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Proc Natl Acad Sci U S A. Apr 30, 2002; 99(9): 6274–6279.
PMCID: PMC122939
From the Cover
Medical Sciences

Characterization of the c-MYC-regulated transcriptome by SAGE: Identification and analysis of c-MYC target genes

Abstract

To identify target genes of the oncogenic transcription factor c-MYC, serial analysis of gene expression (SAGE) was performed after adenoviral expression of c-MYC in primary human umbilical vein endothelial cells: 216 different SAGE tags, corresponding to unique mRNAs, were induced, whereas 260 tags were repressed after c-MYC expression (P < 0.05). The induction of 53 genes was confirmed by using microarray analysis and quantitative real-time PCR: among these genes was MetAP2/p67, which encodes an activator of translational initiation and represents a validated target for inhibition of neovascularization. Furthermore, c-MYC induced the cell cycle regulatory genes CDC2-L1, Cyclin E binding protein 1, and Cyclin B1. The DNA repair genes BRCA1, MSH2, and APEX were induced by c-MYC, suggesting that c-MYC couples DNA replication to processes preserving the integrity of the genome. MNT, a MAX-binding antagonist of c-MYC function, was up-regulated, implying a negative feedback loop. In vivo promoter occupancy by c-MYC was detected by chromatin immunoprecipitation for CDK4, Prohibitin, MNT, Cyclin B1, and Cyclin E binding protein 1, showing that these genes are direct c-MYC targets. The c-MYC-regulated genes/tags identified here will help to define the set of bona fide c-MYC targets and may have potential therapeutic value for inhibition of cancer cell proliferation, tumor-vascularization, and restenosis.

The proto-oncogene c-MYC is at the center of a transcription factor network that regulates cellular proliferation, replicative potential, growth, differentiation, and apoptosis (14). Expression of c-MYC is rapidly induced by a diverse catalog of mitogens, and down-regulated during differentiation (1). Deregulation of c-MYC expression has been implicated in the genesis of diverse human cancers (1). The c-MYC gene encodes a nuclear transcription factor with a C-terminal basic/helix-loop-helix/leucine-zipper (bHLHzip) domain and an N-terminal transactivation domain. The HLHzip motif allows dimerization with the bHLHzip-protein MAX, which is a prerequisite for specific binding to DNA at E-box sequences (5′-CA(C/T)G(T/C)G-3′) in the vicinity of target gene promoters (5). The MAX protein has alternative dimerization partners (MAD-1, -3, and -4, MXI1, and MNT), which represent antagonists of c-MYC function (1, 3). Dimerization with MAX and specific binding to DNA are required for induction of cell cycle progression, apoptosis, and transformation by c-MYC (1, 6), suggesting that c-MYC exerts its oncogenic effects by transactivation of target genes via E-boxes. However, transcriptional repression has also been implicated in transformation by c-MYC (1), although the mechanisms involved are less understood.

The nature of c-MYC target genes is expected to ultimately elucidate why c-MYC is a potent activator of carcinogenesis. Intriguingly, other mitogenic transcription factors (e.g., E2F-1) do not display significant transforming activity, although they have similar effects on cell cycle progression. Furthermore, bona fide c-MYC target genes will be instrumental for understanding the molecular mechanisms of c-MYC-mediated gene regulation, as exemplified by recent studies on c-MYC-mediated changes in histone acetylation (7, 8).

A number of c-MYC-regulated genes have been identified by using microarray analysis of cell lines with conditional c-MYC alleles (912) or tumor cells expressing various c-MYC levels (13). However, identification of c-MYC targets in established cell lines may be obscured by genetic and epigenetic changes, which are selected for during passaging or immortalization and affect c-MYC target gene expression. To identify c-MYC target genes, we therefore decided to analyze global gene expression shortly after adenoviral transfer of an ectopic c-MYC allele into primary human cells.

Materials and Methods

Tissue Culture.

