ABSTRACT
CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.
Subject(s)
Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Binding Sites , Brain/metabolism , Chickens , Cloning, Molecular , Drosophila , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Molecular Sequence Data , Neoplasm Proteins , Phosphorylation , Rats , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , TransfectionABSTRACT
Compounds that could block tumor angiogenesis and induce tumor cell differentiation in malignant gliomas represent a very valuable tool in anticancer treatments. In this paper, we demonstrate that more selective drugs, which interfere with specific cellular targets, could treat glioma more effectively. 8-Cl-cAMP and tiazofurin (TR) are site-specific analogs that selectively inhibit PKAI and IMP dehydrogenase, are directly involved in cell proliferation and apoptosis, and mediate the mitogenic effects of different oncogenes and growth factors. In this study, we have examined influence of 8-Cl-cAMP and TR on the production of an angiogenic factor [vascular endothelial growth factor (VEGF)] by human glioblastoma U251 MG cells, as well as their influence on the expression of a differentiating marker [glial fibrillary acidic protein (GFAP)]. Using a cell proliferation assay, VEGF enzyme-linked immunoassay and GFAP immunocytochemistry we demonstrated the effects of these compounds. Our results demonstrate that 8-Cl-cAMP and TR decrease VEGF production by U251 MG cells, and that under the influence of both agents these cells increase GFAP expression and change their morphology, becoming more differentiated. These findings also suggest that 8-Cl-cAMP and TR may have potential for further investigation of their antiangiogenic and differentiational role in malignant disease such as human gliomas.
Subject(s)
8-Bromo Cyclic Adenosine Monophosphate/analogs & derivatives , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Endothelial Growth Factors/biosynthesis , Glial Fibrillary Acidic Protein/biosynthesis , Glioblastoma/metabolism , Lymphokines/biosynthesis , Ribavirin/analogs & derivatives , Ribavirin/pharmacology , Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Cell Division/drug effects , Endothelial Growth Factors/metabolism , Glioblastoma/pathology , Humans , Lymphokines/metabolism , Tumor Cells, Cultured/drug effects , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
8-Cl-cAMP and tiazofurin (TR) are anti-tumor agents that besides their antiproliferative effect, also induce differentiation of tumor cells. Although, these agents exert a profound effect on the same events of tumor cell life, it is thought that 8-Cl-cAMP and TR act by modulating the signal transduction pathway through distinct mechanisms. We have compared their effect on two human glioma cell lines (U87 MG and U251 MG) and examined if there is selectivity in their action toward normal human astrocytes.