tretinoin and Astrocytoma

In this article we report the results of a study of the relationship between response and progression in 375 patients with recurrent glioma enrolled in phase II chemotherapy trials. We reviewed the records of patients from 8 consecutive phase II trials, including 225 patients with recurrent glioblastoma multiforme and 150 with recurrent anaplastic astrocytoma. Median age was 45 years (range, 15-82) and median Karnofsky performance score was 80 (range, 60-100). Forty-one patients (11%) had more than two prior resections and/or more than two prior chemotherapy regimens. Best response was complete (n = 1) or partial (n = 33) in 34 patients (9%). Median time to response was 14 weeks, and median response duration was 44 weeks. Simon-Makuch estimates for 52-week progression-free survival for patients progression-free at 13 weeks were 48% for response and 28% for nonresponse. When response was treated as a time-dependent covariate in a Cox proportional hazards regression analysis, response was associated with significantly lower failure rates (hazard ratio 0.5; 95% confidence interval 0.3-0.8; P = 0.0016). This study showed that response in recurrent glioma is associated with a significant reduction in progression rates.

Topics: Actuarial Analysis; Adolescent; Adult; Aged; Aged, 80 and over; Alitretinoin; Antineoplastic Combined Chemotherapy Protocols; Astrocytoma; Brain Neoplasms; Carboplatin; Combined Modality Therapy; Disease Progression; Disease-Free Survival; Eflornithine; Female; Fluorouracil; Glioblastoma; Glioma; Humans; Interferon-beta; Male; Menogaril; Middle Aged; Neoplasm Recurrence, Local; Procarbazine; Prognosis; Proportional Hazards Models; Texas; Treatment Outcome; Tretinoin

To evaluate the therapeutic effect of all-trans retinoic acid (ATRA) with and without cytosine arabinoside in relapsing malignant gliomas.. 9 patients (8 male, 1 female, age 53.9 +/- 11.2) with relapsing malignant gliomas (grade IV:6; grade III:3) were treated by ATRA 1 to 21 months after the end of their initial treatment. ATRA was given unceasingly during 2 to 17 months at 90 mg/d. In 6 patients it was associated to cytosine arabinoside (4 g/course, 1 to 9 courses every 4 weeks).. 4 non-responder patients died 2.5 to 4 months after starting therapy. One patient who had been reoperated before receiving ATRA and cytosine arabinoside (5 course) had no sign of tumor recurrence after 17 months of treatment. In 4 responder patients (2 glioblastoma and 2 anaplastic astrocytoma) a clinical and radiological stabilization (time to progression) during 9 +/- 2.5 months was observed. This stabilization was associated in 3 of them with the appearance of intra tumoral calcifications visualized on repeated CT scans and confirmed in one patient by post-mortem examination. All of them had received cytosine arabinoside (1 to 9 courses) with ATRA; however small calcifications were also observed in one non-responder patient who did not receive aracytine.. These results suggest: a) a therapeutic effect of ATRA in combination with cytosine arabinoside in patients with relapsing malignant gliomas b) that intratumoral calcifications are related to the effects of ATRA on differentiation and/or on endothelial t-PA production and that these effects explain the tumor progression arrest in responder patients. The transient efficiency is probably related to the pharmacokinetics of ATRA or to changes of cellular mechanisms that modulate the cell response to the drug and is a critical issue for this therapy.

Topics: Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Astrocytoma; Brain Neoplasms; Calcinosis; Cytarabine; Disease Progression; Female; Glioblastoma; Glioma; Humans; Magnetic Resonance Imaging; Male; Middle Aged; Neoplasm Recurrence, Local; Radiography; Time Factors; Tretinoin

