tretinoin and Neoplasms--Germ-Cell-and-Embryonal

Topics: Carrier Proteins; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 12; Cisplatin; Combined Modality Therapy; Drug Resistance; Gene Expression Regulation, Neoplastic; Genes, ras; Genetic Markers; Humans; Male; Neoplasms, Germ Cell and Embryonal; Receptors, Retinoic Acid; Testicular Neoplasms; Tretinoin

Topics: Acetamides; Cell Differentiation; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Growth Substances; Humans; Neoplasms, Germ Cell and Embryonal; Oncogene Proteins; Proto-Oncogene Proteins; Transforming Growth Factor alpha; Tretinoin

Topics: Anthraquinones; Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Breast Neoplasms; Cisplatin; Dose-Response Relationship, Drug; Female; Genetic Techniques; Hodgkin Disease; Hormones; Humans; Immunotherapy; Interferons; Male; Mechlorethamine; Methotrexate; Mitoxantrone; Neoplasm Metastasis; Neoplasms; Neoplasms, Germ Cell and Embryonal; Nitrosourea Compounds; Osteosarcoma; Prednisone; Procarbazine; Prognosis; Receptors, Cell Surface; Tretinoin; Vincristine

Germ cell tumors (GCTs) are characterized by a capacity to differentiate in vivo and in vitro. The authors' previous work highlighted the finding that retinoid-mediated GCT differentiation in vitro correlates with expression of the retinoic acid receptor-gamma.. Six human GCT cell lines were studied to determine whether their growth and differentiation were linked to the retinoic acid response pathway. The maturation and growth inhibitory responses to all-trans retinoic acid (RA) were studied as well as the expression of retinoic acid receptor-gamma (RAR-gamma). The clinical antitumor activity of RA was studied in a Phase II trial of RA in patients with chemotherapy-refractory GCTs.. The RA response pathway was correlated with the control of growth and maturation in three of the six GCT cell lines studied. In one GCT cell line, RA induced RAR-gamma expression and terminal differentiation. In a second, there was high basal expression of RAR-gamma and poor basal proliferative potential. A third cell line showed a more mature basal phenotype than other cell lines studied and marked growth inhibition when treated with RA. Sixteen patients were treated with RA for an average of 7 weeks in the clinical trial. No complete or partial responses were noted.. A Phase II clinical trial of RA failed to show significant clinical antitumor activity in patients with chemotherapy-refractory GCTs. However in vitro data suggest that the control of growth and maturation in some GCT cell lines involve the RA signaling pathway. Further studies are warranted to define the role that other retinoids with receptor selectivity or more favorable pharmacologic properties may play in the maturation or antitumor responses of GCTs.

Topics: Adolescent; Adult; Cell Differentiation; Cell Division; Female; Gene Expression; Growth Inhibitors; Humans; Male; Middle Aged; Neoplasms, Germ Cell and Embryonal; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4) and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Fibronectins; Homeodomain Proteins; Humans; Male; Membrane Proteins; Nanog Homeobox Protein; Neoplasms, Germ Cell and Embryonal; Nestin; Octamer Transcription Factor-3; Real-Time Polymerase Chain Reaction; Stathmin; Testicular Neoplasms; Tretinoin

PIWI family proteins have recently emerged as essential contributors in numerous biological processes including germ cell development, stem cell maintenance and epigenetic reprogramming. Expression of some of the family members has been shown to be elevated in tumors. In particular, PIWIL2 has been probed as a potential neoplasia biomarker in many cancers in humans. Previously, PIWIL2 was shown to be expressed in most tumours as a set of its shorter isoforms. In this work, we demonstrated the presence of its 60 kDa (PL2L60A) and 80 kDa (PL2L80A) isoforms in testicular cancer cell lines. We also ascertained the transcriptional boundaries of mRNAs and alternative promoter regions for these PIWIL2 isoforms. Further, we probed a range of testicular germ cell tumor (TGCT) samples and found PIWIL2 to be predominantly expressed as PL2L60A in most of them. Importantly, the levels of both PL2L60A mRNA and protein products were found to vary depending on the differentiation subtype of TGCTs, i.e., PL2L60A expression is significantly higher in undifferentiated seminomas and appears to be substantially decreased in mixed and nonseminomatous TGCTs. The higher level of PL2L60A expression in undifferentiated TGCTs was further validated in the model system of retinoic acid induced differentiation in NT2/D1 cell line. Therefore, both PL2L60A mRNA and protein abundance could serve as an additional marker distinguishing between seminomas and nonseminomatous tumors with different prognosis and therapy approaches.

