Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study.

Common fragile sites (CFSs) are regions of the genome prone to breakage by replication inhibitors (extrinsic replication stress). Recently, we and others observed that oncogene-induced replication stress (RS) induces DNA damage from the earliest stages of cancer. Our aim was to perform a genome-wide analysis in precancerous and cancerous experimental models to examine whether allelic imbalance occurs within CFSs. Subsequently, CFSs sequence characteristics were assessed. We used a growth-factor-induced human skin hyperplasia and a H-ras-induced mouse hyperplastic urothelium as preneoplastic models, along with an inducible U2OS-CDT1(Tet-ON) cancer cell line model, all bearing established oncogene-induced RS stimuli. Human DNA was analysed with Affymetrix SNP microarrays, while mouse DNA was analysed with Nimblegen array CGH. We studied 56 aphidicolin-type CFSs and 1914 regions of control, nonfragile DNA. Our theoretical in silico analysis spanned 2.16 billion nonoverlapping bases on human chromosomes 1-22. Our results provide direct experimental evidence indicating that genomic alterations were more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. CFSs were on average less flexible than nonfragile regions, contained more guanine-cytosine (GC) and Alu sequences. Importantly, regions with loss-of-heterozygosity were also less flexible and had a higher Alu percentage.

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.

Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans.

PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.