ER stress-mediated apoptotic pathway induced by Abeta peptide requires the presence of functional mitochondria.

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimer's disease (AD). Previously we found that Abeta induces both mitochondrial and endoplasmic reticulum (ER) dysfunction leading to apoptosis, and now we address the relevance of ER-mitochondria crosstalk in apoptotic cell death triggered by Abeta peptide. Using mitochondrial DNA-depleted rho0 cells derived from the human NT2 teratocarcinoma cell line, characterized by the absence of functional mitochondria, and the parental rho+ cells, we report here that treatment with the synthetic Abeta1-40 peptide, or the classical ER stressors thapsigargin or brefeldin A, increases GRP78 expression levels and caspase activity, two ER stress markers, and also depletes ER calcium stores. Significantly, we show that the presence of functional mitochondria is required for ER stress-mediated apoptotic cell death triggered by toxic insults such as Abeta. We found that the increase in the levels of the pro-apoptotic transcription factor GADD153/CHOP, which mediates ER stress-induced cell death, as well as caspase-9 and -3 activation and increased number of TUNEL-positive cells, occurs in treated parental rho+ cells but is abolished in rho0 cells. Our results strongly support the close communication between ER and mitochondria during apoptotic cell death induced by the Abeta peptide and provide insights into the molecular cascade of cell death in AD.

The road from teratocarcinoma to human embryonic stem cells.

In this article, a brief review of the research that began with the study of murine teratomas of the testis and ultimately led to the culture of human embryonic stem cells is discussed. Most of the space will be devoted to the studies in which the author personally took part, and the discussion will also touch upon some of the crucial experiments important for the understanding of this entire research effort.

Light and electron microscopy of the pagetoid spread of germ cell carcinoma in the rete testis: morphologic evidence suggestive of field effect as a mechanism of tumor spread.

The purpose of this study is to investigate the mechanism of tumor spread in the pagetoid spread of germ cell tumors in the rete testis (PSRT). Twenty consecutive cases of germ cell tumor of the testis (9 seminomas, 3 embryonal carcinomas, and 8 teratocarcinomas) were retrieved to identify the cases with PSRT. The areas of pagetoid spread were examined by the serial sectioning of the entire thickness of the tissue block. Available fresh tissue was submitted for electron microscopic study. Ten cases were associated with PSRT and had focal or extensive areas of intratubular germ cell neoplasia (IGCN) in the proximity of the tumor and the rete testis (RT). In the remaining 10 cases, 6 were associated with IGCN distant from the RT and the last 4 were not associated with IGCN. Seminiferous tubules with IGCN were seen connecting with the RT with pagetoid spread. Isolated single intraepithelial tumor cells also were identified at the periphery of the areas with PSRT. Electron microscopic study of the RT of 4 cases with PSRT (2 seminomas, 1 embryonal carcinoma, and 1 teratocarcinoma) revealed desmosome-type junctions between tumor cells with RT epithelial cells. Direct tumor expansion and cell motility as mechanisms of tumor spread in PSRT does not explain the presence of isolated cells and desmosome-type junctions of the tumor cells as demonstrated in this study. The authors believe that the field effect plays an important part in the pathogenesis of this pagetoid spread in the RT. It is likely that this field effect is induced by the germ cell tumor and is operated through the immature germ cells or undifferentiated epithelial cells in the RT adjacent to the tumor cells.

Sinonasal teratocarcinosarcoma: ultrastructural and immunohistochemical evidence of neuroectodermal origin.

The authors report a case of sinonasal teratocarcinosarcoma in a 37-year-old man, which was located in the anterior skull base and extended to the right nasal cavity and paranasal sinuses. The tumor was surgically resected twice, but it could not be removed completely. Microscopically, it was mainly composed of primitive cell nests within a moderately cellular stroma. The components of squamous cell epithelia with focal teratoid appearance and adenocarcinomatous differentiation were observed. There were many rhabdomyoblasts scattered in the nests and stroma. Ultrastructurally, the primitive cells had many neural processes with parallel microtubules, resembling olfactory neuroblastoma. Rhabdomyoblasts showed various degrees of skeletal muscle differentiation. Some of the stromal spindle cells had actin filaments with dense patches and dense core granules. Immunohistochemically, the primitive cells were positive for epithelial markers, neuron-specific enolase, synaptophysin, and myogenic regulatory proteins. The rhabdomyoblasts showed immunoreactivity for myoid markers, cytokeratin, epithelial membrane antigen, and synaptophysin. Most of the stromal spindle cells were positive for smooth muscle actin, neuron-specific enolase and synaptophysin. The immunohistochemical and ultrastructural findings suggest that primitive cells had the most primitive phenotype of placodes, and support the possibility that sinonasal teratocarcinosarcoma is essentially a neuroectodermal tumor with divergent differentiation.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells.