Topics: Argonaute Proteins; Cell Differentiation; Cell Line, Tumor; Humans; Male; Neoplasms, Germ Cell and Embryonal; Polyadenylation; Protein Isoforms; RNA Splice Sites; RNA, Messenger; Spermatozoa; Testicular Neoplasms; Transcriptome; Tretinoin

Germ cell testicular cancer is understood to arise during embryogenesis, based on the persistence of embryonic germ cell markers in carcinoma in situ and seminoma. In this study, we examine the potential of the seminoma-derived TCam-2 cell line to be used as representative in functional analyses of seminoma. We demonstrate expression of several early germ cell markers, including BLIMP1, OCT3/4, AP2γ, NANOG and KIT. Many TGF-beta superfamily receptors and downstream transcription factors are also present in these cells including the normally foetal ACTRIIA receptor, indicating potential responsiveness to TGF-beta superfamily ligands. Treatment with BMP4 or RA induces a significant increase in ACTRIA, ACTRIIA and ACTRIIB transcripts, whereas activin A decreases ACTRIB. BMP4 and RA each support TCam-2 survival and/or proliferation. In addition, despite increased KIT mRNA levels induced by BMP4, RA and activin A, activin A does not improve survival or proliferation. The capacity for BMP4 and retinoic acid to enhance foetal germ cell survival and proliferation/self-renewal has been demonstrated in mice, but not previously tested in humans. This study is the first to demonstrate a functional response in seminoma cells, using a well-characterized cell line, consistent with their foetal germ cell-like identity.

Topics: Activin Receptors, Type II; Activins; Adaptor Protein Complex 2; Biomarkers; Bone Morphogenetic Protein 4; Cell Line, Tumor; Cell Proliferation; Cell Survival; Germ Cells; Homeodomain Proteins; Humans; Ligands; Male; Nanog Homeobox Protein; Neoplasms, Germ Cell and Embryonal; Octamer Transcription Factor-3; Positive Regulatory Domain I-Binding Factor 1; Proto-Oncogene Proteins c-kit; Repressor Proteins; Seminoma; Signal Transduction; Testicular Neoplasms; Transforming Growth Factor beta; Tretinoin

FERM domain containing protein 7 (FRMD7) mutations are associated with X-linked idiopathic congenital nystagmus (ICN). The purpose of this study is to identify a novel splice variant of FRMD7 (FRMD7-S) in both humans and mice with a shortened exon 4 relative to the original form of FRMD7 (FRMD7-FL),and to detect the role of FRMD7-FL and FRMD7-S in the process of neuronal development.. The splice variant of FRMD7 was identified by PCR. Expression levels of hFRMD7-FL and hFRMD7-S transcripts in developing human fetal brain were tested by RT-PCR, and expression levels in the human pluripotent embryonic carcinoma NTera 2/cl.D1 (NTERA-2; NT2) cell line with all-trans retinoic acid (ATRA) or bone morphogenetic protein-2 (BMP-2) treatment were tested by real-time qPCR. hemaglutinin (HA)-tagged recombinant plasmids DNA encoding hFRMD7-FL and Myc-tagged recombinant plasmids DNA encoding hFRMD7-S were used to transiently transfect the human NT2 cells. Further, immunofluorescence experiments were performed to determine the co-localization of the two fusion proteins. Finally, using co-immunoprecipitation analyses, we demonstrated that FRMD7-FL and FRMD7-S interacted with each other.. A novel splice variant of FRMD7 (FRMD7-S) with a shortened exon 4 relative to the original form of FRMD7 (FRMD7-FL) was identified from the cDNA of the human NT2 cell line and mouse fetal brain. The FRMD7 transcripts showed similar tissue distributions and were upregulated following all trans retinoic acid (ATRA)-induced differentiation of NT2 cells. FRMD7-FL and FRMD7-S co-localized and co-immunoprecipitated with each other. Further, overexpression of FRMD7-FL in NT2 cells resulted in altered neurite development and upregulation of FRMD7-S.. Although the significance of the 45 bp deletion remains unknown, our observations suggest that the FRMD7 isoforms may play a significant role during neuronal differentiation and development.

