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Retinoblastoma Tumor Suppressor Targets dNTP Metabolism to Regulate DNA Replication*

  • Steven P. Angus
    Correspondence
    To whom correspondence should be addressed. Tel.: 513-558-1086; Fax: 513-558-2445;
    Affiliations
    Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267-0521 and
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  • Linda J. Wheeler
    Affiliations
    Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, 97331-7305
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  • Sejal A. Ranmal
    Affiliations
    Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267-0521 and
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  • Xiaoping Zhang
    Affiliations
    Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267-0521 and
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  • Michael P. Markey
    Affiliations
    Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267-0521 and
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  • Christopher K. Mathews
    Footnotes
    Affiliations
    Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, 97331-7305
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  • Erik S. Knudsen
    Footnotes
    Affiliations
    Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267-0521 and
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‖ Supported by National Institutes of Health Grant GM55134 and the National Science Foundation Grant MCB 9916576.
    ** Supported by the National Institutes of Health/National Cancer Institute Grant CA82525 and American Cancer Society Grant RSG-01-254-01-CCG.
Open AccessPublished:September 06, 2002DOI:https://doi.org/10.1074/jbc.M205911200
      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 G2 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.
      The retinoblastoma tumor suppressor (RB)
      The abbreviations used are: RB, retinoblastoma tumor suppressor; MEF, murine embryonic fibroblast; HDAC, histone deacetylase; RNR, ribonucleotide reductase subunits R1/R2; TS, thymidylate synthase; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; BrdUrd, bromodeoxyuridine; HU, hydroxyurea; 5-FU, 5-fluorouracil; CdA, chlorodeoxyadenosine; Dox, doxycycline; DHFR, dihydrofolate reductase; TS, thymidylate synthase; FdU, fluorodeoxyuridine
      1The abbreviations used are: RB, retinoblastoma tumor suppressor; MEF, murine embryonic fibroblast; HDAC, histone deacetylase; RNR, ribonucleotide reductase subunits R1/R2; TS, thymidylate synthase; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; BrdUrd, bromodeoxyuridine; HU, hydroxyurea; 5-FU, 5-fluorouracil; CdA, chlorodeoxyadenosine; Dox, doxycycline; DHFR, dihydrofolate reductase; TS, thymidylate synthase; FdU, fluorodeoxyuridine
      functions as a negative regulator of cell cycle transitions (
      • Wang J.Y.
      • Knudsen E.S.
      • Welch P.J.
      ,
      • Sherr C.J.
      ,
      • Harbour J.W.
      • Dean D.C.
      ,
      • Bartek J.
      • Bartkova J.
      • Lukas J.
      ,
      • Kaelin Jr., W.G.
      ). Due to its frequent inactivation in tumors (>60%), it is highly relevant to determine how RB functions to inhibit cellular proliferation and to elucidate its interaction with chemotherapeutic drugs.
      Biochemically, RB functions as a transcriptional co-repressor that mediates the inhibition of cell cycle progression (
      • Wang J.Y.
      • Knudsen E.S.
      • Welch P.J.
      ,
      • Sherr C.J.
      ,
      • Harbour J.W.
      • Dean D.C.
      ,
      • Bartek J.
      • Bartkova J.
      • Lukas J.
      ,
      • Kaelin Jr., W.G.
      ). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (
      • Dyson N.
      ). In addition to binding E2F, RB also interacts with histone deacetylase (HDAC) and SWI/SNF chromatin remodeling proteins to establish a repressor complex on the promoters of E2F-regulated genes (
      • Harbour J.W.
      • Dean D.C.
      ,
      • Strobeck M.W.
      • Knudsen K.E.
      • Fribourg A.F.
      • DeCristofaro M.F.
      • Weissman B.E.
      • Imbalzano A.N.
      • Knudsen E.S.
      ,
      • Zhang H.S.
      • Gavin M.
      • Dahiya A.
      • Postigo A.A., Ma, D.
      • Luo R.X.
      • Harbour J.W.
      • Dean D.C.
      ). This activity of RB is critical for cell cycle inhibition. In G0 and early G1, RB is hypophosphorylated and forms transcriptional repressor complexes to inhibit cell cycle progression. However, in response to mitogenic signaling, cyclin-dependent kinase (CDK)/cyclin complexes phosphorylate RB (
      • Mittnacht S.
      ). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (
      • Wang J.Y.
      • Knudsen E.S.
      • Welch P.J.
      ,
      • Sherr C.J.
      ,
      • Harbour J.W.
      • Dean D.C.
      ,
      • Bartek J.
      • Bartkova J.
      • Lukas J.
      ,
      • Kaelin Jr., W.G.
      ).
      Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (
      • Dyson N.
      ,
      • DeGregori J.
      • Kowalik T.
      • Nevins J.R.
      ,
      • Ishida S.
      • Huang E.
      • Zuzan H.
      • Spang R.
      • Leone G.
      • West M.
      • Nevins J.R.
      ). Consistent with the role of RB as a repressor of E2F, in disparate settings the expression/activity of cyclin E, cyclin A, and CDK2 have been attenuated during RB-mediated arrest. Because these gene products are required for progression through S phase, it is clear that these targets are important participants in RB-mediated cell cycle inhibition (
      • Sherr C.J.
      ,
      • Reed S.I.
      ,
      • Ohtsubo M.
      • Theodoras A.M.
      • Schumacher J.
      • Roberts J.M.
      • Pagano M.
      ). Throughout S phase, discrete origins of replication fire, and components of the DNA polymerase holoenzyme are sequentially recruited to these sites (
      • Kelly T.J.
      • Brown G.W.
      ). The binding of the sliding clamp protein, proliferating cell nuclear antigen (PCNA), to chromatin enables processive DNA synthesis and represents one of the final stages of this assembly (
      • Waga S.
      • Stillman B.
      ). Consistent with the idea that RB regulates DNA replication, the expression of an active RB allele has been shown to specifically disrupt the association of PCNA with chromatin. Demonstrating the critical nature of CDK2 as a target of RB, PCNA activity was completely restored by the ectopic activation of CDK2 in the presence of active RB (
      • Sever-Chroneos Z.
      • Angus S.P.
      • Fribourg A.F.
      • Wan H.
      • Todorov I.
      • Knudsen K.E.
      • Knudsen E.S.
      ). Interestingly, although replication machinery was restored by CDK2 and some DNA synthesis occurred, replication was incomplete. These observations indicate that RB regulates S phase through an additional mechanism independent of CDK2 activity.
      Here, we define a CDK2-independent pathway through which RB regulates DNA replication by controlling dNTP pools. We show that RB is required to maintain the relative expression of dNTP metabolic enzymes in proliferating cells, as loss of RB results in their deregulated expression and resistance to dNTP pool depletion. Conversely, activated RB completely attenuates enzyme expression, limiting available dNTP pools. The inhibitory effect of RB in this context is analogous to specific antimetabolite chemotherapeutics. Thus, RB impinges on DNA replication not only through canonical CDK/cyclin regulation, but also through the metabolic limitation of DNA precursor molecules.

      DISCUSSION

      RB-mediated cell cycle inhibition occurs in response to antimitogenic signals, DNA damage, and other cellular stresses (
      • Wang J.Y.
      • Knudsen E.S.
      • Welch P.J.
      ,
      • Sherr C.J.
      ,
      • Harbour J.W.
      • Dean D.C.
      ,
      • Bartek J.
      • Bartkova J.
      • Lukas J.
      ,
      • Kaelin Jr., W.G.
      ). The cell cycle arrest invoked by RB is thought to occur through the inhibition of CDK2 activity or the modulation of cell cycle regulatory factors (
      • Sherr C.J.
      ,
      • Reed S.I.
      ,
      • Ohtsubo M.
      • Theodoras A.M.
      • Schumacher J.
      • Roberts J.M.
      • Pagano M.
      ). However, RB-mediated arrest can only be partially subverted by the ectopic expression of the CDK2 activators cyclin E and cyclin A (
      • Sever-Chroneos Z.
      • Angus S.P.
      • Fribourg A.F.
      • Wan H.
      • Todorov I.
      • Knudsen K.E.
      • Knudsen E.S.
      ,
      • Knudsen E.S.
      • Buckmaster C.
      • Chen T.T.
      • Feramisco J.R.
      • Wang J.Y.
      ,
      • Chew Y.P.
      • Ellis M.
      • Wilkie S.
      • Mittnacht S.
      ,
      • Lukas J.
