DAOY Cells Research Articles

The histone deacetylase inhibitor vorinostat induces calreticulin exposure in childhood brain tumour cells in vitro

It has recently been recognised that anticancer chemotherapy can elicit an immunogenic form of apoptosis characterised by the exposure of calreticulin (CRT) on the surface of dying tumour cells, entailing an immune response that contributes to the therapeutic outcome. CRT exposure has been found to be induced by anthracyclins and oxaliplatin, but not by other proapoptotic antineoplastic agents including etoposide, camptothecin and cisplatin. In this study, we examined the histone deacetylase inhibitor vorinostat for its capability to stimulate CRT exposure in tumour cells. Childhood tumour cells, i.e. the brain tumour cell lines PFSK and DAOY and the Ewing's sarcoma cell line CADO-ES-1, were treated with vorinostat, and CRT exposure was determined by flow cytometric analysis of CRT immunofluorescence. Combination effects of vorinostat/TRAIL and vorinostat/bortezomib were also assessed. Vorinostat treatment induced CRT exposure in PFSK and DAOY cells, but not in caspase-8-deficient CADO-ES-1 cells. CRT exposure could be prevented by the pan-caspase inhibitor z-VAD-fmk and by brefeldin A, an inhibitor of Golgi-mediated transport. Vorinostat has the capacity to elicit CRT exposure, suggesting its usefulness as immunogenic antitumour agent.

Differential expression and cellular distribution of γ‐tubulin and βIII‐tubulin in medulloblastomas and human medulloblastoma cell lines

In previous studies, we have shown overexpression and ectopic subcellular distribution of gamma-tubulin and betaIII-tubulin in human glioblastomas and glioblastoma cell lines (Katsetos et al., 2006, J Neuropathol Exp Neurol 65:455-467; Katsetos et al., 2007, Neurochem Res 32:1387-1398). Here we determined the expression of gamma-tubulin in surgically excised medulloblastomas (n = 20) and in the human medulloblastoma cell lines D283 Med and DAOY. In clinical tissue samples, the immunohistochemical distribution of gamma-tubulin labeling was pervasive and inversely related to neuritogenesis. Overexpression of gamma-tubulin was widespread in poorly differentiated, proliferating tumor cells but was significantly diminished in quiescent differentiating tumor cells undergoing neuritogenesis, highlighted by betaIII-tubulin immunolabeling. By quantitative real-time PCR, gamma-tubulin transcripts for TUBG1, TUBG2, and TUBB3 genes were detected in both cell lines but expression was less prominent when compared with the human glioblastoma cell lines. Immunoblotting revealed comparable amounts of gamma-tubulin and betaIII-tubulin in different phases of cell cycle; however, a larger amount of gamma-tubulin was detected in D283 Med when compared with DAOY cells. Interphase D283 Med cells exhibited predominantly diffuse cytoplasmic gamma-tubulin localization, in addition to the expected centrosome-associated distribution. Robust betaIII-tubulin immunoreactivity was detected in mitotic spindles of DAOY cells. Our data indicate that overexpression of gamma-tubulin may be linked to phenotypic dedifferentiation (anaplasia) and tumor progression in medulloblastomas and may potentially serve as a promising tumor marker.

Nitric Oxide Control of Proliferation in Nerve Cells and in Tumor Cells of Nervous Origin

Recent evidence suggests that nitric oxide (NO) has a remarkable anti-proliferative action towards dividing neural precursor cells as well as towards cells giving rise to neural-derived tumors. The present paper summarizes essential literature-derived information on this issue and provides novel experimental evidence for these NO-mediated actions regarding a well characterized population of neuronal precursors, the cerebellar granule cell precursors and a cell line of medulloblastoma, a pediatric tumor originating from these same precursor cells undergoing deregulated proliferation. Evidence is presented regarding the NO-mediated regulation of proliferation of neuronal precursor cells both during developmental and adult neurogenesis. Then, the role of NO in the control of proliferation of neural-derived tumor cells, such as PC12 and neuroblastoma cells, is discussed. Novel experimental data are provided documenting the anti-proliferative action of NO towards basal and mitogen-stimulated division of rat cerebellar granule cell precursors, as well as towards medulloblastoma DAOY cells. Finally, some molecular correlates of NO action on cell cycle regulation are discussed. Overall, the data presented and discussed here highlight similarities at the molecular level between physiologic processes regulating normal proliferation of neural precursors and pathologic deregulation of these processes leading to tumor formation.

