TY - JOUR
T1 - Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents
AU - Mayhew, Christopher N.
AU - Perkin, Lisa M.
AU - Zhang, Xiaoping
AU - Sage, Julien
AU - Jacks, Tyler
AU - Knudsen, Erik S.
N1 - Funding Information:
The authors are grateful to Dr Karen Knudsen, Dr Thérèse Tuohy, David Solomon, Christin Petre-Draviam and Emily Bosco for comments on the manuscript and all members of the Knudsen’s laboratories for thought provoking discussion. This work was supported by ACS grant RSG-01-254-01-CCG to ESK. TJ is an investigator of the Howard Hughes Medical Institute. CNM is supported by National Cancer Institute Training Grant T32 CA 59268.
PY - 2004/5/20
Y1 - 2004/5/20
N2 - The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites tanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.
AB - The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites tanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.
KW - Cell cycle checkpoints
KW - Chemotherapeutics
KW - Immortalization
KW - Retinoblastoma tumor suppressor
KW - Thymidylate synthase
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U2 - 10.1038/sj.onc.1207503
DO - 10.1038/sj.onc.1207503
M3 - Article
C2 - 15064736
AN - SCOPUS:2942558345
SN - 0950-9232
VL - 23
SP - 4107
EP - 4120
JO - Oncogene
JF - Oncogene
IS - 23
ER -