The Radiation Therapy Oncology Group enrolled 30 patients with recurrent malignant astrocytomas onto a phase II study (RTOG 91-13). Patients were treated with all-trans-retinoic acid at a starting dose of 120 mg/m2 per day orally continuously until disease progression. Fourteen patients had glioblastoma, 14 had anaplastic astrocytoma, and 2 had other histologies; 53% were under 50 years of age. All patients had failed radiation therapy and/or at least one chemotherapy regimen. All patients had a Karnofsky performance status score of at least 70, but only 37% had a KPS of 90-100. Forty percent had a neurologic function status of grade 1 (able to work). A minimum of 4 weeks of all-trans-retinoic acid defined adequate treatment. Twenty-five patients received adequate therapy. Most common toxicities were dry skin, cheilitis, anemia, and headache; 3 patients had grade 3 headache requiring suspension of all-trans-retinoic acid. No grade 3 hematologic toxicity was observed. Of 25 adequately treated patients, 3 showed objective regression of tumor on magnetic resonance imaging and computed tomography scans, 3 patients remained stable, and 19 patients had disease progression. The median time to tumor progression was 3.8 months and the median survival time was 5.7 months. This study suggests that this dose of single agent all-trans-retinoic acid has modest clinical activity against recurrent malignant gliomas with tolerable side effects. A response rate of 12% and a stabilization rate of 12% are lower than expected. Future studies with higher dosage or in combination with biological response modifiers or chemotherapy may be warranted.

Topics: Antineoplastic Agents; Astrocytoma; Brain Neoplasms; Disease Progression; Female; Glioblastoma; Humans; Magnetic Resonance Imaging; Male; Middle Aged; Neoplasm Recurrence, Local; Neurologic Examination; Survival Rate; Time Factors; Treatment Failure; Tretinoin

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Topics: Animals; Astrocytoma; Behavior, Animal; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Dopamine; Dopaminergic Neurons; Gene Transfer Techniques; Genetic Complementation Test; Glial Cell Line-Derived Neurotrophic Factor; HEK293 Cells; Homeodomain Proteins; Humans; Oxidopamine; Parkinson Disease; Phenotype; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Transcription Factors; Tretinoin

Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Astrocytoma; Brain Neoplasms; Cell Differentiation; Cell Separation; Cytochrome P-450 Enzyme System; Fatty Acid-Binding Proteins; Flow Cytometry; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Prognosis; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tretinoin

Retinoic acid (Ra) is crucial for the patterning and neuronal differentiation in the central nervous system (CNS). Ra deficiency in animals disrupts the motor activities and memory abilities. The molecular mechanisms underlying these behavior abnormalities remain largely unknown. In the current study, we treated the astrocytoma cells with citral, an inhibitor of Ra synthesis. We analyzed the differences in the protein concentrations between the treated and untreated astrocytoma cells by two-dimensional gel electrophoresis (2-DE), Imagemaster software, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In total, 39 of 46 altered protein spots with significant mascot scores were identified representing 36 proteins, that were involved in significantly altered glutamate metabolism, lipid metabolism, mitochondrial function, and oxidative stress response by Ingenuity Pathway Analysis (IPA). Altered 3-phosphoglycerate dehydrogenase (PHGDH) was also observed in western blot. These data provide some clues for explaining the behavioral changes caused by Ra deficiency, and support the hypothesis that Ra signaling is associated with some symptoms of neurodegenerative disorders and schizophrenia.

Topics: Acyclic Monoterpenes; Animals; Astrocytoma; Cell Line, Tumor; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Humans; Molecular Sequence Data; Monoterpenes; Phosphoglycerate Dehydrogenase; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin

To investigate the impact of all-trans retinoic acid (ATRA) on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma.. The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade II and grade IV astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis.. The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to beta-actin in ATRA-treated and untreated SHG-44 were 14.02+/-0.35 and 21.40+/-0.58 (t = 24.728, P = 0.000), respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% (6/25) and 56.52% (13/23) (chi(2) = 5.298, P = 0.021) in WHO grade II and grade IV astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy.. ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

Topics: Antineoplastic Agents; Astrocytoma; Cell Line, Tumor; Cell Proliferation; Cell Size; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-mdm2; Time Factors; Tretinoin

Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence by the alpha-secretase prevents the formation of toxic Abeta peptides. It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as alpha-secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsalpha. Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.