Human umbilical vein endothelial cells (HUVEC) and their respective media were obtained from Clonetics (San Diego). For serum starvation, HUVEC were kept in media containing 0.25% FBS for 24 h. P493-6 cells, RAT1A fibroblast (subclone TGR-1), and c-Myc−/−-RAT1A (subclone HO15.19) were maintained as described previously (12, 14).

Serial Analysis of Gene Expression (SAGE) and Microarray Analysis.

Total RNA was prepared by using CsCl-gradient ultracentrifugation of guanidinium isothiocyanate-lysed cells. Poly(A) mRNA was obtained by using the Messagemaker kit (GIBCO/BRL). SAGE was performed as described previously (15, 16). For data and statistical analysis, the sage-2000 software was used. For microarray analysis, 600 ng poly(A) mRNA was converted to cDNA with incorporation of Cy3- or Cy5-labeled dNTPs. Hybridization to arrays coated on glass, quality control, and normalization were performed by IncyteGenomics (Palo Alto, CA). The “Human Unigene1” microarray contained probes for 8,392 annotated genes/expressed sequence tag (EST) clusters and 74 nonannotated genes/ESTs (probes are derived from the 5′ cDNA region), whereas the “Human Drug Target” array represented 8,031 unique annotated genes/EST clusters and 207 nonannotated genes/EST clusters (probes are derived from the 3′ cDNA region). Because of an overlap of 1,261 probes, the two arrays represented 15,443 unique human mRNA species.

Northern Blot Analysis.

RNA was isolated by using the RNAgents-kit (Promega). Probes directed against the 3′-untranslated region of the respective mRNAs were generated by PCR using ESTs as templates and subsequent gel purification. Hybridizations were performed in QuickHyb, following the manufacturer's instructions (Stratagene).

Quantitative Real-Time PCR (qPCR).

qPCR was performed by using the LightCycler and the FastStart DNA Master SYBR Green 1 kit (Roche Applied Science). For qPCR of cDNA, primer pairs were designed to generate intron-spanning products of 110–335 bp (except 408 bp for PHB). Primer sequences are available as Table 4 (which is published as supporting information on the PNAS web site, www.pnas.org). cDNA was generated by using the RevertAid First Strand cDNA synthesis kit (MBI Fermentas, St. Leonrod, Germany). The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis. Each primer pair was tested with a logarithmic dilution of a cDNA mix to generate a linear standard curve (crossing point CP plotted vs. log of template concentration), which was used to calculate the primer pair efficiency (E = 10(−1/slope)). Elongation factor 1α (ELF1α) mRNA was used as an external standard because its expression was not altered significantly by c-MYC as detected by SAGE and Northern blot analysis (data not shown). For data analysis, the second derivative maximum method was applied, and induction of a cDNA species (geneX) was calculated according to Pfaffl (17):

equation M1
1

Chromatin Immunoprecipitation.

HeLa cells (4 × 108) were fixed in 1% formaldehyde. Chromatin was sheared to an average size of 500 bp by sonication in the presence of glass beads [3 times for 30 s at continuous maximum power setting; Bandelin Sonopuls HD 70 w. MS73 Sonotrode (3 mm), Berlin, Germany]. Lysates corresponding to 1.33 × 108 HeLa cells were rotated at 4°C for 12 h with 5 μg of polyclonal antibodies specific for c-MYC (sc-764, Santa Cruz Biotechnology), CDC25C (as an irrelevant antibody, sc-327), or no antibody. Washing and reversal of cross-links was performed as described (18). Precipitated DNA fragments were quantified by using qPCR, with primer pairs flanking E-boxes in the respective promoters (PCR-I to -VI; see Fig. Fig.22A) or primer pairs generating fragments located in more than 1.5 kbp distance 5′ or 3′ to the binding sites (PCR-A to -F; Fig. Fig.22A). A genomic fragment corresponding to ELF1α was used as an external standard. Enrichment of a DNA fragment putatively bound to c-MYC (gene X) was calculated as follows:

equation M2
2

Figure 2
In vivo binding of c-MYC to promoter sequences. (A) Maps of c-MYC-induced genes indicating the positions of E-boxes (black rectangles). Exons are represented with ORFs (gray rectangles). Arrows indicate the transcription start site (TSS), which corresponds ...

