To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.
Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.
The relationship between dNTP levels and DNA synthesis was investigated using ␣ factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G 1 /S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G 1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37°C before being released to 25°C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G 1 levels. Accumulation of dNTPs at G 1 /S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25°C were able to replicate when subsequently switched to 37°C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold. Hydroxyurea (HU)1 is a potent inhibitor of the enzyme ribonucleotide reductase (RNR) and inhibits DNA replication in a wide variety of cells, including Saccharomyces cerevisiae (1). The simplest explanation for HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. HU treatment has been shown to reduce the purine dNTP pools in a variety of mammalian cells (2-7); however, conflicting data exist concerning its modulation of pyrimidine dNTP pool levels. Furthermore, even for purine dNTPs, HU has only rarely been shown to cause a complete depletion of the dGTP or dATP pools (2-4). More commonly, HU results in only partial depletion of the purine dNTP pools (5-7). The complicated, often reciprocal, changes in individual dNTP pools that occur in HU-treated mammalian cells may be due to the compensatory activities of deoxyribonucleotide salvage pathways in higher eukaryotes. Budding yeast offers a simpler system in which to study the mechanism by which HU affects replication. Yeast possess no deoxyribonucleoside kinase activities, and thus deoxyribonucleotide synthesis is entirely dependent on ribonucleotide reductase. Also, yeast can easily be synchronized in G 1 using mating pheromone, and the availability of several temperature-sensitive cdc mutations allows cell cycle progression to be reversibly halted at specific points throughout the cell cycle (8). Reciprocal switch experiments in yeast have ordered the execution point of several cdc genes with respect to the HU-sensitive step during the cell cycle (9). Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the gene...
In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.
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 Sphase 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) 1 functions as a negative regulator of cell cycle transitions (1-5). Due to its frequent inactivation in tumors (Ͼ60%), 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 (1-5). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (6). 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 (3,7,8). This activity of RB is critical for cell cycle inhibition. In G 0 and early G 1 , 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 (9). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (1-5).Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (6,10,11). Consistent with the role of RB as a repressor of E2F, in disparate settings the expression/activity of cyclin E, cy...
Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21 CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21 CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21 CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.
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