Topics: Alternative Splicing; Animals; Bone Morphogenetic Protein 2; Brain; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; Exons; Fetus; Gene Expression Regulation, Developmental; Humans; Membrane Proteins; Mice; Neoplasms, Germ Cell and Embryonal; Neurons; Nystagmus, Congenital; Plasmids; Protein Isoforms; Real-Time Polymerase Chain Reaction; Recombinant Fusion Proteins; Transfection; Tretinoin

Metastatic germ cell tumors (GCT) are curable, however GCTs refractory to cisplatin-based chemotherapy have a poor prognosis. This study explores D-type cyclins as molecular targets in GCTs because all-trans-retinoic acid (RA)-mediated differentiation of the human embryonal carcinoma (EC) cell line NT2/D1 is associated with G1 cell cycle arrest and proteasomal degradation of cyclin D1. RA effects on D-type cyclins are compared in human EC cells that are RA sensitive or dually RA and cisplatin resistant (NT2/D1-R1) and in clinical GCTs that have both EC and mature teratoma components. Notably, GCT differentiation was associated with reduced cyclin D1 but increased cyclin D3 expression. RA was shown here to repress cyclin D1 through a transcriptional mechanism in addition to causing its degradation. The siRNA-mediated repression of individual cyclin D species resulted in growth inhibition in both RA sensitive and resistant EC cells. Only repression of cyclin D1 occurred in vitro and when clinical GCTs mature, implicating cyclin D1 as a molecular therapeutic target. To confirm this, the EGFR-tyrosine kinase inhibitor, Erlotinib, was used to repress cyclin D1. This inhibited proliferation in RA and cisplatin sensitive and resistant EC cells. Taken together, these findings implicate cyclin D1 targeting agents for the treatment of GCTs.

Topics: Antineoplastic Agents; Cell Differentiation; Cyclin D1; DNA Fragmentation; Enzyme Inhibitors; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; Humans; Neoplasms, Germ Cell and Embryonal; Quinazolines; Receptors, Retinoic Acid; RNA, Heterogeneous Nuclear; RNA, Small Interfering; Time Factors; Tretinoin

WNT signaling molecules, playing key roles in embryogenesis and carcinogenesis, are potent targets for regenerative medicine and clinical oncology. We have previously cloned and characterized the human orthologue of mouse proto-oncogene Wnt-10b using bioinformatics and cDNA-PCR. Human WNT10B is moderately expressed in MKN45 and MKN74 cells derived from human gastric cancer, and is up-regulated by tumor necrosis factor alpha (TNFalpha) in MKN45 cells. Here, expression and regulation of WNT10B in human cancer other than gastric cancer were investigated using cDNA-PCR. WNT10B mRNA was expressed in the majority of squamous cell carcinoma cell lines derived from esophageal cancer and cervical cancer. WNT10B mRNA was relatively highly expressed in TE3, TE6, TE10, TE11 (esophageal cancer), Hs700T (pancreatic cancer), SKG-IIIa, HeLa S3 (cervical cancer), and T-47D (breast cancer). Expression of WNT10B mRNA was up-regulated by beta-estradiol in MCF-7 cells expressing estrogen receptors. Expression of WNT10B mRNA was down-regulated by all-trans retinoic acid in NT2 cells with the potential of self renewal and neuronal differentiation. WNT10B might be implicated in self renewal of stem cells as well as in carcinogenesis through activation of the WNT - beta-catenin pathway.