Topics: ADAM Proteins; ADAM17 Protein; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Astrocytoma; Blotting, Western; Cattle; Cell Culture Techniques; Cell Differentiation; Cell Extracts; Cell Line; Cell Line, Tumor; Dipeptides; Disintegrins; Endopeptidases; Gene Expression Regulation; Humans; Metalloproteases; Mice; Mice, Transgenic; Mutagenesis; Nerve Tissue Proteins; Neuroblastoma; Protease Inhibitors; Solubility; Tetradecanoylphorbol Acetate; Tretinoin; Up-Regulation

We previously showed that the expression of p16(ink4a) (p16), in conjunction with retinoic acid (RA) treatment in the p16-deficient astrocytoma cell line, U343 MG-A, induced a potent cell cycle arrest in G(1) associated with changes in morphology. In this study, we investigated the effects of p16 expression and RA treatment on the expression and distribution of actin, glial fibrillary acidic protein (GFAP), and vimentin within the U343 MG-A astrocytoma cytoskeleton. Changes in expression and location of the small GTPase, rhoA, were also examined after p16 expression and RA treatment. We showed that p16 expression and RA treatment led to an increase in the expression of GFAP, as well as its reorganization but that it did not significantly affect actin or vimentin expression. p16 induction in combination with RA treatment resulted in a decreased expression and activation of rhoA as determined by immunocytochemistry and Western blot analysis of soluble and insoluble fractions of cell lysates. Endogenous levels of rhoA expression varied among samples in a panel of astrocytoma cell lines as determined by Western blot analysis. Introduction of a dominant active rhoA mutant into p16-induced, RA-treated U343 MG-A astrocytoma cells was associated with the loss of long astrocytic processes and stellate morphology. These data are among the first to report the pattern of rhoA expression in human astrocytoma cell lines. They furthermore suggest that the stellate cell phenotype observed in U343 MG-A astrocytoma cells after cyclin-dependent kinase inhibitor (CKI) induction and RA treatment is accompanied by an inhibition and inactivation of rhoA in this cell system.

Topics: Actin Cytoskeleton; Astrocytoma; Brain Neoplasms; Cell Compartmentation; Cell Differentiation; Cell Membrane; Cell Movement; Cell Size; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Phenotype; Protein Transport; rhoA GTP-Binding Protein; Tretinoin; Tumor Cells, Cultured

Retinoids participate in the onset of differentiation, apoptosis and the inhibition of growth in a wide variety of normal and cancerous cells. Several recent reports have shown that hepatocyte growth factor (HGF), and its receptor, c-Met, are expressed at abnormally high levels in various human malignant gliomas and exert a strong proliferative action in an autocrine fashion. These results, consequently, imply that HGF and its receptor may represent a major contributor to the progression of such malignancies. Since astrocytomas are the most frequently occurring glioma, we have shown here that U87 cells - a well-established, human astrocytoma cell line - express both HGF and c-Met, thereby providing a suitable astrocytic tumor model for studying the potential role of HGF, functioning in an autocrine mode, in astrocytic tumorigenesis. Furthermore, we demonstrated the expression of the retinoic acid receptor (RAR) isoforms, RARalpha, -beta and -gamma, as well as the retinoid x-receptor (RxR) isoforms, RxRalpha and -beta, by RT-PCR and western blot analysis in these cells. Since ligands of the RARs and RxRs are known to exert growth inhibitory effects on various tumor cells which include some astrocytomas, we speculated that such effect of retinoids might be mediated via inhibition of HGF secretion in human astrocytoma cells. Indeed, we have shown that the RAR agonists, all-trans retinoic acid (ATRA) and (E)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB), inhibited HGF secretion with half maximal inhibition occurring at 3.0 microM and 15 nM, respectively, as did the RxR agonists, 9-cis- and 13-cis retinoic acid (9cRA and 13cRA, respectively), which exerted half-maximal inhibitory effects at 40 and 25 nM, respectively. These actions of the RAR and RxR agonists appear to be exerted at the transcriptional level as assessed by Northern blot analysis. Taken together, our results show for the first time that retinoids, acting via the RAR and RxRs, significantly inhibit both the secretion and expression of HGF, thereby interrupting a potentially highly tumorigenic autocrine loop in astrocytoma cells.

Topics: Antineoplastic Agents; Astrocytoma; Autocrine Communication; Benzoates; Brain Neoplasms; Cell Division; DNA Primers; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Neovascularization, Pathologic; Paracrine Communication; Proto-Oncogene Proteins c-met; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.