Nerve growth factor activation of the TrkA receptor induces cell death, by macropinocytosis, in medulloblastoma Daoy cells

Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.

Phytoquinoids and Secoprezizaane‐Type Sesquiterpenes from Illicium arborescens

AbstractA phytochemical investigation of the MeOH extract of Illicium arborescens yielded the two new phytoquinoid epimers, 2,3‐didehydro‐5‐O‐methyl‐11‐epiillifunone E (1) and 2,3‐didehydro‐5‐O‐methylillifunone E (2), as well as five new sesquiterpene lactones (8,9‐secoprezizaane‐type sesquiterpenes). Two of them, i.e., 3 and 4, were minwanensin‐type sesquiterpenes, the other two, i.e., 5 and 6, had the anisatin‐type (or floridanolide type) skeleton, and the fifth, i.e., 7, was a dunnianin‐type sesquiterpene. Their structures were established by analyses of 1D‐ and 2D‐NMR, HR‐MS, and chemical evidence. The in vitro cytotoxic activity of compounds 1–7 was tested against four human tumor cell lines, including HeLa (cervical epitheloid), WiDr (colon), Daoy (medulloblastoma), and Hep2 (liver carcinoma) human‐tumor cells.

P53 regulates LIF expression in human medulloblastoma cells

Medulloblastomas are highly malignant, poorly differentiated childhood tumours arising in the cerebellum. These tumors rarely lose TP53, which is the most commonly mutated gene in cancer. Recent work has shown that the basal level of p53 plays an important role in maternal reproduction by maintaining the expression of LIF in the uterus. Since LIF can maintain the undifferentiated state of stem cells we set out to ask if p53 regulates LIF in the human medulloblastoma cell lines DAOY and D283MED. We also used p53-/- and p53+/+ isogenic HCT116 colorectal carcinoma cell lines, already reported to exhibit p53-dependent expression of the LIF D transcript, to establish the extent of p53-dependency for LIF M and T alternative transcripts. Whilst all three known, full-length alternative transcripts are more abundant in p53+/+ cells, the alternative LIF M and T transcripts appear particularly sensitive to p53. In the p53 wild-type medulloblastoma cell line D283MED chromatin immunoprecipitation experiments showed p53 binding to the LIF gene. The mutant p53 expressed in line DAOY did not bind to this region or to the p21(WAF1) p53 binding site. RNA interference against either WIP1 or SIRT1 stabilized p53 and enhanced the transcription of LIF in D283MED cells. Interestingly, siRNA against WIP1 or SIRT1 also induced increased apoptosis in the medulloblastoma line D283MED and, over a longer time period, in DAOY cells. We speculate that suppression of p53 function by combined WIP1-mediated dephosphorylation and SIRT1 deacetylation enables medulloblastoma cell survival but p53-dependent and independent apoptotic pathways remain intact. Thus small molecule inhibitors of SIRT1 may be useful in treatment of medulloblastoma.

Camptothecin and Fas receptor agonists synergistically induce medulloblastoma cell death: ROS-dependent mechanisms

Medulloblastoma, a common malignant pediatric brain tumor, is highly resistant to death receptor-mediated apoptosis despite death receptor expression by tumor cells. Developing new strategies to overcome this resistance to death receptor activation could positively impact therapeutic outcomes. We explored the modulation of death receptor-induced medulloblastoma cell death by the topoisomerase I inhibitor camptothecin (CPT). CPT significantly increased the human medulloblastoma DAOY cell death response to agonistic anti-Fas antibody (CH-11). Cell death after CPT, CH-11, and CPT+CH-11 treatment was 9, 7, and 33%, respectively. Isobologram analysis showed that CH-11 and CPT act synergistically to induce cell death in DAOY cells. A similar pattern of synergism between CPT and CH-11 was found in ONS-76 medulloblastoma cells. Synergistic cell death was found to be predominantly apoptotic involving both extrinsic and intrinsic pathways as evidenced by annexin V staining, cleavage of caspases (3, 8, and 9), Bid and PARP, and cytoprotection by caspase inhibitors. Flow cytometric analyses showed that expression of cell surface Fas or Fas ligand did not change with drug treatment. Western blot analyses showed that the combination of CH-11+CPT induced a significant decrease in XIAP levels. Furthermore, reactive oxygen species, especially O2, were elevated after CPT treatment, and even more so by the CH-11+CPT treatment. The antioxidants glutathione and N-acetyl-cysteine prevented cell death induced by CPT+CH-11. Moreover, the mitochondrial respiratory chain complex I inhibitor rotenone potentiated CH-11-induced apoptosis in DAOY cells. Taken together, these findings show that CPT synergizes with Fas activation to induce medulloblastoma apoptosis through a mechanism involving reactive oxygen species and oxidative stress pathways.

Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells

Manganese superoxide dismutase (Mn-SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn-SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2-DE, multienzyme digestions were carried out and peptides were analyzed by MALDI-TOF/TOF, Qq-TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp-186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2-oxo-histidine and a previously described intermediate (His+14). Complete sequencing of the two Mn-SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn-SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn-SOD activity.

New Nitrogen‐Containing Sesquiterpenoids from the Taiwanese Soft Coral Cespitularia taeniataMay

Chemical investigation of the EtOH extract of the soft coral Cespitularia taeniata May collected in Taiwan has resulted in the isolation of two new sesquiterpene lactams, taenialactams A and B (1 and 2, resp.), and a new sesquiterpene lactone, taenialactone A (3), together with 8beta-methoxyatractylenolide (4) and atractylenolactam (5). The structures of these compounds were determined on the basis of spectroscopic analyses. The in vitro cytotoxic activities of the isolated metabolites were tested against KB, Daoy, and WiDr cell lines. A biogenetic pathway for these novel compounds was proposed.

Role of Rac1-regulated signaling in medulloblastoma invasion

Medulloblastomas are the most common malignant brain tumors in children. These tumors are highly invasive, and patients harboring these lesions are frequently diagnosed with distant spread. In this study, the authors investigated the role of Rac1, a member of the Rho family of small guanosine triphosphatases, in medulloblastoma invasion. Three established medulloblastoma cell lines were used: DAOY, UW-228, and ONS-76. Specific depletion of Rac1 protein was accomplished by transient transfection of small interfering RNA. Cell invasion through extracellular matrix (Matrigel) was quantified using a transwell migration assay. Mitogen activated protein kinase activation was determined using phospho-MAP kinase-specific antibodies, and inhibition of MAP kinase pathways was achieved by specific small molecule inhibitors. Localization of Rac1 and its expression levels were determined by immunohistochemical analysis using a Rac1-specific antibody, and Rac1 activation was qualitatively assessed by Rac1 plasma membrane association. Small interfering RNA-mediated depletion of Rac1 strongly inhibited medulloblastoma cell invasion. Although depletion of Rac1 inhibited the proliferation of UW-228 cells, and of ONS-76 cells to a lesser extent, it stimulated the proliferation of DAOY cells. Depletion of Rac1 also inhibited the activation of the ERK and JNK MAP kinase pathways, and inhibition of either pathway diminished invasion and proliferation. Immunohistochemical analysis demonstrated that the Rac1 protein was overexpressed in all medulloblastoma tumors examined, and indicated that Rac1 was hyperactive in 6 of 25 tumors. The authors' data show that Rac1 is necessary for the invasive behavior of medulloblastoma cells in vitro, and plays a variable role in medulloblastoma cell proliferation. In addition, these results indicate that Rac1 stimulates medulloblastoma invasion by activating the ERK and JNK pathways. The authors suggest that Rac1 and signaling elements controlled by this guanosine triphosphatase may serve as novel targets for therapeutic intervention in malignant medulloblastomas.

BDNF and PDE4, but not the GRPR, Regulate Viability of Human Medulloblastoma Cells

Medulloblastoma is the most common brain tumor of childhood. Emerging molecular targets in medulloblastoma include neurotrophin and neuropeptide receptors. In the present study, we have examined the influence of brain-derived neurotrophic factor (BDNF)/TrkB receptor- and gastrin-releasing peptide receptor (GRPR)-mediated signaling on the viability of human medulloblastoma cells. The expression of TrkB and GRPR was confirmed by immunohistochemistry and mRNA for both BDNF and GRPR was detected by reverse transcriptase polymerase chain reaction in Daoy, D283, and ONS76 cells. Treatment with BDNF significantly inhibited the viability of Daoy and D283, but not ONS76 cells, measured with the MTT assay. Neither the GRPR agonists GRP and bombesin nor the GRPR antagonist RC-3095 affected cell viability. Because previous findings have indicated that the viability of glioma cells might be enhanced by GRP when combined with the cAMP phosphodiesterase-4 (PDE4) inhibitor rolipram, we also examined the effects of rolipram alone or combined with GRP on cell viability. Rolipram significantly reduced the viability of all three cell lines, and the inhibitory effect of rolipram in Daoy cells was not modified by cotreatment with GRP. The results suggest that BDNF/TrkB and PDE4, but not the GRPR, regulate the viability of medulloblastoma cells.

