Mario Encinas

Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells.

Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron‐like cells from the SH‐SY5Y neuroblastoma cell line. Sequential exposure of SH‐SY5Y cells to retinoic acid and brain‐derived neurotrophic factor in serum‐free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5‐bromo‐2′‐deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain‐derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron‐specific enolase, and growth‐associated protein‐43 as well as neuronal polarity markers such as tau and microtubule‐associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large‐scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Extracellular-Regulated Kinases and Phosphatidylinositol 3-Kinase Are Involved in Brain-Derived Neurotrophic Factor-Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH-SY5Y

Abstract : Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH‐SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA‐predifferentiated SH‐SY5Y cells with brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT‐3), or neurotrophin 4/5 (NT‐4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal‐regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT‐3, or NT‐4/5, but not NGF, promotes cell survival and neurite outgrowth in serum‐free medium. The mitogen‐activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF‐induced neurite outgrowth and growth‐associated protein‐43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3‐kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.

Receptors of the Glial Cell Line-Derived Neurotrophic Factor Family of Neurotrophic Factors Signal Cell Survival through the Phosphatidylinositol 3-Kinase Pathway in Spinal Cord Motoneurons

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promote in vivo and in vitro survival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor α (GRFα) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRα-1, GFRα-2, and GFRα-4 in chicken motoneurons using in situ hybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)–mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.

Proteasome Inhibitors Induce Death but Activate NF-κB on Endometrial Carcinoma Cell Lines and Primary Culture Explants

Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-κB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-κB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKKα/β, phosphorylation and degradation of IκBα, and phosphorylation of the p65 NF-κB subunit on serine 536. Proteasome inhibitor-induced NF-κB activity can be blocked by a non-degradable form of IκBα or dominant negative forms of either IKKα or IKKβ. Lentiviral delivery of shRNAs to either IKKα or IKKβ cause blockade of NF-κB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IκBa degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-κB activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-κB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-κB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-κB activation should be carefully examined in particular cell types.

Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis

The Ret receptor tyrosine kinase mediates physiological signals of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and is essential for postnatal survival in mice. It is implicated in a number of human diseases and developmental abnormalities. Here, we describe our analyses of mice expressing a Ret mutant (RetDN) with diminished kinase activity that inhibits wild-type Ret activity, including its activation of AKT. All RetDN/+ mice died by 1 month of age and had distal intestinal aganglionosis reminiscent of Hirschsprung disease (HSCR) in humans. The RetDN/+ proximal small intestine also had severe hypoganglionosis and reduction in nerve fiber density, suggesting a potential mechanism for the continued gastric dysmotility in postsurgical HSCR patients. Unlike Ret-null mice, which have abnormalities in the parasympathetic and sympathetic nervous systems, the RetDN/+ mice only had defects in the parasympathetic nervous system. A small proportion of RetDN/+ mice had renal agenesis, and the remainder had hypoplastic kidneys and developed tubulocystic abnormalities postnatally. Postnatal analyses of the testes revealed a decreased number of germ cells, degenerating seminiferous tubules, maturation arrest and apoptosis, indicating a crucial role for Ret in early spermatogenesis.

Basic helix-loop-helix proteins bind to TrkB and p21(Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells.

ABSTRACT Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21Cip1 at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21Cip1 promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21Cip1 , whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21Cip1 promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21Cip1 , and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

The prevention of the staurosporine‐induced apoptosis by Bcl‐XL, but not by Bcl‐2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti‐apoptotic strategies in staurosporine‐treated SH‐SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z‐VAD.fmk a broad‐spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH‐SY5Y cells. At higher concentrations, z‐VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over‐expression of Bcl‐2 in SH‐SY5Y protected cells from death at doses of staurosporine up to 1 µm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl‐2 increased the threshold for apoptotic cell death commitment. Over‐expression of Bcl‐XL was far more effective than Bcl‐2. Bcl‐XL transfected cells showed a remarkable resistance staurosporine‐induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 µm staurosporine. In these conditions, SH‐SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP‐2 neuronal specific proteins.

CK2 controls TRAIL and Fas sensitivity by regulating FLIP levels in endometrial carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising antineoplastic agent because of its ability to selectively kill tumoral cells. However, some cancer cells are resistant to TRAIL-induced apoptosis. We have previously demonstrated that in endometrial carcinoma cells such resistance is caused by elevated FLICE-inhibitory protein (FLIP) levels. The present study focuses on the mechanisms by which FLIP could be modulated to sensitize endometrial carcinoma cells to TRAIL-induced apoptosis. We find that inhibition of casein kinase (CK2) sensitizes endometrial carcinoma cells to TRAIL- and Fas-induced apoptosis. CK2 inhibition correlates with a reduction of FLIP protein, suggesting that CK2 regulates resistance to TRAIL and Fas by controlling FLIP levels. FLIP downregulation correlates with a reduction of mRNA and is prevented by addition of the MG-132, suggesting that CK2 inhibition results in a proteasome-mediated degradation of FLIP. Consistently, forced expression of FLIP restores resistance to TRAIL and Fas. Moreover, knockdown of either FADD or caspase-8 abrogates apoptosis triggered by inhibition of CK2, indicating that CK2 sensitization requires formation of functional DISC. Finally, because of the possible role of both TRAIL and CK2 in cancer therapy, we demonstrate that CK2 inhibition sensitizes primary endometrial carcinoma explants to TRAIL apoptosis. In conclusion, we demonstrate that CK2 regulates endometrial carcinoma cell sensitivity to TRAIL and Fas by regulating FLIP levels.

Neuronal survival induced by neurotrophins requires calmodulin

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

Antioxidants block proteasome inhibitor function in endometrial carcinoma cells

We have recently demonstrated that proteasome inhibitors can be effective in inducing apoptotic cell death in endometrial carcinoma cell lines and primary culture explants. Increasing evidence suggests that reactive oxygen species are responsible for proteasome inhibitor-induced cell killing. Antioxidants can thus block apoptosis (cell death) triggered by proteasome inhibition. Here, we have evaluated the effects of different antioxidants (edaravone and tiron) on endometrial carcinoma cells treated with aldehyde proteasome inhibitors (MG-132 or ALLN), the boronic acid-based proteasome inhibitor (bortezomib) and the epoxyketone, epoxomicin. We show that tiron specifically inhibited the cytotoxic effects of bortezomib, whereas edaravone inhibited cell death caused by aldehyde-based proteasome inhibitors. We have, however, found that edaravone completely inhibited accumulation of ubiquitin and proteasome activity decrease caused by MG-132 or ALLN, but not by bortezomib. Conversely, tiron inhibited the ubiquitin accumulation and proteasome activity decrease caused by bortezomib. These results suggest that edaravone and tiron rescue cells of proteasome inhibitors from cell death, by inhibiting blockade of proteasome caused by MG-132 and ALLN or bortezomib, respectively. We also tested other antioxidants, and we found that vitamin C inhibited bortezomib-induced cell death. Similar to tiron, vitamin C inhibited cell death by blocking the ability of bortezomib to inhibit the proteasome. Until now, all the antioxidants that blocked proteasome inhibitor-induced cell death also blocked the proteasome inhibitor mechanism of action.