tretinoin and Neuroectodermal-Tumors--Primitive

Cdk9 is a member of the Cdc2-like family of kinases. Its cyclin partners are members of the family of cyclin T (T1, T2a and T2b) and cyclin K. The Cdk9/Cyclin T complex appears to be involved in regulating several physiological processes. Recently, Cdk9 has been identified as a regulator of the differentiation program of several cell types, such as muscle cells, monocytes and lymphocytes, suggesting that it may have a function in controlling specific differentiative pathways. We analyzed whether Cdk9 and Cyclin T1 may be involved in the regulation of neuron and astrocyte differentiation. Cdk9 and Cyclin T1 expression levels were monitored during the differentiation program of neuroblastoma and astrocytoma cell lines. Our results suggest that Cdk9/Cyclin T1 complex may be required for neuron differentiation induced by retinoic acid, because the expression level of the complex varies during differentiation, but no significant changes were observed in its expression in the astrocytoma cell line. In addition, the expression of Cdk9 and Cyclin T1 was evaluated by immunohistochemistry in samples of neuroblastoma, PNET (Primary Neuroectodermal Tumor) and astrocytoma tumors of different grades, in order to assess whether there was a correlation between Cdk9 expression and tumor grading. Our results show that in neuroblastoma and PNET tumor samples Cdk9 is more expressed the more differentiated the tumor is. Conversely, no significant alteration of Cdk9 expression was observed in astrocytoma tumor samples of different grades, thus confirming the results obtained for the cell lines.

Topics: Astrocytes; Cell Differentiation; Cell Line, Tumor; Cyclin T; Cyclin-Dependent Kinase 9; Cyclins; Humans; Immunohistochemistry; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; RNA, Messenger; Tretinoin

Peripheral primitive neuroectodermal tumor (PNET) and Ewing's sarcoma (ES) constitute a unique group of small round cell tumors in childhood and young adults that are characterized by the same chromosomal translocation t(11;22)(q24;q12). Recently, the expression of neurotrophin receptors has been found in various human tumors including PNET/ES, but the functional significance of these receptor expressions has not been documented in PNET/ES. In the present study, we investigated the biologic effects of trkA neurotrophin receptor activation by nerve growth factor (NGF) in a newly established Askin tumor cell line, JK-GMS, which constitutively expresses a high level of trkA. The activation of trkA induced differentiation and inhibited the growth of JK-GMS cells, which was characteristically associated with down-regulation of c-myc and N-myc mRNA expression. NGF did not exert significant changes in two different PNET/ES cell lines, CADO-ES1 and RD-ES, which did not express detectable levels of trkA. The biologic effects mediated by NGF were abrogated by treatment of the cells with K-252a, and the treatment with brain-derived neurotrophic factor did not affect the biologic behavior of JK-GMS cells, indicating that the effects are trkA specific. The results observed were quite similar to those of neuroblastoma cells, another childhood tumor of neural crest origin. Overall findings strongly suggest that the trkA-mediated signaling pathway plays a crucial role in controlling the basic biologic properties of JK-GMS cells.

Topics: Adolescent; Base Sequence; Bucladesine; Cell Differentiation; Cell Division; DNA Primers; Humans; Karyotyping; Male; Neuroectodermal Tumors, Primitive; Receptor, trkA; Tretinoin; Tumor Cells, Cultured

Ras protooncogenes encode small guanine nucleotide binding proteins (p21ras) activated by phosphorylation. Phosphorylation of p21ras is predominantly regulated by the GTPase activating proteins type 1 GAP120 and neurofibromin. Increased levels of p21ras-GTP (active) have been associated with increased cell growth and malignant transformation. In this study the relationship between p21ras, type 1 GAP120 and neurofibromin with growth and differentiation has been examined in neuroblastoma and peripheral primitive neuroectodermal tumour (pPNET) cell lines. The level of p21ras protein in neuroblastoma and pPNET cells was the same. However, the amount of p21ras-GTP bound was higher in pPNET than in neuroblastoma cells. This most likely reflects the absence of neurofibromin. Retinoic acid (RA)-induced differentiation and growth inhibition of neuroblastoma cells was associated with an increase in type 1 GAP120 and neurofibromin mRNA, and a decrease in p21ras-GTP. In pPNET cells levels of type 1 GAP120 but not neurofibromin mRNA were increased to similar levels to those in neuroblastoma cells. This was not associated with decreased p21ras-GTP, modulation of growth or change in morphology. In summary, constitutive activation of p21ras may have a role in the biology of pPNET cells. This may reflect abnormalities in neurofibromin expression, and could inpart explain why RA did not induce morphological differentiation and growth inhibition in pPNETs.

Topics: Cell Differentiation; Cell Division; Enzyme Activation; Genes, ras; GTPase-Activating Proteins; Humans; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurofibromin 1; Phosphorylation; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; RNA, Messenger; Sarcoma, Ewing; Sequence Analysis, DNA; Tretinoin; Tumor Cells, Cultured

Widespread proliferation of dystrophic neurites in the cerebral cortex represents an important neuroanatomical correlate of dementia in Alzheimer's disease (AD). Increased CNS expression of the 21-kDa neuronal thread protein (NTP) species is also correlated with dementia in AD. Pilot in vitro experiments provided evidence that high-level NTP expression might be linked to neuritic growth. The present study examines retinoic acid (RA) modulation of NTP expression during neurite outgrowth and neuronal differentiation in SH-Sy5y neuroblastoma and PNET2 CNS-derived cells. In both cell lines, RA-induced neuronal differentiation resulted in increased synthesis, expression, and phosphorylation of several NTP species, with high steady-state levels and stepwise hyper-phosphorylation of 21-kDa NTP molecules. With neurite outgrowth, NTP molecules were translocated from the perikarya to long, slender, unbranched cell processes (axons) and growth cones. RA-mediated changes in NTP expression were independent of DNA synthesis. The findings suggest that high-level expression of 21-kDa, and closely related phosphorylated NTP molecules correlates with neuritic growth. Therefore, over-expression of 21-kDa NTP molecules in AD probably reflects the widespread cortical neuritic sprouting associated with dementia. In view of the rapid phosphorylation and cell process translocation of NTP that occurs during neurite outgrowth in vitro, the accumulation of NTP in AD cortical neuronal perikarya suggests a further problem related to post-translational processing and transport of NTP molecules in AD neurodegeneration.

Topics: Alzheimer Disease; Calcium-Binding Proteins; Cell Differentiation; DNA; Humans; Lithostathine; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; Phosphoproteins; 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