Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage

Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2'-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.Natural Sciences and Engineering Research Council of Canada (NSERC); European Union [PCOFUND-GA-2009-246542]; Foundation for Science and Technology of Portugal; Beatrice Hunter Cancer Research Institute; Terry Fox Foundationinfo:eu-repo/semantics/publishedVersio

Radiation-induced G1 arrest is not defective in fibroblasts from Li-Fraumeni families without TP53 mutations

Radiation-induced G1 arrest was studied in four classes of early passage skin fibroblasts comprising 12 normals, 12 heterozygous (mut/wt) TP53 mutation-carriers, two homozygous (mut/–) TP53 mutation-carriers and 16 strains from nine Li-Fraumeni syndrome or Li-Fraumeni-like families in which no TP53 mutation has been found, despite sequencing of all exons, exon–intron boundaries, 3′ and 5′ untranslated regions and promoter regions. In an assay of p53 allelic expression in yeast, cDNAs from these non-mutation strains behaved as wild-type p53. Using two different assays, we found G1 arrest was reduced in heterozygous strains with mis-sense mutations and one truncation mutation, when compared to the range established for the normal cells. Heterozygous strains with mutations at splice sites behaved like normal cells, whilst homozygous (mut/–) strains showed either extremely reduced, or no, arrest. Strains from all nine non-mutation families gave responses within the normal range. Exceptions to the previously reported inverse correlation between G1 arrest and clonogenic radiation resistance were observed, indicating that these phenotypes are not strictly interdependent. © 1999 Cancer Research Campaig

Abrogation of the radiation-induced G2 checkpoint by the staurosporine derivative UCN-01 is associated with radiosensitisation in a subset of colorectal tumour cell lines

Ionising radiation is commonly used in the treatment of colorectal cancer. Tumour cells with mutant p53 undergo cell cycle arrest at G2/M after ionising radiation and evidence suggests that abrogation of this G2 arrest can lead to a premature, aberrant mitosis, thus enhancing ionising radiation-induced cell killing. The G2 checkpoint inhibitor UCN-01 was thus investigated to determine whether it would abrogate the G2 checkpoint induced by 5 Gy ionising radiation in a range of colorectal tumour cell lines. Data presented show that, at doses that are alone non-toxic to the cells, UCN-01 inhibits the ionising radiation-induced G2 checkpoint in five colorectal tumour cell lines with mutant p53. The ability of UCN-01 to sensitise cells to ionising radiation-induced growth inhibition and apoptosis was also investigated and UCN-01 was found to radiosensitise two out of five cell lines. These results were confirmed by long-term colony forming efficiency studies. These results demonstrate that abrogation of the ionising radiation-induced G2 checkpoint is not necessarily associated with sensitisation to ionising radiation, however, some colorectal tumour cell lines can be radiosensitised by UCN-01. Although the mechanism of radiosensitisation is not clear, this may still be an important treatment strategy

Dominant negative knockout of p53 abolishes ErbB2-dependent apoptosis and permits growth acceleration in human breast cancer cells

We previously reported that the ErbB2 oncoprotein prolongs and amplifies growth factor signalling by impairing ligand-dependent downregulation of hetero-oligomerised epidermal growth factor receptors. Here we show that treatment of A431 cells with different epidermal growth factor receptor ligands can cause growth inhibition to an extent paralleling ErbB2 tyrosine phosphorylation. To determine whether such growth inhibition signifies an interaction between the cell cycle machinery and ErbB2-dependent alterations of cell signalling kinetics, we used MCF7 breast cancer cells (which express wild-type p53) to create transient and stable ErbB2 transfectants (MCF7-B2). Compared with parental cells, MCF7-B2 cells are characterised by upregulation of p53, p21WAF and Myc, downregulation of Bcl2, and apoptosis. In contrast, MCF7-B2 cells co-transfected with dominant negative p53 (MCF7-B2/Δp53) exhibit reduced apoptosis and enhanced growth relative to both parental MCF7-B2 and control cells. These data imply that wild-type p53 limits survival of ErbB2-overexpressing breast cancer cells, and suggest that signals of varying length and/or intensity may evoke different cell outcomes depending upon the integrity of cell cycle control genes. We submit that acquisition of cell cycle control defects may play a permissive role in ErbB2 upregulation, and that the ErbB2 overexpression phenotype may in turn select for the survival of cells with p53 mutations or other tumour suppressor gene defects