      • Herzinger T.
      • Hansen K.
      • Moroni M.C.
      • Resnitzky D.
      • Helin K.
      • Reed S.I.
      • Bartek J.
      ). Cyclin overproduction in the presence of active RB restores CDK2 activity and triggers S-phase entry; however, efficient DNA replication is not achieved. Analysis of the replication machinery indicated that PCNA tethering was restored, suggesting that downstream effects on the supply of dNTPs may be limiting. Here, we report that the expression of active RB down-regulates the levels of both RNR subunits and TS. Targeted disruption of RB resulted in deregulation of TS and RNR-R2 protein levels and resistance to antimetabolites that target their enzyme activity. Active RB induced an imbalance of intracellular dNTP pools, concomitant with the inhibition of DNA replication. The effects of RB on cell cycle and dNTP levels were comparable to effects of antimetabolites that target RNR and TS activity. Thus, the RB tumor suppressor pathway regulates DNA replication via CDK2 modulation and the metabolic control of dNTP pools.
      The function of RB to negatively regulate cellular proliferation is attributed to its transcriptional repression of E2F target genes (
      • Harbour J.W.
      • Dean D.C.
      ). These E2F targets encompass a wide variety of cell cycle regulatory and metabolic enzymes (
      • Dyson N.
      ,
      • DeGregori J.
      • Kowalik T.
      • Nevins J.R.
      ,
      • Ishida S.
      • Huang E.
      • Zuzan H.
      • Spang R.
      • Leone G.
      • West M.
      • Nevins J.R.
      ). It has been viewed that the down-regulation of cell cycle regulatory machinery is the primary means by which RB limits cell proliferation. Consistent with this, RB has been shown to inhibit the expression of cyclin E, cyclin A, or CDK2 to impede S-phase progression (
      • Zhang H.S.
      • Gavin M.
      • Dahiya A.
      • Postigo A.A., Ma, D.
      • Luo R.X.
      • Harbour J.W.
      • Dean D.C.
      ,
      • Sever-Chroneos Z.
      • Angus S.P.
      • Fribourg A.F.
      • Wan H.
      • Todorov I.
      • Knudsen K.E.
      • Knudsen E.S.
      ,
      • Knudsen E.S.
      • Buckmaster C.
      • Chen T.T.
      • Feramisco J.R.
      • Wang J.Y.
      ,
      • Chew Y.P.
      • Ellis M.
      • Wilkie S.
      • Mittnacht S.
      ,
      • Zhang H.S.
      • Postigo A.A.
      • Dean D.C.
      ,
      • Lukas J.
      • Sorensen C.S.
      • Lukas C.
      • Santoni-Rugiu E.
      • Bartek J.
      ,
      • Knudsen K.E.
      • Fribourg A.F.
      • Strobeck M.W.
      • Blanchard J.M.
      • Knudsen E.S.
      ). This has been demonstrated through the reduction in the amount of target proteins and subsequent attenuation of CDK2-associated kinase activity. Because CDK2 activity is required for DNA synthesis, this represents a mechanism through which RB inhibits cell cycle progression (
      • Sherr C.J.
      ,
      • Reed S.I.
      ,
      • Ohtsubo M.
      • Theodoras A.M.
      • Schumacher J.
      • Roberts J.M.
      • Pagano M.
      ,
      • Waga S.
      • Stillman B.
      ). Consistent with this idea, ectopic expression of cyclins E or A can partially overcome the inhibition of DNA replication mediated by active RB alleles (
      • Sever-Chroneos Z.
      • Angus S.P.
      • Fribourg A.F.
      • Wan H.
      • Todorov I.
      • Knudsen K.E.
      • Knudsen E.S.
      ,
      • Knudsen E.S.
      • Buckmaster C.
      • Chen T.T.
      • Feramisco J.R.
      • Wang J.Y.
      ,
      • Chew Y.P.
      • Ellis M.
      • Wilkie S.
      • Mittnacht S.
      ,
      • Lukas J.
      • Herzinger T.
      • Hansen K.
      • Moroni M.C.
      • Resnitzky D.
      • Helin K.
      • Reed S.I.
      • Bartek J.
