title: Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.
abstract: The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.
authors.affiliation: Department of Pathology, University of Texas Health Science Center, San Antonio, Texas 78284, USA.
authors.forename: P M
authors.initials: PM
authors.lastname: Ghosh
authors.forename: M L
authors.initials: ML
authors.lastname: Moyer
authors.forename: G E
authors.initials: GE
authors.lastname: Mott
authors.forename: J I
authors.initials: JI
authors.lastname: Kreisberg
mesh_terms: D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins; D018797:Cell Cycle Proteins; D048429:Cell Size / Q000187:drug effects; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D016193:G1 Phase / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008148:Lovastatin / Q000494:pharmacology*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D020742:rhoA GTP-Binding Protein
chemical_list: D000199:Actins; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D008869:Microtubule-Associated Proteins; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008148:Lovastatin; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D006656:Protamine Kinase; D018844:Cyclin-Dependent Kinases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D009355:Neomycin