Ras-related small GTPases RalA and RalB regulate cellular survival after ionizing radiation.
OBJECTIVE
Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases RalA and RalB are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole-body irradiation, but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR.


METHODS
RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expression of short hairpin RNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, gammaH2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (PARP) cleavage for apoptosis.


RESULTS
Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR, and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR.


CONCLUSIONS
Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.
Kidd
Ambrose R
AR
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Snider
Jared L
JL
Martin
Timothy D
TD
Graboski
Sarah F
SF
Der
Channing J
CJ
Cox
Adrienne D
AD
D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects*; D003114:Colony-Forming Units Assay / Q000379:methods; D020690:GTPase-Activating Proteins / Q000502:physiology*; D055785:Gene Knockdown Techniques; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D055029:Inverted Repeat Sequences / Q000502:physiology; D010190:Pancreatic Neoplasms / Q000473:pathology / Q000532:radiotherapy; D011836:Radiation Tolerance / Q000235:genetics / Q000502:physiology*; D020725:ral GTP-Binding Proteins / Q000502:physiology*
D018528:ATP-Binding Cassette Transporters; D020690:GTPase-Activating Proteins; C095637:RALBP1 protein, human; D020725:ral GTP-Binding Proteins