but that CD95/Fas signaling was not necessary for cell killing. Experiments testing the response of chemotherapy-treated cells to agonists of the CD95/Fas receptor established that Dox and CDDP treatment sensitizes cells to CD95/Fas killing. Together, these results are consistent with a model in which caspase-9 is of central importance in the death mechanism utilized by these drugs in S-type cells. Although the death response is not dependent on CD95/Fas, concomitant stimulation of this receptor amplifies the death response in drug-treated cells.

The cellular response to PPARgamma ligands is related to the phenotype of neuroblastoma cell lines.

Neuroblastoma (NB) is a phenotypically heterogeneous tumor, displaying cells of neuronal, melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also present in vitro, where cells of neuroblastic (N)- or stromal (S)-type may be identified. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown to inhibit growth in different tumor cell lines. The purpose of this study was to determine PPARgamma expression and the response to its ligands in NB cell lines with different phenotypes. We used eight NB cell lines with N-, mixed, and S-phenotype. PPARgamma expression was found in all NB cell lines, regardless of their phenotype. Mutational analysis and transactivation assays showed that PPARgamma is not mutated and remains functional in NB cells. Two PPARgamma ligands, 15-deoxy-delta12,14-prostaglandin J2 (PGJ2) and rosiglitazone, inhibited growth of all cell lines, with PGJ, being the most potent agent. PGJ2, but not rosiglitazone, induced arrest of the cells in the G2/M phase as well as apoptosis. The sensitivity to the two ligands appeared to be more related to the phenotype than PPARgamma expression, with the S-type cells being less sensitive than the N-type, partly because of their lower capability of undergoing apoptosis. No synergistic effect on growth inhibition was observed when all cell lines were co-treated with 9-cis retinoic acid (9-cis RA) and rosiglitazone. Our data indicate that PPARgamma expression and function are maintained in phenotipically different NB cell lines. Activation of PPARgamma by its synthetic ligands might have a therapeutic role in advanced NB.

The Effect of Major Nutrient Elements on the Growth and Population Homogeneity of the R, S, and M Dissociants of Pseudomonas aeruginosa and the Glucose Oxidation and Fermentation Pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.

A role for distinct cell types in determining malignancy in human neuroblastoma cell lines and tumors

Human neuroblastoma arises from the developing neural crest. Tumors are categorized clinically by their location, age at diagnosis, spread/metastasis, and degree of cellular maturation and heterogeneity. Our long-term studies have shown the presence in human neuroblastoma cell lines of three distinct cell types: I-type stem cells, N-type neuroblastic/neuroendocrine precursors, and S-type Schwannian/melanoblastic precursors. These distinct cell types can differentiate predictably along specific neural crest lineages in response to particular morphogens. As assessed by tumor formation in nude mice and anchorage-independent growth in soft agar, I-type stem cells are significantly more malignant than either N- or S-type cells. Recent research shows that three similar cell types are also present in human neuroblastoma tumors. Using immunocytochemical, laser-capture microdissection, or short-term culture methods to identify cell types in tumors of different stages and/or different outcomes, these studies have shown that (1) all tumors contain neuroblasts in various differentiation states; (2) presumptive I-type stem cells are present in tumors of all stages; and (3) stromal cells may be tumor-derived, i.e. S-type cells, as well as of normal origin. More importantly, there is a higher incidence of I-type cells in tumors that progress, consistent with the high malignant potential of this cell type in vitro. A better understanding of the cause and consequences of cellular heterogeneity of human neuroblastoma tumors is an important prerequisite to the development of more effective therapies for this often fatal disease.