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp(+/+)) ES cells and Parp(+/-) and Parp(-/-) ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp(-/-) clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp(+/-) clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp(-/-) clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Functional serotonin-2B receptors are expressed by a teratocarcinoma-derived cell line during serotoninergic differentiation.

Among immortalized teratocarcinoma-derived cells, the clone 1C11 is a committed precursor of the neuronal lineage. On day 2 of its serotoninergic differentiation, this clone expresses only one subtype of serotonin [5-hydroxytryptamine (5-HT)] receptor, which is functionally coupled to phosphatidylinositol hydrolysis. The identity of these receptors was established by comparing their properties with those of 5-HT2B receptors expressed by LMTK- fibroblasts stably transfected with the recently cloned murine cDNA NP75 (LM5 cells). In both cell types, the analysis of (+/-)-1-(2,5-dimethoxy-4-[125I]iodophenyl)- 2-aminopropane HCl ([125I]DOI) binding revealed the presence of a single class of sites, the affinity of which was 1 order of magnitude lower than that reported for 5-HT2A receptors. In 1C11 cells differentiated for 2 days, as well as in LM5 cells, DOI binding was decreased by nonhydrolyzable analogs of GTP, indicating that the 5-HT2B receptor is functionally coupled to a G protein. The DOI-induced increase of phosphoinositide hydrolysis, which was correlated with both GTPase activity and binding data, is mediated by a Gq protein. This work demonstrates that the 5-HT2B receptor is functionally expressed before complete serotoninergic differentiation of 1C11 cells. The inducible 1C11 clone thus provides an in vitro model to investigate the possible role of the 5-HT2B receptor in the expression of the serotoninergic phenotype.

Apoptosis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

In this work we have characterized a process of cell death by apoptosis occurring in retinoic acid-induced differentiation of F9 embryonal carcinoma cells. Morphological changes corresponding to apoptosis were observed concomitantly with those of differentiated phenotype, in cell cultures treated for 48 h with physiological concentrations of all-trans-retinoic acid. Internucleosomal DNA fragmentation was also detected using agarose gels. These observations show that retinoic acid can induce both differentiation and apoptosis in an experimental system--the teratocarcinoma stem cell lines--widely used so far in the study of differentiation. We present this system as a tool for a better understanding of the role of retinoic acid in the regulation of cell differentiation and programmed cell death.

Co-expression of laminin and a 67 kDa laminin-binding protein in teratocarcinoma embryoid bodies.

The synthesis of laminin chains is usually correlated to specific functions of laminin during embryo development. In this study we show that the CE44 teratocarcinoma embryoid bodies synthesize B1 and B2 chains of laminin as well as a 67 kDa laminin-binding protein while simultaneously differentiating into parietal endoderm. The intracystic presence of laminin and the 67 kDa cell surface laminin-receptor in teratocarcinoma differentiated cells suggest that the B chains of laminin play an important role in induction and/or mediation of cell differentiation and confirm the importance of laminin A chain in cell polarization and the supramolecular rearrangement of definitive basement membrane.

Ultrastructural analysis of the early development of teratocarcinomas.

Genital ridges of 12-day embryos of strain 129 mice were transplanted into the testes of adult mice of the same strain. Of the transplants that differentiated into fetal testes, 80% contained intratubular teratocarcinomas by the 7th day after transplantation. These teratocarcinomas were studied ultrastructurally, daily from the 7th-14th day following transplantation, to gain information about the histogenesis of the tumors and the mode of development of multipotent cells. The embryonal carcinoma cells, which are the stem cells of teratocarcinomas, so closely resembled primordial germ cells that the former appeared to be derived from the latter. The cytoplasm of these multipotential cells was characterized by large numbers of dispersed ribosomes and polysomes with few membranous organelles other than mitochondria. Among the earliest signs of differentiation was the development of rough-surfaced endoplasmic reticulum and Golgi complexes, followed by modification of the plasma membrane.