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Retinoblastoma tumor suppressor targets dNTP metabolism to regulate DNA replication
Steven P Angus1, Linda J Wheeler, Sejal A Ranmal
1Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Ohio, 45267-0521, USA. Steven.Angus@uc.edu
Abstract:
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.
Insights
The retinoblastoma tumor suppressor (RB) controls cell cycle progression by reducing dNTP synthesis. This metabolic regulation, alongside cell cycle inhibition, highlights RB
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The retinoblastoma tumor suppressor (RB) protein is a critical negative regulator of the cell cycle, frequently inactivated in human cancers.
- RB's role in cell cycle arrest is partly understood through its inhibition of CDK2 activity.
- Previous studies indicated RB-mediated S-phase arrest persists even when CDK2 activity is bypassed, suggesting additional regulatory mechanisms.
Purpose of the Study:
- To investigate the role of RB in regulating DNA replication and S-phase progression beyond its canonical cell cycle inhibitory functions.
- To determine if RB influences the metabolic pathways involved in nucleotide synthesis.
- To elucidate the mechanisms by which RB suppresses tumor formation.
Main Methods:
- Assessed the expression of key enzymes in deoxynucleotide triphosphate (dNTP) synthesis, including dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS), under conditions of RB activation and inactivation.
- Analyzed dNTP pool sizes and cell cycle progression in response to RB modulation and treatment with TS or RNR inhibitors.
- Compared the effects of active RB with those of known antimetabolites.
Main Results:
- Constitutively active RB was found to attenuate the expression of dNTP synthetic enzymes (dihydrofolate reductase, RNR, TS).
- RB inactivation led to increased levels of these metabolic proteins and conferred resistance to inhibitors targeting dNTP synthesis.
- RB-mediated cell cycle arrest was associated with significant depletion of dNTP pools and reduced levels of TS and RNR proteins.
Conclusions:
- RB functions as a transcriptional repressor that reduces available dNTP pools, thereby controlling S-phase progression.
- RB employs both canonical cell cycle regulation (via CDK2) and metabolic regulation (via dNTP synthesis) to inhibit proliferation.
- These dual mechanisms underscore the potent tumor-suppressive capacity of RB.