Topics: Breast Neoplasms; Cell Line; Down-Regulation; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Neoplasms, Germ Cell and Embryonal; Proto-Oncogene Mas; Proto-Oncogene Proteins; Tretinoin; Up-Regulation; Wnt Proteins

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

Topics: Antineoplastic Agents; Apoptosis; Cell Survival; DNA Nucleotidylexotransferase; Dopamine; Humans; Neoplasms, Germ Cell and Embryonal; Neurons; Stem Cells; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Exposure of aggregated murine P19 embryonal carcinoma cells to dimethylsulfoxide (DMSO) induces mesoderm and both embryonic cardiac and skeletal muscle differentiation, while retinoic acid (RA) is an inducer of neuroectodermal differentiation. P19 cells constitutively express the retinoic acid receptor alpha (RAR alpha) and RAR gamma mRNAs while RAR beta expression is induced by RA through a consensus RA-response element in the RAR beta promoter. In the present study we show that the RAR beta transcript is strongly expressed in both P19 cells and in a RA-nonresponsive derivative of P19 cells, called RAC65, during DMSO-induced mesoderm and muscle differentiation. Reverse transcriptase-polymerase chain reaction analysis indicated that RAR beta 2 is the predominant isoform expressed in DMSO-differentiated cells, providing the first evidence for RA-independent regulation of RAR beta 2 transcript levels. Immunoblot analysis showed a 3-fold increase in the RAR beta protein expression over basal levels in differentiated cells, and immunohistochemistry indicated that all cells in the culture including muscle reacted positively for the RAR beta protein. RAR beta 2 transcript expression was differentiation-dependent and occurred without transactivation of a transfected RARE beta 2 reporter gene. Little transcription of the RAR beta gene was detected in nuclear run-off assays of undifferentiated P19 cells and only a small increase in transcription was observed in nuclei from DMSO-treated cells. RA treatment of P19 cells stably transfected with the RA-responsive element from the RAR beta gene showed that RAR beta 2 mRNA expression during DMSO differentiation was associated with increased sensitivity to RA. Together these data show that RAR beta 2 is expressed spontaneously in an apparently RA-independent manner in differentiating mesoderm and mesoderm derivatives, resulting in increased sensitivity to RA in these cells.

Topics: Animals; Blotting, Northern; Blotting, Southern; Cell Differentiation; Densitometry; Dimethyl Sulfoxide; DNA Primers; Gene Expression; Immunoblotting; Immunohistochemistry; Mesoderm; Mice; Neoplasms, Germ Cell and Embryonal; Plasmids; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT-PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4-14 days after induction with retinoic acid. The RT-PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the pred

Topics: Acetamides; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Drosophila Proteins; Frizzled Receptors; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neurons; Receptors, G-Protein-Coupled; Sequence Homology, Nucleic Acid; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Wnt Proteins

The expression patterns of the class I homeogenes HOXB and HOXC clusters in the presence of retinoic acid (RA) were studied in two human small-cell lung cancer (SCLC) cell lines and compared to that of NT2/D1 embryonal carcinoma cells. Contrasting with the sequential 3'-5' induction of the HOX genes observed after RA treatment of embryonic NT2/D1 cells, in the SCLC cells the responding genes (induced or down-regulated) were interspersed with insensitive genes (expressed or unexpressed), while no genomic alteration affected the corresponding clusters. These findings imply that HOX gene regulatory mechanisms are altered in non-embryonic SCLC cells, perhaps reflecting their ability to respond to more diversified stimuli, in relation with their origin from adult tissues.