Topics: Animals; Antineoplastic Agents; Aquaporin 4; Aquaporins; Astrocytes; Astrocytoma; Cell Differentiation; Cerebral Cortex; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Embryonal Carcinoma Stem Cells; Gene Expression; Mice; Mice, Inbred Strains; Neoplastic Stem Cells; Rats; Rats, Wistar; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

This work explores the hypothesis that perturbations caused by ethanol on the regulatory role of retinoids in brain development may be a mechanism involved in the neuropathology of fetal alcohol syndrome. The interaction of ethanol and retinoic acid (RA) on RA receptor (RAR) beta and glial fibrillary acidic protein (GFAP) mRNA expression is evaluated. In the U-373 MG astrocytoma, mRNA expression of RAR beta was increased and GFAP was decreased by RA. Ethanol decreased the expression of RAR beta mRNA, but increased that of GFAP. The RA-stimulated increase in RAR beta was not affected by the presence of ethanol. RA prevented the ethanol-induced increase in GFAP mRNA. Cycloheximide abolished only the GFAP response to ethanol. This work shows that an interrelationship between ethanol and RA exists in the astrocyte.

Topics: Astrocytoma; Cycloheximide; Dose-Response Relationship, Drug; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Gene Expression; Glial Fibrillary Acidic Protein; Humans; Pregnancy; Protein Synthesis Inhibitors; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.

Topics: Antineoplastic Agents; Astrocytoma; Biological Transport; Cell Differentiation; Cells, Cultured; Humans; Inositol; Myo-Inositol-1-Phosphate Synthase; Neuroblastoma; Neurons; Phlorhizin; Stem Cells; Teratocarcinoma; Tretinoin; Water; Water-Electrolyte Balance

Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti-tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP-1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors. TSP-1-adherent U-251MG cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins alpha3beta1 and alpha5beta1 in clusters in lamellipodia and filopodia. The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36. Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins alpha3beta1 and alpha5beta1 mediated TSP-1 attachment of SK-N-SH cells, and integrins alpha3beta1, alpha5beta1, and alphavbeta3 mediated TSP-1 attachment of U-251MG cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1. Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells.

Topics: Astrocytes; Astrocytoma; Brain; CD36 Antigens; Cell Adhesion; Cell Differentiation; Chondroitin ABC Lyase; Chondroitin Sulfates; Cytoskeleton; Endothelium; Ganglioneuroblastoma; Ganglioneuroma; Glioblastoma; Heparin; Humans; Integrin alpha3beta1; Integrins; Neuroblastoma; Neurons; Oligopeptides; Peptide Fragments; Polysaccharide-Lyases; Receptors, Fibronectin; Recombinant Proteins; Thrombospondin 1; Tretinoin; 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

We have characterized the expression and activity of the cell cycle regulatory machinery and the organization of the cytoskeleton of the p16(Ink4a)-deficient astrocytoma cell line, U343 MG-a (U343), following retinoic acid (RA) treatment. RA causes cell cycle arrest at low cell density and significant morphological changes in U343 cells, reflected by reorganization of the intermediate filament, GFAP, and actin. RA-induced cell cycle arrest is also associated with induction of p27Kip1 expression, inhibition of cdk2-associated kinase activity and alteration of the phosphorylation state of the pRB-family proteins. We next determined the effect of inducing expression of the cyclin dependent kinase inhibitors (CKI's), p16(Ink4a), p21Cip1/Waf1 or p27Kip1 on the proliferation and morphology of these malignant astrocytoma cells in the absence and presence of RA. Induction of p16, p21 or p27, using the tetracycline repressor system, potently inhibits proliferation of U343 cells. However, rather than resembling RA-treated cells, CKI-induced U343 cells become flat with abundant cytoplasm and perinuclear vacuolization. CKI-induced morphological alterations are accompanied by a significant reorganization of glial filaments within the cytoplasm. Interestingly, when U343 cells are growth arrested by p16, p21 or p27 induction and treated simultaneously with RA, a dramatic morphological change occurs, cells acquiring multiple long, tapering processes reminiscent of primary astrocytes. This rearrangement is accompanied by reorganization of GFAP, vimentin and actin. Vimentin specifically relocalizes to the tips of the long processes which form. The arrangement of intermediate filaments in these cells is, in fact, indistinguishable from their arrangement in primary human astrocytes. These data demonstrate that when a strong proliferative block, produced by CKI expression, occurs in conjunction with the morphogenic signals generated by RA, these p16-deficient malignant astrocytoma cells are induced to phenotypically resemble normal astrocytes.

