Retinoblastoma tumor suppressor targets dNTP metabolism to regulate DNA replication.
The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G(2) phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control S-phase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.
Angus
Steven P
SP
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Ohio, 45267-0521, USA. Steven.Angus@uc.edu
Wheeler
Linda J
LJ
Ranmal
Sejal A
SA
Zhang
Xiaoping
X
Markey
Michael P
MP
Mathews
Christopher K
CK
Knudsen
Erik S
ES
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D005434:Flow Cytometry; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D016160:Retinoblastoma Protein / Q000502:physiology*; D016196:S Phase; D013997:Time Factors; D014158:Transcription, Genetic
D019927:Cyclin E; D016160:Retinoblastoma Protein; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases