Oncogenic RAS induces accelerated transition through G2/M and promotes defects in the G2 DNA damage and mitotic spindle checkpoints.
Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.
Knauf
Jeffrey A
JA
Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, OH 45267, USA.
Ouyang
Bin
B
Knudsen
Erik S
ES
Fukasawa
Kenji
K
Babcock
George
G
Fagin
James A
JA
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D004249:DNA Damage; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D016195:G2 Phase; D042822:Genomic Instability; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D051381:Rats; D008941:Spindle Apparatus / Q000502:physiology*; D018631:ras Proteins / Q000502:physiology*
D019869:Phosphatidylinositol 3-Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins