Fragile sites and cancer : Isolation of two calyculin A-induced hotspots of breakages at the human common fragile sequence FRA11D

Abstract

Cancer cells are characterized by their ability to grow and divide at an unregulated, quickened and indefinite rate. Point mutations in tumor suppressor genes and protooncogenes, changes in chromosomes copy number and chromosomes structure are various forms of genome instability which constitute a hallmark of cancer cells. Fragile sites, the nonrandomly located gaps or breaks observed on metaphase chromosomes, are located within genes that are frequently deleted in human cancers. Two parameters are used in their classification: frequency in the population and chemistry of induction. Less than 5% of individuals possess rare fragile sites which are mostly folate stress sensitive. On the other hand, common fragile sites are found in all individuals and they are induced by low doses of aphidicolin, a partial inhibitor of replication or calyculin A, a specific inhibitor of serine/threonine protein phosphatases type 1 and type 2A that leads to a premature chromosome condensation in all phases of the cell cycle (G1, S, G2, and M). Over half of cancer-specific translocations containing breakpoints colocalize with fragile sites. This finding highlights the importance of fragile sites in cancer development. It also suggests that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. In this study, our objective is to isolate at the molecular level the hotspots of breakages at FRA11D, the cytogenetically mapped fragile site to the chromosome band 11p14. To induce fragile sites, lymphocytes from peripheral blood of three healthy donors were treated by calyculin A at 60 ng/ml. Using Fluorescent In Situ Hybridization technique and several probes corresponding to our region of interest, we located 2 specific hotspots of breakages at the level of the human genomic clones 1L12 and 283H3. The first clone is localized at the interface between the two chromosomal band 11p14.1 and 11p14.2. The second clone covers the mucin 15 gene, a member of a family widely associated with cancer. This molecular characterization is an essential step to understand the origin of DNA rearrangements observed in this region and in the entire genome.