      ). However, replication is incomplete; cells accumulate with S-phase DNA content and punctate BrdUrd labeling is observed. Investigation of DNA replication machinery under these conditions indicated that PCNA is still associated with chromatin. PCNA is a component of the processive DNA polymerase holoenzyme and is one of the last regulatory effectors of DNA replication (
      • Waga S.
      • Stillman B.
      ,
      • Sever-Chroneos Z.
      • Angus S.P.
      • Fribourg A.F.
      • Wan H.
      • Todorov I.
      • Knudsen K.E.
      • Knudsen E.S.
      ). Thus, the sustained inhibition achieved by PSM-RB in the presence of cyclin E represents a very late step in DNA replication and suggests that a specific action of RB may be to act downstream of the replication machinery to inhibit DNA synthesis. One of the few previously identified mechanisms through which replication is inhibited with PCNA tethered to chromatin is through the depletion of dNTP pools through the use of HU (
      • Bravo R.
      • Macdonald-Bravo H.
      ).
      The relative levels of dNTPs and the regulation of their synthesis play a critical role in DNA replication (
      • Mathews C.K.
      ,
      • Reichard P.
      ,
      • Mathews C.K.
      • Ji J.
      ). As such, expression of dNTP synthetic enzymes is cell cycle-regulated, with enhanced expression in S-phase. Even subtle changes in the levels of dNTPs can have a dramatic effect on DNA replication (
      • Mathews C.K.
      • Ji J.
      ). For example, inhibition of RNR activity by 50% using CdA leads to marked inhibition of cell cycle progression (
      • Griffig J.
      • Koob R.
      • Blakley R.L.
      ). Additionally, dNTP levels vary within S-phase of the cell cycle (
      • Leeds J.M.
      • Slabaugh M.B.
      • Mathews C.K.
      ); these variations may be responsible for changes in the rate of DNA replication during S-phase (
      • Collins J.M.
      ).
      S. A. Martomo and C. K. Mathews, manuscript in preparation.
      Consistent with the idea that the attenuation of dNTP metabolism could be a mechanism through which RB inhibits DNA replication, E2F can modify the transcription of several metabolic enzymes (
      • DeGregori J.
      • Kowalik T.
      • Nevins J.R.
      ,
      • Ishida S.
      • Huang E.
      • Zuzan H.
      • Spang R.
      • Leone G.
      • West M.
      • Nevins J.R.
      ,
      • Slansky J.E.
      • Farnham P.J.
      ,
      • Dou Q.P.
      • Zhao S.
      • Levin A.H.
      • Wang J.
      • Helin K.
      • Pardee A.B.
      ). Specifically, it has been demonstrated that ectopic expression of E2F can stimulate the expression of DHFR, RNR-R1, RNR-R2, TS, and thymidine kinase in quiescent cells (
      • DeGregori J.
      • Kowalik T.
      • Nevins J.R.
      ). In fact, recent chromatin immunoprecipitation analyses have detected RB on the DHFR promoter at the G1/S transition (
      • Wells J.
      • Boyd K.E.
      • Fry C.J.
      • Bartley S.M.
      • Farnham P.J.
      ). Thus, E2F activity is believed to maintain the relative levels of enzyme mRNA during cell cycle progression. Here, we evaluated whether RB could specifically attenuate the expression of metabolic targets as part of a program to inhibit DNA replication. We find that RB reduces the mRNA levels of dNTP synthetic enzymes, with RNR-R2 being the most strongly repressed and DHFR being weakly repressed. We show that active RB targets the protein levels of RNR-R1, RNR-R2, DHFR and TS to effectively limit their abundance. As may be expected for metabolic enzymes, the kinetics of DHFR attenuation were slow and did not correlate with cell cycle inhibition achieved by active RB. However, the RNR-R2, RNR-R1 and TS enzymes were significantly attenuated, concurrent with cell cycle inhibition. In addition, activation of endogenous pocket proteins by ectopic p16ink4a expression led to the loss of RNR-R2, TS, and DHFR. Thus, the depletion of metabolic enzymes mediated by active RB could participate in the inhibition of DNA replication by virtue of altered dNTP pools.
      In keeping with the significant role of dNTP metabolism in replication control, a number of therapeutic drugs are utilized that target dNTP synthetic enzymes (
      • Schweitzer B.I.
      • Dicker A.P.
      • Bertino J.R.
