Retinoblastoma Tumor Suppressor Targets dNTP Metabolism to Regulate DNA Replication
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m205911200
ISSN1083-351X
AutoresSteven P. Angus, Linda J. Wheeler, Sejal A. Ranmal, Xiaoping Zhang, Michael P. Markey, Christopher K. Mathews, Erik S. Knudsen,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe 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, 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) 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 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, fluorodeoxyuridinefunctions as a negative regulator of cell cycle transitions (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). 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 (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). 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 (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 7Strobeck M.W. Knudsen K.E. Fribourg A.F. DeCristofaro M.F. Weissman B.E. Imbalzano A.N. Knudsen E.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7748-7753Crossref PubMed Scopus (209) Google Scholar, 8Zhang H.S. Gavin M. Dahiya A. Postigo A.A., Ma, D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). 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 (9Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (333) Google Scholar). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar).Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 10DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (834) Google Scholar, 11Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar). 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 (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 12Reed S.I. Cancer Surv. 1997; 29: 7-23PubMed Google Scholar, 13Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1043) Google Scholar). Throughout S phase, discrete origins of replication fire, and components of the DNA polymerase holoenzyme are sequentially recruited to these sites (14Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar). 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 (15Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (659) Google Scholar). 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 (16Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar). 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.DISCUSSIONRB-mediated cell cycle inhibition occurs in response to antimitogenic signals, DNA damage, and other cellular stresses (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). 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 (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 12Reed S.I. Cancer Surv. 1997; 29: 7-23PubMed Google Scholar, 13Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1043) Google Scholar). However, RB-mediated arrest can only be partially subverted by the ectopic expression of the CDK2 activators cyclin E and cyclin A (16Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar, 21Knudsen E.S. Buckmaster C. Chen T.T. Feramisco J.R. Wang J.Y. Genes Dev. 1998; 12: 2278-2292Crossref PubMed Scopus (190) Google Scholar, 25Chew Y.P. Ellis M. Wilkie S. Mittnacht S. Oncogene. 1998; 17: 2177-2186Crossref PubMed Scopus (74) Google Scholar, 26Lukas J. Herzinger T. Hansen K. Moroni M.C. Resnitzky D. Helin K. Reed S.I. Bartek J. Genes Dev. 1997; 11: 1479-1492Crossref PubMed Scopus (324) Google Scholar). 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 (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar). These E2F targets encompass a wide variety of cell cycle regulatory and metabolic enzymes (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 10DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (834) Google Scholar, 11Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar). 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 (8Zhang H.S. Gavin M. Dahiya A. Postigo A.A., Ma, D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 16Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar, 21Knudsen E.S. Buckmaster C. Chen T.T. Feramisco J.R. Wang J.Y. Genes Dev. 1998; 12: 2278-2292Crossref PubMed Scopus (190) Google Scholar, 25Chew Y.P. Ellis M. Wilkie S. Mittnacht S. Oncogene. 1998; 17: 2177-2186Crossref PubMed Scopus (74) Google Scholar, 41Zhang H.S. Postigo A.A. Dean D.C. Cell. 1999; 97: 53-61Abstract Full Text Full Text PDF PubMed Google Scholar, 42Lukas J. Sorensen C.S. Lukas C. Santoni-Rugiu E. Bartek J. Oncogene. 1999; 18: 3930-3935Crossref PubMed Scopus (68) Google Scholar, 43Knudsen K.E. Fribourg A.F. Strobeck M.W. Blanchard J.M. Knudsen E.S. J. Biol. Chem. 1999; 274: 27632-27641Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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 (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 12Reed S.I. Cancer Surv. 1997; 29: 7-23PubMed Google Scholar, 13Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1043) Google Scholar,15Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (659) Google Scholar). Consistent with this idea, ectopic expression of cyclins E or A can partially overcome the inhibition of DNA replication mediated by active RB alleles (16Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar, 21Knudsen E.S. Buckmaster C. Chen T.T. Feramisco J.R. Wang J.Y. Genes Dev. 1998; 12: 2278-2292Crossref PubMed Scopus (190) Google Scholar, 25Chew Y.P. Ellis M. Wilkie S. Mittnacht S. Oncogene. 1998; 17: 2177-2186Crossref PubMed Scopus (74) Google Scholar, 26Lukas J. Herzinger T. Hansen K. Moroni M.C. Resnitzky D. Helin K. Reed S.I. Bartek J. Genes Dev. 1997; 11: 1479-1492Crossref PubMed Scopus (324) Google Scholar). 