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 G₁/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS^V12 accelerated the G₂/M phase by ∼4 h and promoted bypass of the G₂ DNA damage and mitotic spindle checkpoints. Accelerated passage through G₂/M and bypass of the G₂ 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 G₂ DNA damage checkpoint, because cells arrested appropriately in G₂ despite conditional expression of HRAS^V12,S35 or BRAF^V600E. By contrast to the MAPK requirement for radiation-induced G₂ 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 G₂ 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 G₂/M.