Synthesis, Shedding, and Intercellular Transfer of Human Medulloblastoma Gangliosides: Abrogation by a New Inhibitor of Glucosylceramide Synthase

Abstract: The biological activity of human medulloblastoma tumor gangliosides very likely involves the interaction of these molecules with host cells in the tumor microenvironment. To trace the hypothesized intercellular transfer of shed medulloblastoma gangliosides, we used an in vitro dual-chamber culture system in which the tumor cells, the shed gangliosides, and the target cells to which they might bind would not be perturbed during the transfer process. We observed that under these unmanipulated conditions, gangliosides were shed by the Daoy medulloblastoma cell line (∼300 pmol/10⁸ cells/h), traversed the chamber membrane, and stably bound to the target fibroblasts at the very high density of 10⁷ molecules per cell within 48 h. To determine if this substantial intercellular transfer of shed gangliosides, with its potential of modifying target cell function, could be blocked, we evaluated a new inhibitor of glucosylceramide synthase, dl - threo -1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP (1.0 µ M ) reduced (90%) Daoy cell ganglioside content strikingly, without causing toxicity or inhibiting cell proliferation. Subsequently, ganglioside shedding by the medulloblastoma cells was diminished significantly (to ∼50 pmol/10⁸/h), and binding of radiolabeled shed medulloblastoma gangliosides to target fibroblasts was consequently almost completely abrogated. We conclude that the shedding and transfer of potentially biologically active human medulloblastoma gangliosides can be diminished effectively by PPPP.     

Biophysical Properties of Human Medulloblastoma Cells

Medulloblastoma is a pediatric high-grade cerebellar malignancy derived from neuronal precursors. Although electrophysiologic characteristics of cerebellar granule neurons at all stages of cell development have been well described, such characterization has not been reported for medulloblastoma. In this study we attempt to characterize important electrophysiologic features of medulloblastoma that may distinguish it from the surrounding cerebellum. Using patient-derived cell lines and tumor tissues, we show that medulloblastoma cells have no inward Na+ current or transient K+ current involved in action potential generation and propagation, typically seen in granule neurons. Expression and function of calcium-activated, large-conductance K+ channels are diminished in medulloblastoma, judged by electrophysiology and Western analysis. The resting membrane potential of medulloblastoma cells in culture is quite depolarized compared to granule neurons. Interestingly, medulloblastoma cells express small, fast-inactivating calcium currents consistent with T-type calcium channels, but these channels are activated only from hyperpolarized potentials, which are unlikely to occur. Additionally, a background acid-sensitive K+ current is present with features characteristic of TASK1 or TASK3 channels, such as inhibition by ruthenium red. Western analysis confirms expression of TASK1 and TASK3. In describing the electrophysiologic characteristics of medulloblastoma, one can see features that resemble other high-grade malignancies as opposed to normal cerebellar granule neurons. This supports the notion that the malignant phenotype of medulloblastoma is characterized by unique changes in ion channel expression.     

Gangliosides of Human Cerebral Astrocytomas

Abstract: Ganglioside content and composition were examined in a series of 25 human gliomas, which were graded histologically by the criteria of Kerno-han. The concentration of total gangliosides (lpid-bound sialic acid [LBSA]) was decreased with respect to normal brain tissue in nearly all tumors and the extent of reduction correlated with the stage of tumor anaplasia. The distribution of individual gangliosides was altered in glial tumor tissue with an increase in proportion of the structurally simple gangliosides and reduction of polysialogangliosides. The most consistent and significant difference was the elevation of proportion of ganglioside GD3 from 4–5% of total LBSA in normal brain to 20% in the astrocytoma grade IV. The proportions of gangliosides GM2 and GD2 were also found to be elevated in all grades of the tumors. The simplification of ganglioside composition seems to be associated with transformation of the astrocyte with the accumulation of the simpler gangliosides, since the changes resemble those reported with in vitro transformation rather than those of analyses of preparations of purified glial cells.     