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 (15Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (659) Google Scholar, 16Sever-Chroneos Z. Angus S.P. Fribourg A.F. Wan H. Todorov I. Knudsen K.E. Knudsen E.S. Mol. Cell. Biol. 2001; 21: 4032-4045Crossref PubMed Scopus (51) Google Scholar). 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 (44Bravo R. Macdonald-Bravo H. EMBO J. 1985; 4: 655-661Crossref PubMed Scopus (224) Google Scholar).The relative levels of dNTPs and the regulation of their synthesis play a critical role in DNA replication (30Mathews C.K. Prog. Nucleic Acids Res. Mol. Biol. 1993; 44: 167-203Crossref PubMed Scopus (33) Google Scholar, 32Reichard P. Annu. Rev. Biochem. 1988; 57: 349-374Crossref PubMed Scopus (625) Google Scholar, 45Mathews C.K. Ji J. Bioessays. 1992; 14: 295-301Crossref PubMed Scopus (55) Google Scholar). 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 (45Mathews C.K. Ji J. Bioessays. 1992; 14: 295-301Crossref PubMed Scopus (55) Google Scholar). For example, inhibition of RNR activity by 50% using CdA leads to marked inhibition of cell cycle progression (40Griffig J. Koob R. Blakley R.L. Cancer Res. 1989; 49: 6923-6928PubMed Google Scholar). Additionally, dNTP levels vary within S-phase of the cell cycle (46Leeds J.M. Slabaugh M.B. Mathews C.K. Mol. Cell. Biol. 1985; 5: 3443-3450Crossref PubMed Scopus (79) Google Scholar); these variations may be responsible for changes in the rate of DNA replication during S-phase (47Collins J.M. J. Biol. Chem. 1978; 253: 8570-8577Abstract Full Text PDF PubMed Google Scholar). 3S. 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 (10DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (834) Google Scholar, 11Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar, 48Slansky J.E. Farnham P.J. Bioessays. 1996; 18: 55-62Crossref PubMed Scopus (80) Google Scholar, 49Dou Q.P. Zhao S. Levin A.H. Wang J. Helin K. Pardee A.B. J. Biol. Chem. 1994; 269: 1306-1313Abstract Full Text PDF PubMed Google Scholar). 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 (10DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (834) Google Scholar). In fact, recent chromatin immunoprecipitation analyses have detected RB on the DHFR promoter at the G1/S transition (50Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell. Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (207) Google Scholar). 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 (28Schweitzer B.I. Dicker A.P. Bertino J.R. FASEB J. 1990; 4: 2441-2452Crossref PubMed Scopus (303) Google Scholar, 29Bertino J.R., Li, W.W. Lin J. Trippett T. Goker E. Schweitzer B. Banerjee D. Mt. Sinai J. Med. 1992; 59: 391-395PubMed Google Scholar, 39Pinedo H.M. Peters G.F. J. Clin. Oncol. 1988; 6: 1653-1664Crossref PubMed Scopus (792) Google Scholar). 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 (38Almasan A. Yin Y. Kelly R.E. Lee E.Y. Bradley A., Li, W. Bertino J.R. Wahl G.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5436-5440Crossref PubMed Scopus (279) Google Scholar, 51Li W. Fan J. Hochhauser D. Banerjee D. Zielinski Z. Almasan A. Yin Y. Kelly R. Wahl G.M. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10436-10440Crossref PubMed Scopus (99) Google Scholar). Specifically, Almasan et al. showed that mRNA levels of both TS and DHFR were elevated in asynchronously proliferatingRb −/− MEFs compared with wild-type MEFs (38Almasan A. Yin Y. Kelly R.E. Lee E.Y. Bradley A., Li, W. Bertino J.R. Wahl G.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5436-5440Crossref PubMed Scopus (279) Google Scholar). 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 (38Almasan A. Yin Y. Kelly R.E. Lee E.Y. Bradley A., Li, W. Bertino J.R. Wahl G.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5436-5440Crossref PubMed Scopus (279) Google Scholar, 51Li W. Fan J. Hochhauser D. Banerjee D. Zielinski Z. Almasan A. Yin Y. Kelly R. Wahl G.M. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10436-10440Crossref PubMed Scopus (99) Google Scholar). 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 (52Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar, 53Chabes A. Domkin V. Thelander L. J. Biol. Chem. 1999; 274: 36679-36683Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). 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. The retinoblastoma tumor suppressor (RB) 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 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, fluorodeoxyuridinefunctions as a negative regulator of cell cycle transitions (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). 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 (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). 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 (3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 7Strobeck M.W. Knudsen K.E. Fribourg A.F. DeCristofaro M.F. Weissman B.E. Imbalzano A.N. Knudsen E.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7748-7753Crossref PubMed Scopus (209) Google Scholar, 8Zhang H.S. Gavin M. Dahiya A. Postigo A.A., Ma, D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). 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 (9Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (333) Google Scholar). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (1Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 4Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 5Kaelin Jr., W.G. Cancer Invest. 1997; 15: 243-254Crossref PubMed Scopus (37) Google Scholar). Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (6Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 10DeGregori J. Kowalik T. Nev
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