A high resolution genomic portrait of bladder cancer: correlation between genomic aberrations and the DNA damage response.

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho-epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (γH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n=319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (P<0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6-3.8) and higher T stage (P<0.05). Univariate analysis confirmed this association (P=0.006) in an independent cohort (n=241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

Genomic instability in induced stem cells.

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.

Evaluation of claspin as a proliferation marker in human cancer and normal tissues.

Claspin is a nuclear protein involved in DNA replication and the DNA damage response. Its structural and functional properties suggest that it may represent a potentially useful proliferation marker. To this end, a monoclonal antibody was generated and the expression of claspin was investigated in normal fibroblasts and various cancer cell lines, as well as in tumour and normal tissues from patients with primary epithelial carcinomas. Immunoblotting analysis confirmed the specificity of the antibody, while immunohistochemistry demonstrated its applicability in archival material. In normal cells and tissues, claspin expression was weak, whereas increased levels were observed in cancer cell lines and tumour specimens. Claspin staining correlated strongly with Ki67 staining in both normal (p < 0.001) and tumour tissues (p < 0.001). However, the labelling index (LI) of claspin was consistently lower than that of Ki67, suggesting that claspin expression may be limited to a narrower part of the cell cycle. Co-localization assays with cyclin A and cell synchronization experiments indicated that claspin expression coincides with the S phase. Interestingly, the relative increase of the claspin LI in tumour samples compared with normal tissues was significantly higher (14-fold) than that of the Ki67 LI (five-fold), suggesting that claspin may be a more sensitive marker of aberrant proliferation.

Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest.

Ionizing radiation (IR) induces an increase in the levels and activity of the p53 tumor suppressor protein. The increased activity is attributed to IR-induced posttranslational modifications, some of which regulate the interaction of p53 with other proteins. One of these modifications is dephosphorylation of Ser(376), which leads to association of p53 with 14-3-3 proteins. To establish the significance of this interaction, we examined the function of mutant p53 proteins that do not interact with 14-3-3 proteins in vivo. These p53 mutants retained sequence-specific DNA binding activity. However, their ability to activate transcription of the endogenous p21/waf1/cip1 gene and to induce G(1) arrest was compromised, suggesting that the dephosphorylation of Ser(376) and the association of p53 with 14-3-3 proteins contribute to the activation of p53 in response to IR.

Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases.

Cellular DNA damage causes stabilization and activation of the tumor suppressor and transcription factor p53, in part by promoting multiple covalent modifications of the p53 protein, including acetylation. We investigated the importance of acetylation in p53 function and the mechanism by which acetylation influences p53 activity. Acetylation site substitutions reduced p53-dependent transcriptional induction and G1 cell cycle arrest. Chromatin immunoprecipitation analysis of the endogenous p21 promoter showed increased association of p53, coactivators (CBP and TRRAP), and acetylated histones following cell irradiation. Results with acetylation-defective p53 demonstrate that the critical function of acetylation is not to increase the DNA binding affinity of p53 but rather to promote coactivator recruitment and histone acetylation. Therefore, we propose that an acetylation cascade consisting of p53 acetylation-dependent recruitment of coactivators/HATs is crucial for p53 function.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.

Chfr defines a mitotic stress checkpoint that delays entry into metaphase.

Chemicals that target microtubules induce mitotic stress by affecting several processes that occur during mitosis. These processes include separation of the centrosomes in prophase, alignment of the chromosomes on the spindle in metaphase and sister-chromatid separation in anaphase. Many human cancers are sensitive to mitotic stress. This sensitivity is being exploited for therapy and implies checkpoint defects. The known mitotic checkpoint genes, which prevent entry into anaphase when the chromosomes are not properly aligned on the mitotic spindle, are, however, rarely inactivated in human cancer. Here we describe the chfr gene, which is inactivated owing to lack of expression or by mutation in four out of eight human cancer cell lines examined. Normal primary cells and tumour cell lines that express wild-type chfr exhibited delayed entry into metaphase when centrosome separation was inhibited by mitotic stress. In contrast, the tumour cell lines that had lost chfr function entered metaphase without delay. Ectopic expression of wild-type chfr restored the cell cycle delay and increased the ability of the cells to survive mitotic stress. Thus, chfr defines a checkpoint that delays entry into metaphase in response to mitotic stress.

Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.

Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G(1) arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1).

p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.

p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5-15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs.

Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage.

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53-Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.

Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.

Involvement of a p53-dependent pathway in rubella virus-induced apoptosis.

In light of the important role of apoptotic cell death in the pathogenesis of several viral infections, we asked whether the cytopathogenicity evoked by rubella virus (RV) might also involve apoptotic mechanisms. The To-336 strain of RV induced apoptosis in Vero and RK-13 cells, but not in fibroblast cell lines. UV-inactivated RV virions did not elicit the apoptotic response, indicating that productive infection is required for the induction of cell death. Both p53 and p21 protein levels were highly elevated in RV-infected Vero cells. The level of p21 mRNA was increased, while expression of the p53 gene was unaffected by RV infection. A dominant-negative p53 mutant (p53(W248)) conferred partial protection from RV-induced apoptosis. These data implicate a p53-dependent apoptotic pathway in the cytopathogenicity of RV, thereby suggesting a mechanism by which RV exerts its teratogenic effects.

p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage.

The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53's ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression.

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins.

The p53 tumour-suppressor protein is a sequence-specific DNA-binding transcription factor that induces cell cycle arrest or apoptosis in response to genotoxic stress. Activation of p53 by DNA-damaging agents is critical for eliminating cells with damaged genomic DNA and underlies the apoptotic response of human cancers treated with ionizing radiation (IR) and radiomimetic drugs. The molecular mechanisms by which DNA damage activates p53 have not been elucidated. Both the levels of p53 protein and its affinity for specific DNA sequences increase in response to genotoxic stress. In vitro, the affinity of p53 for DNA is regulated by its carboxy-terminus. We therefore examined whether this region of p53 is targeted by DNA-damage signalling pathways in vivo. In nonirradiated cells, serines 376 and 378 of p53 were phosphorylated. IR led to dephosphorylation of Ser376, creating a consensus binding site for 14-3-3 proteins and leading to association of p53 with 14-3-3. In turn, this increased the affinity of p53 for sequence-specific DNA. Consistent with the lack of p53 activation by IR in ataxia telangiectasia (AT; refs 14,15), neither Ser376 dephosphorylation, nor the interaction of p53 with 14-3-3 proteins occurred in AT cells.

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding.

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393. The C-terminal residues 364-393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.