Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.
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 flanking 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.
Mayhew
Christopher N
CN
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, USA.
Perkin
Lisa M
LM
Zhang
Xiaoping
X
Sage
Julien
J
Jacks
Tyler
T
Knudsen
Erik S
ES
D000782:Aneuploidy; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism*; D005472:Fluorouracil / Q000494:pharmacology; D016193:G1 Phase / Q000502:physiology; D017353:Gene Deletion; D017930:Genes, Reporter; D016161:Genes, Retinoblastoma / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016196:S Phase / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D016159:Tumor Suppressor Protein p53; D005472:Fluorouracil