Deletions of the region 17p11-13 in advanced melanoma revealed by cytogenetic analysis and fluorescence in situ hybridization

The significance of the p53 tumour-suppressor gene in the oncogenesis of a variety of malignant tumours has been demonstrated over recent years. However, the role of p53 in human malignant melanoma is still unclear. Therefore, we investigated melanoma metastases from 11 patients cytogenetically and with fluorescence in situ hybridization (FISH) after short-term culture, employing a p53 region-specific probe for 17p13.1 and a probe detecting the centromere of chromosome 17. Furthermore, paraffin-embedded tissue samples from nine of these patients were investigated immunohistochemically for expression of the p53 protein. Deletions of the short arm of chromosome 17 were seen in six melanomas in cytogenetic analysis. With FISH, three malignant melanomas had clones with only one p53-allele and an additional four malignant melanomas showed a reduced number of signals at the p53 tumour-suppressor gene locus compared with signals for the centromeric region of chromosome 17. This was confirmed by immunohistochemistry. Our results suggest that the 17p11–13 region is frequently deleted in malignant melanomas and that p53 or other genes located on this band might contribute to the malignant potential of advanced melanoma. © 1999 Cancer Research Campaig

Elderly Japanese women with cervical carcinoma show higher proportions of both intermediate-risk human papillomavirus types and p53 mutations

The p53 mutation has been found only in 0–6% of cervical carcinomas. In light of recent studies demonstrating that mutation of p53 gene was found in over 20% of the patients with vulvar carcinoma a disease of elderly women and a known human papillomavirus (HPV)-related malignancy, we analysed mutation of the p53 gene in 46 women with cervical carcinomas at the age of 60 or more (mean; 71 years, range; 60–96 years). The presence of HPV and its type were analysed by polymerase chain reaction (PCR)-based assay using the consensus primers for L1 region. Mutation of the p53 gene was analysed by PCR-based single-strand conformation polymorphism and DNA sequencing technique. Point mutation of the p53 gene was detected in 5 out of 46 (11%) cervical carcinomas: 1 of 17 (6%) samples associated with high-risk HPVs (HPV 16 and HPV 18) and 4 of 27 samples (15%) with intermediate-risk HPVs (P = 0.36) whereas no mutation was found in 2 HPV negative cases. The mutated residues resided in the selective sequence known as a DNA-binding domain. The immunohistochemistry revealed the overexpression in cancer tissues positive for p53 mutation. All of the observed mutations of the p53 gene were transition type, suggesting that the mutation may be caused by endogenous mutagenesis. Although falling short of statistical significance reduces the strength of the conclusion, data presented here imply that p53 gene mutation, particularly along with intermediate-risk HPV types, may constitute one pathogenetic factor in cervical carcinoma affecting elderly women. © 1999 Cancer Research Campaig

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease

p53 status and response to radiotherapy in rectal cancer: a prospective multilevel analysis

The aim of this study was to evaluate, in a prospective study, the predictive role of p53 status analysed at four different levels in identifying the response to preoperative radiotherapy in rectal adenocarcinoma. Before treatment, 70 patients were staged and endoscopic forceps biopsies from the tumour area were taken. p53 status was assessed by total cDNA sequencing, allelic loss analysis, immunohistochemistry, and p53 antibodies. Neoadjuvant treatment was based on preoperative radiotherapy or radiochemotherapy. Response to therapy was evaluated after surgery by both pathologic downstaging and histologic tumour regression grade. In all, 35 patients (50.0%) had p53 gene mutations; 44.4% of patients had an allelic loss; nuclear p53 overexpression was observed in 39 patients (55.7%); and p53 antibodies were detected in 11 patients (16.7%). In the multilevel analysis of p53 status, gene mutations correlated with both nuclear protein overexpression (P<0.0001) and loss of heterozygosity (P=0.013). In all, 29 patients (41.4%) were downstaged by pathologic analysis, and 19 patients (29.2%) were classified as tumour regression grade 1. Whatever the method of evaluation of treatment response, no correlation between p53 alterations and response to radiotherapy was observed. Our results do not support the use of p53 alterations alone as a predictive marker for response to radiotherapy in rectal carcinoma

Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1