Topics: Carcinoma, Small Cell; DNA Primers; Electrophoresis, Agar Gel; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Homeodomain Proteins; Humans; Neoplasms, Germ Cell and Embryonal; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Germ cell tumors are empirically divided into seminomas and nonseminomatous germ cell tumors (NSGCT). Some authorities consider seminomas to be the precursors of NSGCT, whereas others consider them as distinct and unrelated neoplasms. Here, we report that the human NSGCT-derived stem cell line, NCCIT has hybrid features of seminoma and embryonal carcinoma, and suggest that this cell line could be useful for studying the relationship of seminoma to NSGCT.. NCCIT, a developmentally pluripotent permanent cell line derived from a mediastinal NSGCT was karyotyped and characterized morphologically, immunochemically, and biochemically. The cells were grown under standard tissue culture conditions and were also exposed to retinoic acid to induce differentiation.. The dividing NCCIT stem cell populations consist of vimentin-positive, keratin-negative cells that do not express desmoplakin or cadherin E (uvomorulin) and are not interconnected with one another. These cells have a high nucleocytoplasmic ratio and contain few cytoplasmic organelles, except for free ribosomes and a small number of mitochondria. Lacto- and globoseries oligosaccharide antigens recognized with antibodies to murine stage specific antigens 1, 3 and 4 (SSEA-1, SSEA-3 and SSEA-4), and human teratocarcinoma mucin-like antigen TRA-1-60 and TRA-1-81 are coexpressed on the cell membranes of a considerable number of stem cells. On most cells alkaline phosphatase can be detected by enzyme histochemistry. The placental isoenzyme of alkaline phosphatase was demonstrated by Western blotting in cell extracts. The liver/bone/kidney isoenzyme of alkaline phosphatase is immunochemically detected on 40% of cells. The culture supernatants also contain chorionic gonadotropin and alpha-fetoprotein, presumably derived from trophoblastic and yolk sac-like cells. The cells are hyperdiploid (chromosome range from 54 to 64) and show prominent structural chromosomal aberrations, mostly deletions and isochromosomes. Retinoic acid treatment inhibited the growth of NCCIT cells and induced stem cell differentiation into keratin, glial fibrillary acid protein, and neurofilament-positive somatic cells. The differentiation was associated with the disappearance of oligosaccharide surface antigens typical of the undifferentiated stem cells; a loss of proteins typical of undifferentiated cells and the appearance of new proteins; and the deposition of extracellular matrix.. NCCIT is a developmentally pluripotent cell line that can differentiate into derivatives of all three embryonic germ layers (i.e., ectoderm, mesoderm, and endoderm) and extraembryonic cell lineages. We suggest that this cell line could be a malignant replica of human cleavage stage embryonic cells with features intermediate between seminoma and embryonal carcinoma.

Topics: Adult; Antigens, Surface; Antigens, Tumor-Associated, Carbohydrate; Cell Differentiation; Cell Division; Chromosome Aberrations; Embryonal Carcinoma Stem Cells; Humans; Immunohistochemistry; Keratins; Male; Neoplasm Transplantation; Neoplasms, Germ Cell and Embryonal; Neoplastic Stem Cells; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

Neuroectodermal tumours express hormones which are post-translationally processed and inactivated by the action of specific proteases and peptidases. The data reported here show the presence of a novel thermolysin-like metallo-endopeptidase activity in several human cell lines. The soluble fractions of neuroblastoma, melanoma and a glioblastoma tumour cell lines are able, with different degrees, to cleave the Ser12-Phe13 bond of a DVDERDVRGFAS decreases FLNH2 substrate. The inhibition pattern suggests a metallo-endopeptidase thermolysin-like character, with the involvement of thiol group(s), clearly distinct from neutral endopeptidase (NEP; EC 3.4.24.11). This metallo-endopeptidase activity is down regulated during retinoic acid(RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells but not in the RA-resistant BE(2)-M17 cells, suggesting that the down regulation is related to neuronal differentiation and not a direct effect of RA on the enzymatic activity.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Glioma; Humans; Kinetics; Melanoma; Metalloendopeptidases; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neprilysin; Neuroblastoma; Oligopeptides; Recombinant Proteins; Skin; Substrate Specificity; Thermolysin; Tretinoin; Tumor Cells, Cultured; Xenopus laevis