Results

SAGE After Ectopic Expression of c-MYC.

SAGE libraries were generated from RNA isolated 12 h after viral infection of serum-starved HUVEC with Ad-MYC or as a control, Ad-GFP. At this time point, levels of ectopic c-MYC protein had reached maximum levels (data not shown). With SAGE, unique 10-bp tags are isolated from cDNA, concatenated, and sequenced (16). The abundance of a given tag in the SAGE library corresponds to the expression level of the corresponding gene. Combined, the two SAGE libraries (Ad-MYC-lib.: 55,430 tags; Ad-GFP-lib.: 37,048 tags) represented 27,283 unique tags, which corresponded to ≈8,500 different mRNAs, when only tags that occurred at least twice were taken into account. Because c-MYC is not sufficient to induce cell cycle reentry in HUVEC (15), the detected changes in gene expression are not secondary to c-MYC-induced cell cycle progression. According to a statistical analysis using a Monte Carlo simulation, 476 tags were significantly, differentially regulated (P < 0.05), with 216 tags induced and 260 tags repressed by c-MYC. Examples of previously described c-MYC target genes corresponding to induced tags include HSP70 (43:0 tags), ODC (14:4 tags), CAD (9:2 tags), LDH-A (9:2 tags), eIF4E (11:2 tags), and α-prothymosin (36:12 tags). The complete set of detected SAGE tags is provided online at http://www.biochem.mpg.de/hermeking/mycsage.html.

Validation of SAGE Results.

To confirm the SAGE results, two microarray analyzes were performed (see Materials and Methods). Of 153 annotated cDNAs, which were significantly induced by c-MYC according to SAGE, 102 were represented by probes on the microarrays. Forty-two of these transcripts showed a significant induction as detected by microarray analysis (Table (Table1).1). This result most likely represents an underestimation of correct tag-to-gene assignments, which may be due to a lower sensitivity of microarray analysis vs. the SAGE method. For a subset of interesting genes, the expression data obtained by SAGE were confirmed by using qPCR, because the sensitivity of qPCR exceeds that of microarray hybridizations (Fig. (Fig.11A, Table Table1).1). In the case of MSH2 and BRCA1, qPCR analysis demonstrated that a negative result by microarray analysis does not exclude regulation by c-MYC.

Table 1
Identification and confirmation of c-MYC-induced genes
Figure 1
Analysis of c-MYC-induced genes by qPCR. Serum-starved HUVEC were infected with Ad-MYC, Ad-MADMYC, or Ad-GFP adenovirus. For restimulation, serum was added 4 h after infection. RNA was isolated 12 h after adenoviral infection or restimulation with serum. ...

Classification of c-MYC Target Genes.

The 53 confirmed tag-to-gene assignments were grouped into functional classes (Table (Table1).1). The complete set of 476 tags significantly, differentially regulated by c-MYC is listed in Tables 2 and 3 (which are published as supporting information on the PNAS web site), with assignment to cDNAs and functional classes. Most of the functional classes are in agreement with previous studies aimed to identify c-MYC target genes (913). However, the genes listed in Table 1–3 significantly increase the number of known c-MYC-regulated genes involved in these processes and also indicate pathways not previously linked to c-MYC: e.g., the induction of a number of genes involved in DNA repair (BRCA1, MSH2, and APEX) was unexpected and points to a role of c-MYC in the coordination of synthesis and repair of DNA.