Topics: Actins; Antineoplastic Agents; Astrocytoma; Cell Cycle Proteins; Cell Division; Cyclin G; Cyclin G1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytoskeleton; G1 Phase; Glial Fibrillary Acidic Protein; Humans; Microtubule-Associated Proteins; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vimentin

Apolipoprotein E (apoE), one of the major plasma lipoproteins, also is expressed in a variety of cell types, including the glial cells of the nervous system. apoE is involved in processes of degeneration and regeneration after nerve lesions as well as in the pathogenesis of Alzheimer's disease (AD). Glial synthesis of apoE is activated in response to injury both in the peripheral and central nervous system. We now report that the activity of the proximal apoE promoter in astrocytes is upregulated by cAMP and retinoic acid, which act synergistically. Sequence analysis of the apoE promoter indicated the presence of several AP-2 consensus sequences that could mediate the stimulatory effect of cAMP and retinoic acid. The possible functional role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with an apoE promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated apoE promoter activity. DNase I footprinting technique revealed the existence of two binding sites for recombinant AP-2 in regions from -48 to -74 and from -107 to -135 of the apoE promoter. Mutations in these regions markedly impaired the trans-stimulatory effect of AP-2. These results indicate the existence of functional AP-2 sites in the promoter region of apoE that could contribute to the complex regulation of this gene in developmental, degenerative, and regenerative processess of the nervous system.

Topics: Apolipoproteins E; Astrocytoma; Base Sequence; Carcinoma, Hepatocellular; Consensus Sequence; Cyclic AMP; DNA-Binding Proteins; Gene Expression Regulation; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Transcription Factor AP-2; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Monolayer and agar culture techniques were used to examine the antiproliferative activities and morphological alterations of glioblastoma-astrocytoma (G1-As-14) cells induced by 20 mumol retinoic acid (RA). RA treated cells assumed flattened appearance and formed multilayers no longer. Most of the cells formed cross-bridges with one another. RA treatment caused time-dependent, dose-dependent and cell seeding-dependent reduction of growth in both monolayer and in agar cultures. RA-induced growth inhibition was also affected by concentration of fetal bovine serum in the culture medium. All these effects could be reversed within 48 h after withdrawal of RA from the growth medium. The results demonstrated that the respective cell line was sensitive to RA-induced growth inhibition and morphological alterations which were generally associated with reduced expression of malignant phenotype.

Topics: Astrocytoma; Brain Neoplasms; Cell Count; Cell Division; Culture Media; Glioblastoma; Humans; Tretinoin; Tumor Cells, Cultured

p36 is a calcium/lipid-binding phosphoprotein that is expressed at high levels in proliferating and transformed cells, and at low levels in terminally differentiated cells, such as CNS neurons. The calcium-dependent binding to membrane phospholipids, and its capacity to interact with intermediate filament proteins suggest that p36 may be involved in the transduction of extracellular signals. The present work examines p36 gene expression in the mature CNS, primary primitive neuroectodermal tumors (PNETs), and transformed PNET cell lines. p36 immunoreactivity was not observed in normal adult human brain, but low levels of the protein were detected by Western blot analysis. Following acute anoxic cerebral injury, the mean levels of p36 protein were elevated two-fold, and injured neurons exhibited increased p36 immunoreactivity. This phenomenon was likely to have been mediated by post-transcriptional mechanisms since there was no corresponding change in the level p36 mRNA. p36 immunoreactivity was detected in 8 of 9 primary PNETs, and in 3 of 3 neurofilament-expressing PNET cell lines. The levels of p36 protein in PNET cell lines were 5-fold higher than in adult human brain tissue. Although p36 gene expression was generally high in proliferating PNET cells, the levels of p36 mRNA and protein were not strictly correlated with DNA synthesis. Instead, p36 gene expression was modulated in both proliferating and non-proliferating PNET cell cultures by treatment with 50 mIU/ml of insulin, 100 mM ethanol, or 5 microM retinoic acid. The frequent discordances observed experimentally and in vivo between p36 mRNA and p36 protein expression suggest that the steady-state levels of p36 protein in neuronal cells may be regulated primarily by post-transcriptional mechanisms.