Gangliosides in Human Fetal Brain

The ganglioside concentration and composition were determined in 42 human fetal brains from gestational week 10 to 22, a period that is morphologically characterized by rapid neuroblast proliferation and migration. The ganglioside concentration was constant during this period, approximately 1 mumol of ganglioside sialic acid/g of fresh tissue weight. At gestational week 10 the ganglioside pattern was dominated by gangliosides of the ganglio b series, with the major ganglioside being GT1b, contributing 40% of total ganglioside sialic acid, whereas GD1b and GD3 contributed only 15 and 10%, respectively. The proportion of b series ganglioside decreased to gestational week 22, with the most pronounced relative reduction affecting GD3, but also GT1b and GD1b to a lesser extent. The ganglioside GQ1b increased in content from gestational week 10 and peaked around week 16. The proportion of GD1a increased markedly between gestational week 12 and 14 and slowly between week 14 and 18 and then increased rapidly from week 20. Ganglioside GM1 underwent a similar change. Gangliosides of the lacto series contributed 6-10% of ganglioside sialic acid between gestational week 10 and 15, and thereafter the proportion slowly decreased. 3'-isoLM1 decreased rapidly in content from gestational week 10 (20 nmol/g of fresh weight) to week 22 (less than 0.5 nmol/g of fresh weight), whereas the gangliosides of the neolacto series (3'-LM1 and 3',8'-LD1) showed a slower and less marked decline in level. The biological significance of the ganglioside changes is discussed.     

外源性神经节苷脂对人－肝癌培养细胞内钙的作用及其方式

本工作采用荧光探针Ｆｕｒａ－２ＡＭ观察了外源性神经节苷脂ＧＭ３和ＧＤ３对ＳＭＭＣ－７７２１人肝癌培养细胞钙的影响,证明ＧＭ３和ＧＤ３均能升高细胞内钙浓度（［Ｃａ２＋］ｉ）,但程度上有极大差异。１０ｎｍｏｌ／ｍＬＧＭ３或１．０ｎｍｏｌ／ｍＬＧＤ３可使［Ｃａ２＋］ｉ上升高是明显,与对照相比［Ｃａ２＋］ｉ分别增加２１５～２５０％和４２％。进一步用Ｖｅｒａｐａｍｉｌ阻断钙通道和内质网钙释放、去除细胞外Ｎａ＋以抑制Ｎａ＋－Ｃａ２＋交换以及去除细胞外Ｃａ２＋在无外钙内流等系统观察了ＧＭ３和ＧＤ３的作用方式,结果提示ＧＭ３升高［Ｃａ２＋］ｉ的机制是一个同时增加内质网钙释放、激活钙通道并伴有质膜Ｃａ２＋－ＡＴＰ酶激活的综合结果;而ＧＤ３则主要抑制Ｎａ＋－Ｃａ２＋交换系统。     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Determination of Gangliosides in Six Human Primary Medulloblastomas

The ganglioside composition of six human medulloblastomas was analyzed. The characterization was performed by thin-layer chromatography, sialidase hydrolysis, and immunological staining with a panel of characterized antiganglioside monoclonal antibodies. The total ganglioside content ranged from 60 to 1,130 nmol of ganglioside sialic acid/g wet weight. Neuronal gangliosides (gangliotetraose series) were found in varying amounts in all medulloblastomas. Gangliosides of the neolactotetraose series (3'-LM1 and LD1) were present in all specimens, and the lactotetraose series ganglioside 3'-isoLM1 was found in all cases showing astrocytic differentiation. This supports our previous findings that 3'-isoLM1 is a marker for proliferating astroglial cells.     

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma^1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker⁵, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.Download video file.^{(31M, mov)}     

Isolation,Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively¹. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development ^2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma ^5-9.A recent report has shown that a subset of medulloblastoma cells derived from Patched1^LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors ¹⁰. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (¹¹, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.Download video file.^{(40M, mov)}     

Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells

It has been reported that Annexin A2 (ANXA2) is up-regulated in hepatocellular carcinoma (HCC), but the roles of ANXA2 in the migration and invasion of HCC cells have not been determined. In this study, we found that ANXA2-specific siRNA (si-ANXA2) significantly inhibited the migration and invasion of HCC cells co-cultured with fibroblasts in vitro. In addition, the production of MMP-2 by fibroblasts cultured in supernatant collected from si-ANXA2-transfected HCC cells was notably down-regulated. ANXA2 was also found to be co-localized and co-immunoprecipitated with CD147. Further investigation revealed that the expression of ANXA2 in HCC cells affected the shedding of CD147-harboring membrane microvesicles, acting as a vehicle for CD147 in tumor-stromal interactions and thereby regulating the production of MMP-2 by fibroblasts. Together, these results suggest that ANXA2 enhances the migration and invasion potential of HCC cells in vitro by regulating the trafficking of CD147-harboring membrane microvesicles.     

Shedding Light on Mammary Stem Cells and Tumorigenesis

No abstract available.     

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

1. Download high-res image (178KB)
2. Download full-size image

     

Topographical Atlas of the Gangliosides of the Adult Human Brain

Forty different brain samples, consisting of neocortical, archicortical, and paleocortical areas; telencephalic, diencephalic, and mesencephalic subcortical nuclei; and the cerebellum as well as some of the corresponding white matter bundles were analyzed with respect to total content of ganglioside-sialic acid and the ganglioside pattern. The total content of gangliosides seems to depend mainly on the proportions of gray and white matter. Thus, neocortical areas, which are rich in gray matter, have a four- to fivefold higher ganglioside content (per milligram of protein) than white matter-rich samples such as optic chiasm, capsula interna, or corpus callosum. White matter-rich regions, although very heterogeneous in ganglioside composition, are further characterized by appreciable amounts of the myelin-enriched GM4. In the neocortex a remarkable degree of regional pattern differences was revealed. In the frontal and parietal areas there is a moderate, and in the temporal region a strong preponderance of sialic acid bound to gangliosides of the a-pathway (GD1a, GM1). In contrast, the occipital cortex favors the b-pathway of ganglioside synthesis (GQ1b, GT1b, GD1b). A predominance of "b-gangliosides" was found in all structures that are related to the visual system (optic chiasm, pulvinar-thalamus, superior colliculi, visual cortex) as well as in the cerebellum and the nucleus ruber. All diencephalic nuclei tend to favor slightly "b-gangliosides," while the mesencephalic nuclei are very heterogeneous in their ganglioside composition. A preponderance of "a-gangliosides" was found in the periamygdalar cortex, putamen, inferior colliculi, substantia nigra, frontal white matter, internal capsule, globus pallidus, basal nucleus of Meynert, and corpus callosum as well as in the frontal, parietal, and temporal cortices. An exceptional predominance of GM1 and GD1a was revealed for the hippocampal archicortex and the amygdala, suggesting a possible functional correlation to glutaminergic synaptic transmission.     

兔肾体外灌注细胞脱落规律及其形态学改变

目的观察室温下21～25℃DMEM、血浆和血液 DMEM有氧灌注对离体兔肾的形态学影响,探讨离体肾在体外灌注情况下的细胞脱落规律。方法50只大耳白兔随机分为正常对照(5只)、DMEM组(15只)、血浆组(15只)、血液 DMEM组(15只),实验组观察时相点为灌注后1、3、6h。各组离体肾分别用相应的灌注液进行有氧灌注,通过大体观察、含水率、病理切片、脱落细胞记数、原位标记法(TerminaldeoxynucleotidyltransferaseTdTmediatedbiotindUTPnickendlabeling,TUNEL)凋亡染色进行灌注肾脏的细胞脱落规律分析。结果各组灌注后肾脏外观明显水肿,正常肾脏含水率为786%,灌注后肾脏含水率显著升高,DMEM组肾脏灌注6h后含水率为848%。光镜下见肾脏间质水肿、肾小球肾小管上皮细胞脱落、细胞核崩解、细胞密度减小。TUNEL染色正常对照凋亡细胞率为23%,DMEM组灌注1h细胞凋亡率即达159%。灌注液中脱落细胞数随灌注时间进行性增加。结论室温下离体肾脏有氧灌注后有较严重的肾脏上皮细胞脱落现象,凋亡和坏死均参与了其发生机制。     

Durotaxis by Human Cancer Cells

Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2–7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration.     