Requirement of c-MYC for Serum-Induced Expression.

c-MYC is an immediate early growth response gene and mediates changes in gene expression observed after serum stimulation. Therefore, we asked whether the c-MYC-regulated genes identified here would be induced in a c-MYC-dependent manner after restimulation of mitogen-deprived HUVEC. As shown in Fig. Fig.11B, restimulation of serum-starved cells led to induction of all genes tested as detected by qPCR. Furthermore, a dominant-negative mutant of c-MYC (MADMYC), in which the transactivation domain was replaced by a histone-deacetylase recruiting Sin3-domain (according to ref. 19), was sufficient to block the serum induction of these genes, implying that the c-MYC gene mediates the effect of serum on the expression of these genes (Fig. (Fig.11B).

Detection of in Vivo Promoter Occupation by c-MYC.

For selected, c-MYC-induced genes, analysis of the genomic sequence revealed several E-boxes upstream of their respective transcription start sites (TSS; Fig. Fig.22A). To determine whether the endogenous c-MYC protein binds to these E-boxes and therefore directly regulates these genes, in vivo chromatin immunoprecipitation (ChIP) assays were performed (Fig. (Fig.22B). The previously described occupancy of the CAD promoter by c-MYC (18) served as a positive control: a DNA fragment spanning two E-boxes was enriched after coimmunoprecipitation of chromatin with a c-MYC-specific antibody in three independent ChIP assays, whereas a fragment located 9,595 bp upstream of the CAD TSS was not enriched (Fig. (Fig.22 A and B). Cyclin dependent kinase (CDK4) has been identified and characterized as a c-MYC-regulated gene previously (15). However, in vivo promoter occupancy by c-MYC has not been shown for CDK4. The precipitation with a c-MYC-specific antibody led to an enrichment of a CDK4 promoter fragment spanning three E-boxes, whereas sequences 9,066 bp downstream of the TSS were not enriched (Fig. (Fig.22 A and B). These results demonstrate that c-MYC is present at the CDK4 promoter in vivo and directly regulates CDK4 expression. In the MNT gene, three candidate c-MYC binding sites are present (Fig. (Fig.22A). For the E-box at −828 bp, occupancy with c-MYC was detected, implying that c-MYC directly regulates the expression of MNT (Fig. (Fig.22B). Consistent with a direct regulation by c-MYC the PHB, CEB1, and Cyclin B1 genes harbor E-boxes in the vicinity of their promoters, which are occupied by c-MYC in vivo (Fig. (Fig.22 A and B).

Regulation by c-MYC in Heterologous Systems.

To prove that putative c-MYC targets are bona fide, global target genes of c-MYC, it is necessary to show that regulation by c-MYC occurs in different cell types and species. Therefore, we tested whether some of the identified genes were regulated in the human B cell line P493-6, which harbors a tetracycline-regulated c-MYC allele (described in ref. 12). As shown in Fig. Fig.33A, removal of tetracycline led to induction of c-MYC mRNA. The induction of B23 and PHB mRNA lagged behind by 2–4 h, which is consistent with direct regulation by the newly synthesized c-MYC protein. Eight c-MYC-regulated genes identified in HUVEC were analyzed in the conditional B cell system by using qPCR (Fig. (Fig.33B). Only BRCA1 and 14-3-3epsilon showed no further induction by c-MYC (Fig. (Fig.33B). One possible explanation may be that these genes are already up-regulated by other factors in the P493-6 cells.

Figure 3
Analysis of c-MYC-regulated genes identified in HUVEC in a human B cell line. P493-6 cells, which harbor a c-MYC gene under control of a tetracycline-responsive element (12), were cultured in the presence of tetracycline (0.1 μg/ml). After ...

Another system that allows the validation of regulation by c-MYC are RAT1A fibroblasts in which c-Myc has been removed by homologous recombination (14, 20). In the parental RAT1A cells, induction of PHB, MSH2, and BRCA1 mRNA was detectable 6–9 h after restimulation whereas, in c-Myc-deficient RAT1A cells, no significant increase in mRNA levels was detectable up to 24 h after restimulation (Fig. (Fig.4).4). Therefore, c-Myc is required for normal induction of PHB, MSH2, and BRCA1 by serum. In summary, c-MYC regulation of most genes analyzed was conserved between species and cell-types, suggesting that the majority of the genes identified here are general targets of c-MYC.

