DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.

Numerous upstream stimulatory and inhibitory signals converge to the pRb/E2F pathway, which governs cell-cycle progression, but the information concerning alterations of E2F-1 in primary malignancies is very limited. Several in vitro studies report that E2F-1 can act either as an oncoprotein or as a tumour suppressor protein. In view of this dichotomy in its functions and its critical role in cell cycle control, this study examined the following four aspects of E2F-1 in a panel of 87 non-small cell lung carcinomas (NSCLCs), previously analysed for defects in the pRb-p53-MDM2 network: firstly, the status of E2F-1 at the protein, mRNA and DNA levels; secondly, its relationship with the kinetic parameters and genomic instability of the tumours; thirdly, its association with the status of its transcriptional co-activator CBP, downstream target PCNA and main cell cycle regulatory and E2F-1-interacting molecules pRb, p53 and MDM2; and fourthly, its impact on clinical outcome. The protein levels of E2F-1 and its co-activator CBP were significantly higher in the tumour area than in the corresponding normal epithelium (p<0.001). E2F-1 overexpression was associated with increased E2F-1 mRNA levels in 82% of the cases examined. The latter finding, along with the low frequency of E2F-1 gene amplification observed (9%), suggests that the main mechanism of E2F-1 protein overexpression in NSCLCs is deregulation at the transcriptional level. Mutational analysis revealed only one sample with asomatic mutation at codon 371 (Glu-->Asp) and one carrying a polymorphism at codon 393 (Gly-->Ser). Carcinomas with increased E2F-1 positivity demonstrated a significant increase in their growth indexes (r=0.402, p=0.001) and were associated with adverse prognosis (p=0.033 by Cox regression analysis). The main determinant of the positive association with growth was the parallel increase between E2F-1 staining and proliferation (r=0.746, p<0.001), whereas apoptosis was not influenced by the status of E2F-1. Moreover, correlation with the status of the pRb-p53-MDM2 network showed that the cases with aberrant pRb expression displayed significantly higher E2F-1 indexes (p=0.033), while a similar association was noticed in the group of carcinomas with deregulation of the p53-MDM2 feedback loop. In conclusion, the results suggest that E2F-1 overexpression may contribute to the development of NSCLCs by promoting proliferation and provide evidence that this role is further enhanced in a genetic background with deregulated pRb-p53-MDM2 circuitry.

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.