transforming-growth-factor-beta and Teratocarcinoma

The transforming growth factor-β (TGFβ) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFβ family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFβ family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFβ family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFβ family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

Topics: Animals; Cell Differentiation; Cell Self Renewal; Humans; Male; Neoplastic Stem Cells; Pluripotent Stem Cells; Signal Transduction; Teratocarcinoma; Testicular Neoplasms; Transforming Growth Factor beta

Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.. We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).. Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.. Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.

Topics: Adenocarcinoma; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Macrophages; Mice; Neoplasm Invasiveness; Teratocarcinoma; Transforming Growth Factor beta

Genes required to maintain pluripotency in human embryonic stem (hES) cells are largely unknown, with the exception of OCT-4, a homolog of mouse Oct-4, which is critical for the establishment of the embryonic inner cell mass and the generation of totipotent mouse embryonic stem (mES) cell lines. In the current study, we identified two genes with expression similar to OCT-4, in that they are largely restricted to pluripotent hES cells, premeiotic germ lineage cells, and testicular germ cell tumor cells. Furthermore, we determined that upon hES cell differentiation, their expression is downregulated. The genes we identified in the current study include the human stella-related (STELLAR) gene, which encodes a highly divergent protein (with just 32.1% identity to mouse stella over the 159 amino acid sequence) that maps to human chromosome 12p13. Notably, human STELLAR is located distal to a previously uncharacterized homeobox gene, which is the human homolog of the recently identified murine gene, Nanog, and proximal to the GDF3 locus, whose transcription is restricted to germ cell tumor cells. Our characterization of STELLAR, NANOG, and GDF3 suggests that they may play a similar role in humans as in mice, in spite of their remarkable evolutionary divergence.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Cells, Cultured; Chromosomes, Human, Pair 12; DNA-Binding Proteins; Down-Regulation; Embryo, Mammalian; Gene Expression Regulation, Developmental; Germ Cells; Growth Differentiation Factor 3; Homeodomain Proteins; Humans; Mice; Molecular Sequence Data; Nanog Homeobox Protein; Octamer Transcription Factor-3; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Stem Cells; Teratocarcinoma; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

Topics: Astrocytes; Astrocytoma; Blotting, Western; Brain Neoplasms; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Glioblastoma; Humans; Membrane Proteins; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Presenilin-1; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Prion diseases are a group of neurodegenerative disorders characterized by intracerebral accumulation of a protease-resistant prion protein (PrP(Sc)) that causes extensive neuronal degeneration and astrogliosis. The regulation of prion protein (PrP) gene expression by a panel of glial and neuronal cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-10, and TGF-beta1) was investigated in human neural cell lines by reverse transcription-polymerase chain reaction and Northern blot analysis. The constitutive expression of PrP mRNA was identified in all human neural cell lines and tissues examined including Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG astrocytoma, KG-1-C glioma, NTera2 teratocarcinoma, NTera2-derived differentiated neurons (NTera2-N), peripheral nerve, and cerebral and cerebellar tissues. In SK-N-SH cells, a 48 hour (h) treatment with 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, or 100 nM phorbol 12-myristate 13-acetate induced a 2.7- to 4.2-fold increase in the level of PrP mRNA, while the exposure to 100 ng/ml IFN-gamma resulted in a 50% decrease. By contrast, none of these cytokines significantly altered the levels of PrP mRNA in IMR-32, NTera2-N, or U-373MG cells. These results indicate that the PrP gene expression is constitutive in a wide range of human neural cell lines and tissues where it is controlled by cell type-specific regulatory mechanisms.

Topics: Astrocytoma; Brain; Brain Neoplasms; Cell Line; Cerebellum; Cytokines; Eye Neoplasms; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-1; Interleukin-10; Neuroblastoma; Neuroglia; Neurons; Polymerase Chain Reaction; Prions; Retinoblastoma; RNA, Messenger; Teratocarcinoma; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Contact is a vital mechanism used by cells to interact with their environment. Contact with living and nonliving elements adjacent to a cell is the basis for many common biological events ranging from growth regulation to metastasis to embryonic pattern formation. We describe the cloning and characterization of a novel density-regulated protein (drp) whose expression is increased in cultured cells at high density compared with cells at low density. A drp cDNA was isolated from the human teratocarcinoma cell line PA-1. Northern analysis with a drp probe revealed transcripts of 2.8 and 3.2 kb. The drp RNA was expressed in a variety of tissues, with the highest amounts in skeletal and cardiac muscle. Using antipeptide antisera, increasing amounts of a 70-kDa protein were detected using several experimental approaches in several cells lines as cell density is increased. Conditioned medium from high-density cells was unable to induce expression of drp in cells growing at low density. Similarly, growth arrest by serum starvation or transforming growth factor-beta (TGF-beta) treatment failed to elicit drp expression. We conclude that drp is a novel protein whose expression is increased at high cell density but not growth arrest.