Figure 4
Requirement of c-Myc for normal induction of BRCA1, MSH2, and PHB after serum stimulation. c-Myc+/+ and c-Myc−/− RAT1A cells were maintained in DMEM containing 0.25% calf serum for 48 h. After stimulation with 8% ...

Discussion

The detection of a large number of genes regulated by c-MYC by using SAGE supports the notion that c-MYC is a central regulator that activates diverse cellular processes associated with progression through the cell cycle and synthesis of cellular components in preparation of cell division. In this study, we focused on genes whose induction by c-MYC could be confirmed by microarray and/or qPCR analysis. However, many of the additional genes detected by SAGE, which are depicted in Tables 2 and 3, may be directly regulated by c-MYC.

Because target gene expression in established cell lines may be obscured by genetic and epigenetic changes acquired during ex vivo passaging, primary human cells were used for the SAGE screen. An example justifying this rationale may be the CDK4 gene, which is expressed at high levels in NIH 3T3 cells and cannot be induced further after activation of a conditional MycER allele in this cell line (15). However, CDK4 is induced by c-MYC in primary HUVECs, conditionally transformed B cells (15), and RAT1A cells (9, 15). Moreover, endogenous c-MYC binds to the E-boxes present in the CDK4 promoter (this study), showing that CDK4 is a direct target gene of c-MYC.

The comparison of SAGE with microarray analysis revealed a limited overlap in the results. This overlap may be explained by the inherent constraints of microarray hybridizations: e.g., nonspecific cross-hybridization of homologous gene family members. Presumably, the higher sensitivity of SAGE further increased the difference between the assays: in general, tags with low numbers could be verified by qPCR and rarely showed induction in the microarray analysis (e.g., Cyclin B1, CDC2-L1, PCAF, and MNT).

The genes identified by SAGE point to a number of interesting new biological functions of c-MYC and further substantiate the role of c-MYC in previously identified pathways regulated by c-MYC: direct induction of CDC2-L1 and Cyclin B1 by c-MYC is consistent with an activating role of c-MYC not only in G1/S progression, but also in the G2 phase of the cell cycle. Consistent with such a role, c-Myc-deficient RAT1A cells show a prolonged G2 phase (14). Although the function of the c-MYC-induced CEB1 gene is still obscure, the reported association between CEB1 and Cyclin E suggests a role during cell cycle progression (21). Consistent with the direct induction of MNT by c-MYC reported here, it has been shown previously that expression of MNT correlates with c-MYC expression (22). Because MNT is a functional antagonist of c-MYC, the induction of MNT may constitute a negative feedback loop that serves to restrict the transactivation activity of c-MYC. Another negative feedback mechanism may be represented by the induction of hnRNP-D, which mediates the degradation of mRNAs encoding cytokines and proto-oncogenes (e.g., c-MYC) through AU-rich elements in their 3′-untranslated regions (23). Conversely, induction of TIP48 by c-MYC suggests the existence of a feed-forward loop, because binding of the RNA-helicase TIP48 to a conserved region in the c-MYC transactivation domain (MYC-BOXII) is necessary for c-MYC-mediated transformation and transactivation (24). In addition, TIP48 may be a cofactor for other transcription factors that are activated during cell cycle progression. c-MYC presumably mediates the induction of transcriptional cofactors and components of DNA-Polymerase II (e.g., PolII subunits F and B) to facilitate the overall increase in transcriptional activity during G1/S transition. Induction of p300/CBP-associated factor (PCAF), a transcriptional cofactor with histone acetyltransferase activity that binds to p300/CBP (25), may also contribute to an overall increase in transcriptional activity. Lactate dehydrogenase A (LDHA) is a known c-MYC target gene (Table (Table1)1) that links c-MYC to the activation of glycolysis (26). Shim et al. (26) suggested that deregulated c-MYC expression is central to the activation of glycolysis, which is found in most tumor cells (also known as the Warburg effect). Here, three additional c-MYC-induced genes encoding enzymes of the glycolytic pathway were identified: LDHB, which forms the tetrameric LDH-complex together with two LDHA molecules, phosphofructokinase and glucose phosphate isomerase.

