title: Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide.
abstract: BACKGROUND
  Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported.
  
  
  METHODS
  C6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml) for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP.
  
  
  RESULTS
  Treatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation.
  
  
  CONCLUSIONS
  Our results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle arrest at G1 and M phases and cAMP-dependent cell differentiation.
authors.affiliation: Department of Neural and Behavioral Sciences, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
authors.forename: Xijun
authors.initials: X
authors.lastname: Liu
authors.forename: Jin-Ming
authors.initials: JM
authors.lastname: Yang
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Zhang
authors.forename: Xin-Yuan
authors.initials: XY
authors.lastname: Liu
authors.forename: David X
authors.initials: DX
authors.lastname: Liu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016193:G1 Phase / Q000187:drug effects*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D016196:S Phase / Q000187:drug effects*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D005904:Glial Fibrillary Acidic Protein; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D000242:Cyclic AMP