Tri‐ and Bicyclic Taxoids from the Taiwanese Yew Taxus sumatrana

AbstractFive new taxoids, including a new 2(3→20)‐abeo‐taxane with a 6/10/6‐membered ring system and four 3,8‐seco‐taxanes having a 6/12‐membered ring system, were isolated from an acetone extract of the leaves and twigs of the Taiwanese yew (Taxus sumatrana, Taxaceae). The structures were established as 2α,7β,10α‐triacetoxy‐5α‐hydroxy‐2(3→20)‐abeo‐taxa‐4(20),11‐dien‐9,13‐dione (1), (3E,8E)‐2α,9,10β, 13α,20‐pentaacetoxy‐7β‐hydroxy‐3,8‐secotaxa‐3,8,11‐trien‐5‐one (2), (3E,8E)‐2α,9,10β,13α,20‐pentaacetoxy‐5α,7β‐dihydroxy‐3,8‐secotaxa‐3,8,11‐triene (3), (3E,8E)‐9,10β,13α‐triacetoxy‐2α,7β,20‐trihydroxy‐5α‐[(2E)‐cinnamoyloxy]‐3,8‐secotaxa‐3,8,11‐triene (4), and (3E,8E)‐2α,5α,7β,9,10β,13α‐hexaacetoxy‐20‐hydroxy‐3,8‐secotaxa‐3,8,11‐triene (5), respectively, on the basis 1D‐ and 2D‐NMR spectral analyses. The in vitro cytotoxic activity of compounds 1–5 against four human tumor cell lines, including HeLa (cervical epitheloid), WiDr (colon), Daoy (medulloblastoma), and Hep2 (liver carcinoma) tumor cells was evaluated. Whereas compounds 1–3 were inactive, the novel taxanes 4 and 5 showed significant cytotoxicity.

Antitumor effect in medulloblastoma cells by gefitinib: Ectopic HER2 overexpression enhances gefitinib effects in vivo

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in three medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial primitive neuroectodermal tumor cell line (PFSK), and four MB primary cultures. Cell lines showed diverse expression of EGFR and human epidermal receptor 2 (HER2), with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines and was not related to expression of HER receptors or receptor baseline activation. Gefitinib induced G(0)/G(1) arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (half-maximal inhibitory concentration [IC(50)], 3.8 microM) and lower (IC(50), 6.6 microM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines but was ineffective in constitutive activation of HER2 in D341 cells. Phosphorylated AKT inhibition paralleled that of phosphorylated EGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/AKT pathway. On the whole, EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, which correlated with the lower sensitivity to gefitinib's antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts treated with gefitinib at 150 mg/kg per day was inhibited by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib's antitumor effects in vivo (tumor volume inhibition = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB.

Imatinib blocks migration and invasion of medulloblastoma cells by concurrently inhibiting activation of platelet-derived growth factor receptor and transactivation of epidermal growth factor receptor

Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells phosphorylates extracellular signal-regulated kinase (ERK) and promotes migration. We sought to determine whether blocking PDGFR activity effectively inhibits signaling required for medulloblastoma cell migration and invasion. DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate (Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR phosphorylation and expression, respectively. PDGFR cell signaling, migration, invasion, survival, and proliferation following PDGF-BB stimulation, with and without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor receptor (EGFR), which correlated with enhanced migration, survival, and proliferation. Imatinib (1 μmol/L) treatment of DAOY and D556 cells inhibited PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively, and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but increased PTEN expression and activity. Imatinib treatment also induced DAOY cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h. siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR transactivation, indicating that the effects of imatinib treatment are specific to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase activity is critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR; thus, imatinib may represent an important novel therapeutic agent for the treatment of medulloblastoma.