The regulation of BRCA1 and MSH2 by c-MYC is consistent with the following observations: BRCA1 is up-regulated during G1/S transition of restimulated cells (27) and expression of MSH2 in epithelia is limited to the proliferating compartment (28). BRCA1 and MSH2 proteins are components of a large DNA damage repair complex, BASC (BRCA1-associated genome surveillance complex), that contains several other tumor-suppressor-gene products (29). For p53, which is a major sensor of DNA damage and mediator of responses to genotoxic insults, transcriptional and posttranscriptional effects mediated by c-MYC have been described previously (30, 31). Taken together, c-MYC seems to couple DNA synthesis, which presumably elevates the frequency of endogenous DNA damage, to the induction of genes involved in DNA repair, some of which function as tumor suppressor genes (BRCA1, MSH2, and P53). This finding is in agreement with observations from Saccharomyces cerevisiae, where DNA repair genes are required during DNA replication to preserve the integrity of the genome (32). Genetic inactivation of genes involved in DNA repair in the presence of an activated c-MYC gene may therefore lead to the bypass of DNA damage checkpoints, accumulation of mutations, and emergence of tumor cells.

c-MYC induces genes encoding components of the permeability transition pore complex (PTPC): up-regulation of the benzodiazipin-receptor gene was detected in this study, whereas induction of VDAC and cyclophilin F by c-MYC was identified previously (9, 11) and in this study (Table (Table1).1). The coordinated up-regulation of components of the PTPC may lower the threshold for cytochrome c release from mitochondria and presumably contributes to the sensitization toward apoptotic stimuli, which is generally observed after c-MYC expression (reviewed in refs. 1 and 4). In agreement with this scenario, studies with c-Myc-deficient cells provide evidence that c-MYC affects the competence for apoptosis at the mitochondrial level (33).

Prohibitin (PHB) has been implicated in tumor suppression and senescence (34). PHB protein seems to function as a mitochondrial chaperone (35). An antiproliferative capacity has been assigned to the 3′-nontranslated region of the 1,826-nt PHB transcript (36). Interestingly, PHB is consistently expressed at higher levels in tumor cells (34), which may be due to the transactivation by c-MYC observed in this study. The up-regulation of the nuclear transport factor 2 (NTF2) gene suggests that nuclear import of nuclear localization signal-containing proteins is augmented after activation of c-MYC. c-MYC has been implicated in the control of translation previously because it directly induces eIF4E and eIF-2α (37). Our results imply, that c-MYC mediates the serum induction of MetAP2/p67. After mitogenic stimulation, MetAP2/p67 is induced and binds to eIF-2, thereby protecting it from inhibitory phosphorylation (38). Interestingly, the anti-angiogenic substance fumagillin and its derivative TNP-470 both inhibit neovascularization through irreversibly binding to MetAP2 (39). Because TNP-470 has proven to be effective in animal model studies, phase III antitumor clinical trials have been initiated. This example shows that certain target genes of c-MYC may represent valuable therapeutic targets for interfering with endothelial cell proliferation during tumor vascularization and restenosis. Because of the widespread activation of c-MYC in cancer, c-MYC target genes may be useful candidates for inhibition of tumor cell proliferation in general.

Supplementary Material

Supporting Tables:

Acknowledgments

We thank Bert Vogelstein, Ken Kinzler, and Axel Ullrich for their support, and Carlo Rago for expert technical assistance. We also thank Dirk Eick and John Sedivy for providing cell lines, Peggy Farnham and Julie Wells for technical advice, and Gregory J. Cost and members of the lab for comments.

Abbreviations

SAGE
serial analysis of gene expression
HUVEC
human umbilical vein endothelial cell
EST
expressed sequence tag
qPCR
quantitative real-time PCR
TSS
transcription start site

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

See commentary on page 5757.

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