Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells.
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.
Dirks
P B
PB
Hospital for Sick Children, Department of Pathology, University of Toronto, Ontario, Canada.
Patel
K
K
Hubbard
S L
SL
Ackerley
C
C
Hamel
P A
PA
Rutka
J T
JT
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001254:Astrocytoma / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000187:drug effects* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D056746:Cyclin G; D056766:Cyclin G1; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000187:drug effects / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000187:drug effects / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000187:drug effects / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014746:Vimentin / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D000970:Antineoplastic Agents; C534702:CCNG1 protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D056746:Cyclin G; D056766:Cyclin G1; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014212:Tretinoin