Topics: Amino Acid Sequence; Base Sequence; Cell Count; Cell Cycle Proteins; Cloning, Molecular; Culture Media, Conditioned; Eukaryotic Initiation Factors; G1 Phase; Gene Expression Regulation; Humans; Molecular Sequence Data; Neoplasm Proteins; Teratocarcinoma; Transforming Growth Factor beta; Tumor Cells, Cultured

Pluripotent human teratocarcinoma stem cells cultured in vitro provide a resource for the study of early embryonic development in man, as well as a means for discovery of novel factors controlling cell differentiation and commitment. We previously reported that the human teratocarcinoma stem cell line GCT 27X-1 could be induced to differentiate into an endodermal progenitor cell by treatment with high doses of retinoic acid. A search for polypeptide inducers of differentiation in this system has identified bone morphogenetic protein-2 (BMP-2) as a potent inducer of differentiation. In cell line GCT 27X-1, treatment with BMP-2 reduces proliferation, induces morphological changes similar to obtained following treatment with retinoic acid, and causes a decrease in the expression of transcripts for the stem cell markers CD30 and Oct-4. Preliminary immunochemical studies indicate that the differentiated cells produced by BMP-2 are endodermal precursors with a pattern of marker expression similar to that found in retinoic acid treated cells. Models of endoderm differentiation in humans will be useful for identifying the molecules which mediate cell interactions in development, and in achieving directed differentiation of cells for use in transplantation.

Topics: Animals; Antigens, Differentiation; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Embryonal Carcinoma Stem Cells; Embryonic and Fetal Development; Endoderm; Humans; Mice; Models, Biological; Neoplastic Stem Cells; Stem Cells; Teratocarcinoma; Transforming Growth Factor beta; Tumor Cells, Cultured

We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Laminin; Mice; Receptors, Retinoic Acid; Signal Transduction; Teratocarcinoma; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Repair of epithelial injury in the gastrointestinal tract is initially accomplished by migration of epithelial cells from the wound edge ("restitution"). To assess expression and function of the extracellular matrix (ECM) in the restitution phase after epithelial injury, in vitro studies using wounded monolayers or a rat intestinal epithelium-derived cell line (IEC-6) were undertaken. IEC-6 cells expressed fibronectin (FN) mRNA and protein in large amounts and lesser quantities of laminin-beta 1 (LN beta 1) and LN gamma 1. Collagen IV (Col IV) was weakly expressed, and LN alpha 1 was not detected. After wounding a significant decrease in FN, LN beta 1, LN gamma 1, and Col IV alpha 1 mRNA steady-state levels was observed; mean content 24 h after wounding was reduced by 75-90%. FN, LN, and Col IV proteins were also reduced. The downregulation of these ECM transcripts and proteins could be substantially prevented by transforming growth factor-beta 1, a restitution-promoting growth factor. In addition to changes of expression, the distribution of FN and LN was also altered in migrating cells after wounding, as assessed by immunofluorescence. Arg-Gly-Asp peptides that recognize the major cell attachment site on FN and antibodies recognizing the main noncollagenous domain of Col IV inhibited cell migration, but immunoneutralizing anti-LN antisera did not affect restitution. In conclusion, although paradoxically downregulated after wounding, ECM proteins, in particular FN and Col IV molecules, are able to enhance intestinal epithelial restitution.

Topics: Animals; Cell Adhesion; Cell Line; Cell Movement; Collagen; DNA Primers; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Fluorescent Antibody Technique, Indirect; Gene Expression; Intestinal Mucosa; Intestine, Small; Laminin; Mice; Polymerase Chain Reaction; Rats; RNA, Messenger; Teratocarcinoma; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Wound Healing

The balance between different cell populations in the developing organism is controlled by regulating the rates of multiplication, differentiation or death of its constituent cells. The human teratocarcinoma derived cell line Tera 2, which in several aspects mirrors early embryonic cells, can be induced to undergo programmed cell death (apoptosis) by depriving cell cultures of serum. This study demonstrates that this process can be reversed by replacing serum with physiological concentrations of insulin like growth factor I (IGF I). As a result, IGF I enhances the rate of Tera 2 cell proliferation in serum free medium. In contrast, Transforming Growth Factor beta1 did not exert any effect on growth or apoptosis in Tera 2 cells. The results indicate that one effect of growth factors on pluripotential cells is to regulate the balance between cell proliferation and cell death.

Topics: Apoptosis; Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; Culture Media, Serum-Free; DNA, Neoplasm; Humans; Insulin-Like Growth Factor I; Mitotic Index; Teratocarcinoma; Transforming Growth Factor beta; Tumor Cells, Cultured