Fragiles sites and cancer : molecular mapping of aphidicolin-induced hotspots of breakages at the human common fragile site FRA11D

Abstract

Cancer is attributed to the genomic rearrangements accumulated in a normal cell leading to its limitless replication potential. There are different causes of cancer including radiation, smoking and alcohol consumption that can induce different kind of mutations such as substitution, insertion, duplication, translocation and deletion. Positive mutations in protooncogenes and negative mutations in tumor suppressor genes are at the origin of the oncogenesis process. The frequencies of these mutations vary considerably along nucleotide sequences but some mutations often concentrate at certain positions called hotspots. More than 50 % of hotspots of breakages in cancer colocalize with fragile sites (FS). These sites are non random and non stained gaps and breaks observed in specific loci of metaphase chromosomes when cells are cultured under replication stress conditions. They are classified into two groups: common and rare. Rare fragile sites (RFS) are present in low frequencies in the human population and are induced by folate stress conditions and non-folate stress such as distamycin A, BrdU and FUdR. The most important RFS is FRAXA which is associated with the fragile X syndrome. Common fragile sites (CFS) are present in all individuals; FRA3B and FRA16D are well known examples that are related to several types of Cancer. CFS are induced by calyculin A, folate and thymidylate stress, 5- azacytidine and low doses of aphidicolin (APC), an inhibitor of DNA polymerases α, δ and ε. The relationship between CFS and cancer highlights the importance of these sites in cancer development and suggests that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. We aim in our study to localize at the molecular level the hotspots of breakages at the human common fragile site FRA11D, cytogenetically mapped to the chromosome band 11p14, induced by APC using Fluorescent in situ hybridization technique (FISH). Lymphocytes isolated from three healthy volunteers were treated with different concentrations of APC to determine the optimal concentration that induces efficiently FS in these donors. The APC concentration of 0.2 µg/ml showed the highest number of breakages on metaphase chromosomes without severe loss of the genetic material. Bacterial Artificial Chromosome clones to be used as probes in the FISH experiments and corresponding to our region of interest were verified by Polymerase Chain Reaction, extracted and labeled. In the FISH results, we isolated two hotspots of breakage at the level of the clones 283H3 and 1L12 with 62.9 % and 61.3 % mean percentages of "in" signals respectively. The first clone 283H3 covers the mucin 15 gene, a member of a family widely associated with cancer. The second clone 1L12 is localized at the interface of the late and early replicating bands 11p14.1 and 11p14.2 respectively, a result that meets previous study correlating the location of CFS with the junction of chromosomal bands showing different replication patterns. This molecular characterization of FRA11D is an essential step to determine the origin of genomic rearrangements observed in this region in several clinical cases.