The potential of a combination of differentiation induction and chemotherapy was analyzed. Treatment of the murine embryonal carcinoma (EC) cell line PCC4 in vitro with all-transretinoic acid (RA) was followed by exposure to cisplatin (CDDP) or etoposide (VP-16). The expression of EC-cell-specific markers decreased upon 96 hr exposure to 10(-9), 10(-8), 10(-7) and 10(-6) M RA, indicating a loss of embryonal phenotype of the cells, whereas expression of markers specific for cytokeratins and neurofilaments was increased after this treatment. These data suggest early somatic differentiation of PCC4 cells upon treatment with RA. Cellular growth rate of PCC4 cells was not affected by preincubation with RA at 10(-9) M for 96 hr, but was reduced at 10(-8) and 10(-7) M RA and inhibited at 10(-6) M RA. Culture of PCC4 cells in the presence of 10(-7) M RA for 96 hr led to accumulation in G1 of the cell cycle, whereas at 10(-8) M RA cell-cycle distribution was not affected. RA-treated and -untreated PCC4 cells were compared for CDDP and VP-16 sensitivity. Pre-treatment with 10(-9), 10(-8) and 10(-7) M RA increased CDDP sensitivity, resulting in a 1.9-, 2.7- and 2.6-fold decrease in the concentrations inhibiting survival by 50% (IC50s) respectively. Pre-treatment with 10(-8) and 10(-7) M RA increased VP-16 sensitivity 2.5- and 3.0-fold, respectively. Enhanced CDDP sensitivity at RA concentrations not affecting cell-cycle distribution was not attributable to changes in cellular platinum (Pt) accumulation, or to changes of Pt-DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antigens, Differentiation; Cell Cycle; Cell Differentiation; Cell Survival; Cisplatin; DNA, Neoplasm; Drug Synergism; Etoposide; Mice; Neoplasms, Germ Cell and Embryonal; Tretinoin; Tumor Cells, Cultured

A new clone of the mouse embryonal carcinoma cell line 1003 (EC 1003.16) can be maintained in an undifferentiated state in serum-containing medium. Shifting these cells to serum-free, hormonally defined medium causes them to differentiate morphologically and acquire a number of molecular properties characteristic of neurons. Whereas undifferentiated cells lack the NILE/L1 glycoprotein, expression of this neuronal cell adhesion molecule is induced in the differentiating cells. Message for NILE/L1 becomes detectable after 5 days in serum-free medium, and cell-surface NILE/L1 can first be seen at this same time. Changes in two other cell adhesion molecules occur in parallel with the induction of NILE/L1. Fibronectin receptor is present on undifferentiated cells, but is down-regulated by the differentiating neurons. The neural cell adhesion molecule (N-CAM) undergoes a shift from the very adhesive adult form to the less adhesive, highly sialylated embryonic form. These changes would appear to emphasize the role of NILE/L1 in adhesive interactions involving differentiating neurons. Some changes in ganglioside expression also occur during EC 1003.16 differentiation. Undifferentiated cells express the D 1.1 ganglioside but lack gangliosides that are reactive with the monoclonal antibody A2B5. Differentiating cells lose D 1.1 and become A2B5-positive. Since D 1.1 is characteristic of undifferentiated neuroepithelial cells and A2B5 reactivity is a marker for several types of differentiated neurons, these changes in vitro appear to mimic events that occur in vivo.

Topics: Adrenal Gland Neoplasms; Animals; Biomarkers; Blotting, Northern; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Down-Regulation; Fluorescent Antibody Technique; Gene Expression; In Vitro Techniques; Lewis X Antigen; Membrane Glycoproteins; Mice; Neoplasms, Germ Cell and Embryonal; Neural Cell Adhesion Molecule L1; Neurons; Pheochromocytoma; Rats; Receptors, Fibronectin; Receptors, Immunologic; RNA; Tretinoin; Tumor Cells, Cultured; Vimentin

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.