A new dimeric phenylpropanoid and cytotoxic norditerpene constituents from Podocarpus nakaii

A new dimeric phenylpropanoid namely podonaka A (1), along with the 13 known compounds including diterpenes (2 and 3), norditerpenes (4 and 5), benzenoids (6–10), steroids (11 and 12), chalcone (13), and megastigmane (14), was isolated from the EtOH extract of Podocarpus nakaii Hayata. The structure of 1 was elucidated on the basis of spectroscopic analysis including 1D and 2D NMR and MS techniques. Biological evaluation showed that norditerpenes, inumakilactone B (4), and podolactone E (5) have potent cytotoxic activities against Daoy, WiDr, KB, and HeLa tumor cell lines. Moreover, low dosage of 5 may induce early apoptosis in KB cells before 12 h.

C-Met activation in medulloblastoma induces tissue factor expression and activity: effects on cell migration

Met, the receptor for hepatocyte growth factor (HGF), is a receptor tyrosine kinase that has recently emerged as an important contributor to human neoplasia. In physiological and pathological conditions, Met triggers various cellular functions related to cell proliferation, cell migration and the inhibition of apoptosis, and also regulates a genetic program leading to coagulation. Since medulloblastomas (MBs) express high levels of tissue factor (TF), the main initiator of blood coagulation, we therefore examined the link between Met and TF expression in these pediatric tumors. We observed that stimulation of the MB cell line DAOY with HGF led to a marked increase of TF expression and procoagulant activity, in agreement with analysis of clinical MB tumor specimens, in which tumors expressing high levels of Met also showed high levels of TF. The HGF-dependent increase in TF expression and activity required Src family kinases and led to the translocation of TF to actin-rich structures at the cell periphery, suggesting a role of the protein in cell migration. Accordingly, addition of physiological concentrations of the TF activator factor VIIa (FVII) to HGF-stimulated DAOY cells promoted a marked increase in the migratory potential of these cells. Overall, these results suggest that HGF-induced activation of the Met receptor results in TF expression by MB cells and that this event probably contribute to tumor proliferation by enabling the formation of a provisional fibrin matrix. In addition, TF-mediated non-hemostatic functions, such as migration toward FVIIa, may also play a central role in MB aggressiveness.

The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G(2)/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, after Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein and the neuronal marker synaptophysin in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma.

The Stem Cell Marker CD133 (Prominin-1) is Phosphorylated on Cytoplasmic Tyrosine-828 and Tyrosine-852 by Src and Fyn Tyrosine Kinases

CD133 (prominin-1) is a transmembrane glycoprotein expressed at the surface of normal and cancer stem cells, progenitor cells, rod photoreceptor cells, and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to fundamental properties of stem cells such as self-renewal and differentiation remains unknown. CD133 contains a short C-terminal cytoplasmic domain with five tyrosine residues, including a consensus tyrosine phosphorylation site that has not yet been investigated. In this study, we show that CD133 is phosphorylated in human medulloblastoma D283 and Daoy cells, in a Src family kinase-dependent manner. The cytoplasmic domain of CD133 is tyrosine phosphorylated in Daoy cells overexpressing Src and Fyn tyrosine kinases, as well as in vitro using recombinant proteins. Deletion of the C-terminal cytoplasmic domain of CD133 considerably reduced its phosphorylation by Src. To identify the tyrosine phosphorylation sites in CD133, we used matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI Q-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of tyrosine-phosphorylated CD133 by mass spectrometry and site-directed mutagenesis identified tyrosine-828 and the nonconsensus tyrosine-852 as the major tyrosine phosphorylation sites both in vitro and in intact cells. Identification of CD133 as a substrate for Src-family tyrosine kinases suggests that the cytoplasmic domain of CD133 might play an important role in the regulation of its functions.

A GLI1-p53 inhibitory loop controls neural stem cell and tumour cell numbers

How cell numbers are determined is not understood. Hedgehog-Gli activity is involved in precursor cell proliferation and stem cell self-renewal, and its deregulation sustains the growth of many human tumours. However, it is not known whether GLI1, the final mediator of Hh signals, controls stem cell numbers, and how its activity is restricted to curtail tumourigenesis. Here we have altered the levels of GLI1 and p53, the major tumour suppressor, in multiple systems. We show that GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro. In contrast, p53 inhibits GLI1-driven neural stem cell self-renewal, tumour growth and proliferation. Mechanistically, p53 inhibits the activity, nuclear localisation and levels of GLI1 and in turn, GLI1 represses p53, establishing an inhibitory loop. We also find that p53 regulates the phosphorylation of a novel N' truncated putative activator isoform of GLI1 in human cells. The balance of GLI1 and p53 functions, thus, determines cell numbers, and prevalence of p53 restricts GLI1-driven stem cell expansion and tumourigenesis.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.