Constitutively Active NFκB Is Required for the Survival of S-type Neuroblastoma

The NFkappaB transcription factors can both promote cell survival and induce apoptosis depending on cell type and context. Neuroblastoma (NB) cells display two predominant culture phenotypes identified as N- and S-types. Malignant S-type cells express neither high levels of MYCN nor Bcl-2, suggesting that other survival mechanisms are important. We characterized NFkappaB activity in S-type cells and determined its role in their survival. S-type lines (SH-EP1 and SK-N-AS) were treated with pyrrolidine dithiocarbamate (PDTC), a NFkappaB inhibitor, or l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a serine protease inhibitor that blocks IkappaBalpha degradation. Both agents induced cell death, suggesting that constitutive NFkappaB activity is required for survival. The transient expression of a super-repressor IkappaBalpha mutant killed S-type cells. The inhibition of NFkappaB produced an apoptotic response characterized by the collapse of the mitochondrial transmembrane electrochemical gradient, caspase-9 activation, and apoptotic DNA changes. Constitutive NFkappaB DNA binding activity specifically involving p65 and p50 was demonstrated in S- but not N-type cells by electromobility supershift and gene reporter assays. This study demonstrates a role for NFkappaB in the survival of S-type NB tumor cells and suggests that NFkappaB activity and function differ according to NB tumor cell phenotype.

Doxorubicin-induced death in neuroblastoma does not involve death receptors in S-type cells and is caspase-independent in N-type cells.

Death induced by doxorubicin (dox) in neuroblastoma (NB) cells was originally thought to occur via the Fas pathway, however since studies suggest that caspase-8 expression is silenced in most high stage NB tumors, it is more probable that dox-induced death occurs via a different mechanism. Caspase-8 silenced N-type invasive NB cell lines LAN-1 and IMR-32 were investigated for their sensitivity to dox, and compared to S-type noninvasive SH-EP NB cells expressing caspase-8. All cell lines had similar sensitivities to dox, independently of caspase-8 expression. Dox induced caspase-3, -7, -8 and -9 and Bid cleavage in S-type cells and death was blocked by caspase inhibitors but not by oxygen radical scavenger BHA. In contrast, dox-induced death in N-type cells was caspase-independent and was inhibited by BHA. Dox induced a drop in mitochondrial membrane permeability in all cell lines. Dox-induced death in S-type cells gave rise to apoptotic nuclei, whereas in N-type cells nuclei were non-apoptotic in morphology. Transfection of SH-EP cells with a dominant negative FADD mutant inhibited TRAIL-induced death, but had no effect on dox-induced apoptosis. These results suggest that S-type cells undergo apoptosis after dox treatment independently of death receptors, whereas N-type cells are killed by a caspase-independent mechanism.

Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.

Requirement for HDM2 Activity in the Rapid Degradation of p53 in Neuroblastoma

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.

MDM2 Mediated Nuclear Exclusion of p53 Attenuates Etoposide-Induced Apoptosis in Neuroblastoma Cells

The p53 gene in neuroblastoma tumors (NB) is rarely mutated but the protein accumulates in the cytoplasm. Because p53 can mediate the cytotoxic effects of chemotherapeutic agents, it is important to determine whether accumulation of p53 in the cytoplasm impairs p53 function. Data presented here indicate that hyperactive nuclear export of p53 suppresses etoposide-induced apoptosis but does not prevent growth arrest. We compared p53 function in a pair of NB subclones. Our data show etoposide induces complete trans-location of p53 to the nucleus and activation of apoptosis in the neuroblastic NB cell line SH-SY5Y (N-type), which expresses low levels of MDM2. However, in Schwann cell-like SH-EP1 cells (S-type), which have up to 10-fold higher levels of MDM2, p53 accumulates in the cytoplasm and the cells are extremely resistant to etoposide-induced apoptosis. Notably, when MDM2 expression is inhibited in S-type cells, with a phosphorothioated antisense oligonucleotide (AS5), then p53 accumulates in the nucleus and the SH-EP1 cells undergo apoptosis. Surprisingly, induction of p21 and G1-arrest are not attenuated in S-type cells, despite the predominantly cytoplasmic location of p53. Whereas, G1-arrest is attenuated in the SH-SY5Y cells, which have high levels of nuclear p53. Taken together, these findings suggest attenuation of G1-arrest is related to the differentiation status of neuroblastomas and occurs downstream of p53 nuclear accumulation. These results demonstrate for the first time that hyperactive nuclear export of p53 attenuates chemotherapy-induced apoptosis in NB cells, and our findings suggest that inhibitors of MDM2 may enhance the therapeutic efficacy of etoposide by promoting apoptosis rather than trans-differentiation.