Topics: Acetamides; Adenovirus Early Proteins; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; DNA, Neoplasm; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasms, Germ Cell and Embryonal; Oncogene Proteins, Viral; Testicular Neoplasms; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) treatment of F-9 embryonal carcinoma cells resulted in cell flattening and increased production of laminin B1 chain, both indicating differentiation to endoderm-like cells. In addition, RA caused a time- and dose-dependent decrease in growth rate in monolayer culture and a dose-dependent decrease in the ability of the cells to form colonies in soft agarose. Differentiation was accompanied by an increase in the fucosylation of specific high-molecular-weight cellular and cell-surface glycoproteins. The fucosylation of glycoproteins of Mr 175,000 (gp175), 250,000 (gp250), and 400,000 (gp400) increased as early as 24 hr after the addition of 5 x 10(-6) M RA to the culture medium. These changes preceded both growth inhibition and the induction of laminin B1 expression, which were detected 48 to 72 hr after addition of RA. The increased fucosylation of these glycoproteins showed a distinct dose-response relationship. Both gp175 and gp250 showed the greatest increase in fucosylation at 10(-5) M, which was also the dose at which RA induced laminin maximally, while the fucosylation of gp400 was greatest at 10(-8) M RA and declined at higher concentrations. The overall synthesis of large fucosylated glycopeptides decreased in RA-treated cells, in spite of the increases in the fucosylation of specific cellular glycoproteins. RA-induced differentiation of F-9 cells was also accompanied by a time- and dose-dependent increase in fucosyltransferase activity. Although the functions of these glycoproteins are not currently known, the early increase in their fucosylation can be considered as a marker of differentiation in this system.

Topics: Animals; Carcinoma; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fucose; Fucosyltransferases; Glycopeptides; Glycoproteins; Glycosylation; Immunoblotting; Mice; Molecular Weight; Neoplasm Proteins; Neoplasms, Germ Cell and Embryonal; Precipitin Tests; Time Factors; Tretinoin; Tumor Cells, Cultured

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.

Topics: Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Gene Expression; Humans; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasms, Germ Cell and Embryonal; Phosphotransferases; Receptors, Cell Surface; Receptors, Somatomedin; Teratoma; Tretinoin; Tumor Cells, Cultured

The murine embryonal carcinoma cell line P19S18O1A1 develops into neuronlike cells after treatment with retinoic acid (Edwards and McBurney, 1983). We have analyzed the expression of cell surface carbohydrate antigens and intracellular cytoskeletal antigens in differentiating O1A1 cells in order to identify the cell types present in the cultures and to characterize the differentiation process. Undifferentiated O1A1 cells express the SSEA-1 antigen, GD3 ganglioside, and the D1.1 ganglioside antigen, carbohydrate markers that are found on early embryonic cells and neuroepithelial germinal cells in vivo. The cells also bind tetanus toxin, cholera toxin, and monoclonal antibody A2B5, probes that bind to gangliosides found on the surfaces of neurons and immature astrocytes in vivo and in vitro. They contain vimentin-type intermediate filament antigens but have no detectable neurofilament or glial filament protein antigens. After aggregation of the cells in medium containing retinoic acid followed by growth in a serum-free chemically defined medium, over 80% of the cells differentiate into neurons as determined by immunofluorescent labeling with antibodies against neurofilament protein antigens. The differentiated cells no longer express either the embryonic or neuroepithelial carbohydrate antigens, but they continue to express the cell surface markers characteristic of neurons. These changes in the expression of cell surface antigens are accompanied by changes in ganglioside metabolism, including a shift towards the synthesis of more complex gangliosides. Thus, the retinoic acid-induced changes in O1A1 cells in vitro resemble the in vivo development of neurons. This establishes the O1A1 cell line as a relevant model system for studies of the molecular basis of neuronal differentiation and development.

Topics: Antibodies, Monoclonal; Antigens, Surface; Cell Differentiation; Cell Line; Epithelium; Fluorescent Antibody Technique; Gangliosides; Models, Biological; Neoplasms, Germ Cell and Embryonal; Neurons; Tretinoin

To a limited extent, we corroborated a previous report of human neuroblastoma sensitivity to 13-cis-retinoic acid. Seven cultured human neuroblastoma, two primitive neuroectodermal tumor, and one melanoma cell line were exposed to 0.001 to 10.0 microM 13-cis-retinoic acid for six to fourteen days. The neuroblastoma cell line, SK-N-DZ, was the only cell line lysed by all concentrations of 13-cis-retinoic acid. The other cell lines were refractory to concentrations as high as 10 microM. Increased cell process formation was observed in three neuroblastoma, SK-N-SH, SK-N-BE, SK-N-LE, and one melanoma cell line. We conclude that sensitivity to 13-cis-retinoic acid is unevenly distributed among histogenetically similar tumors from different patients.