The Histone Deacetylase Inhibitor Sodium Butyrate Promotes Cell Death and Differentiation and Reduces Neurosphere Formation in Human Medulloblastoma Cells

Increasing evidence suggests that alterations in epigenetic mechanisms regulating chromatin state play a role in the pathogenesis of medulloblastoma (MB), the most common malignant brain tumor of childhood. Histone deacetylase (HDAC) inhibitors, which increase chromatin relaxation, have been shown to display anticancer activities. Here we show that the HDAC inhibitor sodium butyrate (NaB) markedly increases cell death and reduces colony formation in human MB cell lines. In addition, NaB increased the mRNA expression of Gria2, a neuronal differentiation marker, in D283 and DAOY cells and reduced the number of neurospheres in D283 cell cultures. Finally, NaB reduced the viability of D283 cells when combined with etoposide. These data show that NaB displays pronounced inhibitory effects on the survival of human MB cells and suggest that NaB might potentiate the effects of etoposide. In addition, our study suggests that HDAC inhibition might promote the neuronal differentiation of MB cells and provides the first evidence that an HDAC inhibitor might suppress the expansion or survival of MB cancer stem cells.     

Shedding Light on the Nature of Seminal Round Cells

Introduction

In this investigation we assess the incidence of round cells (RCs) in semen samples in our infertile patient population and their significance on intracytoplasmic sperm injection (ICSI) cycle outcomes. We also evaluate the usefulness of RCs as indicators of bacterial infection and highlight the origin of this cell-type, as well as its role in the human ejaculate.

Patients and Methods

In a prospective fashion, a total of 4,810 ejaculated samples were included in the study during a period of 24 months. RCs were characterized for white blood cell (WBC) components versus exfoliated germ cells by testing for multiple markers of ploidy as well as protamine assays. Cases displaying ≥ 2 x 10⁶/ml RCs were screened for bacteria. Raw specimens containing RC were processed by peroxidase and other leukocyte assays, specific stains for protamines were used to identify spermiogenic stage, aneuploidy (FISH) assessment was carried out, and the presence of various Sertoli-cell cytoplasmic remnants was analyzed to identify and characterize immature germ cells. The effect of RC on clinical outcome was assessed in specimens used for ICSI.

Results

The average age of the men involved was 39.2 ± 7 years. Semen samples had a mean concentration of 40.7 ± 31 x 10⁶/ml, motility of 42.6 ± 35%, and morphology of 2.3 ± 2%. RCs were identified in 261 specimens, representing a proportion of 5.4%. Men with RCs had comparable age but lower sperm concentration and morphology than the control group (P<0.001). The aneuploidy rate of 4.3% in RCs group was remarkably higher than the control group (2.3%; P<0.001). Sperm aneuploidy rate positively correlated with the number of RCs (P<0.001). Of 44 men, 17 of them in 18 cycles had up to 1.9 x 10⁶/ml RCs without affecting fertilization and clinical pregnancy rates when compared to controls (n = 365 cycles). In 27 men undergoing 33 ICSI cycles with ≥ 2 x 10⁶/ml RCs, the fertilization rate trended lower and the miscarriage rate was significantly increased (P = 0.05). There was lack of correlation between RC and bacteriological growth. Specific markers indicated that seminal RCs are mostly immature germ cells encased in the remnants of Sertoli cell cytoplasm. Moreover, their modest protamine content and their haploid status confirm that they are post-meiotic. Sequential observation in the same man showed that RC episodes were followed by an amelioration of semen parameters, and interestingly, the episodic occurrence of RCs often coincides with flu season peaks.

Conclusions

Seminal RCs are not a marker of infectiousness but rather a transient indicator of spermatogenic insult that possibly occurs in most men following a mild and transient ailment such as the flu.     

Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Receptor Dimerization in Human Glioma U-1242MG and Swiss 3T3 Cells

Abstract: We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [³⁵S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.