Topics: Adult; Animals; Annexin A2; Astrocytoma; Brain; Brain Ischemia; Brain Neoplasms; Cell Differentiation; Ethanol; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Hypoxia, Brain; Insulin; Membrane Lipids; Neoplasm Proteins; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; Rats; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The ets-1 protein has been primarily studied as a sequence-specific transcriptional regulator that is predominately expressed in lymphoid cells. In this report, we show that ets-1 is also expressed in astrocytes and astrocytoma cells and is regulated during both signal transduction and differentiation. Both isoforms of ets-1, p51 and p42, were found in astrocytes and astrocytoma cells, but whereas expression of p51 was strong, p42, the alternate splice product previously shown to lack the phosphorylation domain, was difficult to detect and was present at a level 10- to 40-fold lower than that of p51. This differed by roughly an order of magnitude from the ratio generally observable in T cells and thymocytes. In two astrocytoma lines of human origin, CCF and 1321N1, ets-1 phosphorylation was stimulated by bradykinin and carbachol, respectively. Glutamate, norepinephrine, and bradykinin elicited phosphorylation of p51 in cultures of primary rat type 1 astrocytes. ets-1 phosphorylation was dramatically blocked by KT5926, an inhibitor of myosin light-chain kinase, suggesting that this kinase may be involved in phosphorylation of ets-1 in vivo. Investigations of retinoic acid-induced differentiation in P19 cells provided further support for a strong correlation of ets-1 with the pathway for astrocyte differentiation.

Topics: Alkaloids; Animals; Astrocytes; Astrocytoma; Carbachol; Carbazoles; Cell Differentiation; Cell Line; Cells, Cultured; Cerebral Cortex; Gene Expression; Humans; Indoles; Methionine; Myosin-Light-Chain Kinase; Phosphates; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Rats; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

We studied the effect of retinoids on the growth and differentiation of a cell line (U 343 MG-A) derived from a human malignant astrocytoma. Cultures treated with all-trans or 13-cis retinoic acid showed a dose-dependent inhibition of proliferation and a marked reduction in the mean cell number at the plateau phase of growth (3.5 x 10(6) vs. 1 x 10(7) cells/25 cm2) compared with untreated cultures. At confluence, cells treated with all-trans or 13-cis retinoic acid were contact-inhibited, whereas control cultures showed crowding, piling, and overgrowth. All-trans retinol or retinyl acetate did not inhibit growth. Astrocytoma cultures treated with all-trans retinoic acid (10(-6) M) for 5 days were modestly growth-inhibited but by day 16 had the same numbers of cells as controls; cultures that received all-trans retinoic acid for 9 days were markedly growth-inhibited for 7 days after the drug was removed. All-trans and 13-cis retinoic acid (10(-6) M) prevented the EDTA-induced cell detachment seen in control cultures. Strongly adherent all-trans retinoic-acid-treated astrocytoma cells grew at a slower rate than did readily detached all-trans retinoic-acid-treated or control cells. Cell spreading, an increased cytoplasmic:nuclear ratio, and greater numbers of broadly bipolar cells, some bearing thin cytoplasmic processes, were seen in cultures treated with 10(-6) M all-trans or 13-cis retinoic acid. Small tightly packed cuboidal cells and large broadly bipolar cells were seen in astrocytoma cultures from which all-trans retinoic acid was removed on days 5 and 9. Indirect immunofluorescence revealed more intense staining with antiserum to glial fibrillary acidic protein in cultures treated with 10(-6) M all-trans retinoic acid than in control cultures; electron-microscope examination of similarly treated cultures revealed more abundant 8-10 nm intermediate filaments than in control cultures. An enzyme-linked immunosorbent assay showed that all-trans or 13-cis retinoic acid caused a dose-dependent increase in the quantity of glial fibrillary acidic protein in the astrocytoma cells.

Topics: Astrocytoma; Cell Adhesion; Cell Division; Glial Fibrillary Acidic Protein; Humans; Male; Middle Aged; Retinoids; Thymidine; Tretinoin; Tumor Cells, Cultured