Topics: Cell Line; Cell Survival; Humans; Melanoma; Neoplasms, Germ Cell and Embryonal; Neuroblastoma; Tretinoin; Tumor Stem Cell Assay

Topics: Adolescent; Adult; Drug Evaluation; Humans; Isotretinoin; Male; Neoplasms, Germ Cell and Embryonal; Testicular Neoplasms; Tretinoin

Antibodies reactive with a murine teratocarcinoma cell line (F9) were detected by immunofluorescence staining in sera from patients bearing ovarian germ cell tumour. Immunochemical studies revealed that antibodies binding to the cell surface of F9 cells react with large glycopeptides which are known to be components of F9 antigens defined by murine anti-F9 antibodies. In addition, treatment of F9 cells with retinoic acid, which induces differentiation of embryonal carcinoma cells, distinctly reduced both the ability of these antibodies to stain F9 cells and the biosynthesis of large glycopeptides by the cells. These findings indicate that the large glycopeptides precipitable by the patients' antibodies are differentiation associated antigens on characteristic embryonic cells.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Cell Differentiation; Cell Line; Female; Fluorescent Antibody Technique; Glycopeptides; Humans; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Teratoma; Tretinoin

PCC4azal embryonal carcinoma tumors were grown in strain 129 mice by s.c. transplantation. When palpable, the tumors were treated with a combination of retinoic acid and dimethylacetamide. In vitro, this embryonal carcinoma cell line shows minimal spontaneous differentiation and is exquisitely sensitive to retinoic acid and/or dimethylacetamide induction of differentiation. Ten daily 20-microliter intratumor injections of a solution of 10 mg retinoic acid per ml of dimethylacetamide resulted in nearly complete induction of morphological differentiation mainly into neuroepithelial and glandular derivatives. Control tumors showed minor spontaneous differentiation. Differentiation was associated with decreased tumor growth rate, decreased mitotic index, decreased extent of necrosis, and increased survival time of the hosts. In 4 of 18 cases, long-term survival of the hosts was effected by a complete differentiation of the malignant embryonal carcinoma tumors into benign teratomas. Retinoic acid:dimethylacetamide was also effective in inducing differentiation with the same dosage and schedule when administered systemically, i.e., i.p. or s.c.

Topics: Acetamides; Animals; Cell Differentiation; Drug Evaluation, Preclinical; Mice; Mitotic Index; Neoplasm Transplantation; Neoplasms, Experimental; Neoplasms, Germ Cell and Embryonal; Teratoma; Tretinoin

On the basis of our studies, we conclude that RA is a potent promoter of differentiation of EC cells. We have demonstrated that the tumorigenicity of PCC4-azalR EC cultures can be effectively reduced following exposure of the cells to RA. This is presumably a result of differentiation of the EC cells to nontumorigenic derivatives. However, even after several weeks of exposure to RA, there remains in the culture a subpopulation of unresponsive EC cells. The reason why these cells do not differentiate in the presence of RA is currently under investigation. We have also derived by mutagen treatment and clonal selection EC cells that fail to respond to RA. Preliminary indications are that these cells have lost the capacity to differentiate when subjected to other manipulations which stimulate differentiation of the parental EC cells. This is an important observation since the mutants were selected only by their lack of response to RA. Unlike the parental cells, which have relatively large amounts of RABP, dif- cells appear either to lack RABP or to possess an altered binding protein which has a greatly reduced affinity for RA. These observations are consistent with the view that some function of the RA-RABP complex is critical in the sequence of events leading to differentiation of EC cells. However, further studies are required before we can establish the generality of this proposal. We are presently investigating whether the level and/or function of RA-RABP complexes in cells from the EC lines listed in TABLE 1 can explain their varying tendencies to differentiate.

Topics: Animals; Cell Differentiation; Cell Line; Mice; Neoplasms, Germ Cell and Embryonal; Tretinoin

Topics: Animals; Cell Membrane; Cells, Cultured; Fibroblasts; Membrane Fluidity; Mice; Neoplasms, Experimental; Neoplasms, Germ Cell and Embryonal; Tretinoin; Viscosity