      ,
      • Bertino J.R., Li, W.W.
      • Lin J.
      • Trippett T.
      • Goker E.
      • Schweitzer B.
      • Banerjee D.
      ,
      • Pinedo H.M.
      • Peters G.F.
      ). These antimetabolites generally function as pseudo-substrates that poison their specific target enzymes, leading to the depletion of dNTPs and subsequent inhibition of DNA replication. One mechanism through which resistance to antimetabolites is achieved is through overexpression of the target enzymes. We found that Rb −/− MEFs significantly overproduced RNR-R2, TS, and DHFR protein. Our data are consistent with prior studies demonstrating that loss of RB leads to deregulation of metabolic enzyme mRNA (
      • Almasan A.
      • Yin Y.
      • Kelly R.E.
      • Lee E.Y.
      • Bradley A., Li, W.
      • Bertino J.R.
      • Wahl G.M.
      ,
      • Li W.
      • Fan J.
      • Hochhauser D.
      • Banerjee D.
      • Zielinski Z.
      • Almasan A.
      • Yin Y.
      • Kelly R.
      • Wahl G.M.
      • Bertino J.R.
      ). Specifically, Almasan et al. showed that mRNA levels of both TS and DHFR were elevated in asynchronously proliferatingRb −/− MEFs compared with wild-type MEFs (
      • Almasan A.
      • Yin Y.
      • Kelly R.E.
      • Lee E.Y.
      • Bradley A., Li, W.
      • Bertino J.R.
      • Wahl G.M.
      ). We observed that Rb −/− cells were resistant to increasing doses of the TS inhibitor 5-FU that are known to block DNA synthesis. Furthermore, the increase in RNR-R2 seen in the absence of RB resulted in resistance to the specific RNR inhibitor, HU. These results complement prior studies demonstrating the resistance of RB-deficient cells to MTX and FdU (
      • Almasan A.
      • Yin Y.
      • Kelly R.E.
      • Lee E.Y.
      • Bradley A., Li, W.
      • Bertino J.R.
      • Wahl G.M.
      ,
      • Li W.
      • Fan J.
      • Hochhauser D.
      • Banerjee D.
      • Zielinski Z.
      • Almasan A.
      • Yin Y.
      • Kelly R.
      • Wahl G.M.
      • Bertino J.R.
      ). Thus, RB regulates the relative expression levels of a coordinate set of dNTP synthetic enzymes, thereby rendering cells resistant to a variety of antimetabolites.
      Finally, to directly assess the effect of RB on replication precursors, we analyzed dNTP pools. Surprisingly, no prior study has implicated a mammalian signal-transduction cascade involved in cell cycle control to the level of dNTP and inhibition of DNA replication. InSaccharomyces cerevisiae, several studies have demonstrated the involvement of SML1, an inhibitor of RNR, in the replicative response to DNA damage (
      • Zhao X.
      • Muller E.G.
      • Rothstein R.
      ,
      • Chabes A.
      • Domkin V.
      • Thelander L.
      ). As would be expected from the dramatic effects on protein expression, we find that dNTP pools are significantly reduced through the action of RB. The changes mediated by RB are comparable in magnitude to the changes elicited by antimetabolites that inhibit key enzymes involved in dNTP metabolism. Importantly, the inhibition of replication observed by the use of these antimetabolites was accompanied by the retention of PCNA on chromatin. Thus, cells arrested by antimetabolites behave in a manner analogous to those inhibited for DNA replication with both PSM-RB and cyclin E.
      In summary, our findings reveal dual roles for RB in DNA replication control: concurrent regulation of CDK2 activity and metabolic enzyme activity through transcriptional regulation.

      Acknowledgments

      We thank Drs. Karen Knudsen, Chris Mayhew, and Peter Stambrook for thought-provoking discussion and critical reading of the manuscript. We are grateful to Dr. Masakazu Fukushima (Taiho Pharmaceutical) for the provision of TS polyclonal antibody. We thank Dr. George Babcock and Sandy Schwemberger (Shriner's Hospital for Children) for expert flow cytometric analyses. Recombinant cyclin E adenovirus was a kind gift from Dr. Gustavo Leone (Ohio State University). We are grateful to Dr. Timothy Kowalik (University of Massachusetts) for providing recombinant p16ink4a adenovirus.

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