B. Dutrillaux

Common fragile sites: mechanisms of instability revisited

Common fragile sites (CFSs) are large chromosomal regions prone to breakage upon replication stress that are considered a driving force of oncogenesis. CFSs were long believed to contain sequences blocking fork progression, thus impeding replication completion and leading to DNA breaks upon chromosome condensation. However, recent studies show that delayed completion of DNA replication instead depends on a regional paucity in initiation events. Because the distribution and the timing of these events are cell type dependent, different chromosomal regions can be committed to fragility in different cell types. These new data reveal the epigenetic nature of CFSs and open the way to a reevaluation of the role played by these sites in the formation of chromosome rearrangements found in tumors from different tissues.

Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using a BrdU treatment.

Replication times for all important chromosome bands, of both types R and Q (277 structures) are analysed. — The R-bands form a group of structures whose DNA replicates during the early S-phase, while the DNA situated in the Q-bands replicates during the late S-phase. — There may not exist overlapping between replication times of these two types of structures. — The widest R-bands are those which are the earliest to replicate; in general, the most intense Q-bands are those which are the latest to replicate. Especially among these last ones, a certain asynchronism exists between the replication times. Finally the heterochromatin of chromosomes 1, 16 and Y and of the short arms of the acrocentrics could contain two types of DNA which replicate at different times.

The cell cycle of lymphocytes in Fanconi anemia

SummaryBrdU-incorporation techniques were used to study the cell cycle in 18 cases of Fanconis anemia (FA).By comparison with controls, a significant slowing of the cell cycle of lymphocytes in vitro was observed in all FA patients, and possibly in FA heterozygotes, although to a lesser degree. It is probable that the demonstration of the slowing is dependent on the culture conditions. No slowing was observed in other patients affected by at least one of the symptoms of FA. The slow cell cycle of FA cells is mostly due to a very long G2-phase. A relationship between slow cell cycle and chromatid anomalies exists, the slower cells being significantly more frequently carriers of radial figures than the faster cells, in the same patient.

Common Fragile Site Profiling in Epithelial and Erythroid Cells Reveals that Most Recurrent Cancer Deletions Lie in Fragile Sites Hosting Large Genes

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

Segmentation of human chromosomes induced by 5-ACR (5-azacytidine)

SummaryThe 5-ACR (5-azacytidine) introduced in human lymphocyte cultures induces a lack or a delay of condensation of some chromosome segements corresponding to the G-bands. The resulting R-banding is very similar to that obtained with a 7-h treatment by BrdU, although the segmentation may be much stronger (pulverization) with high doses. However, the 5-ACR does not induce chromatid asymmetry, as BrdU does. This constitutes a new argument for considering that the segmentation and the asymmetry of chromatids depend, at least partly, on two different mechanisms, where proteins are probably involved. Another effect of 5-ACR is to increase chromosome associations by satellites, secondary constrictions, and telomeric regions.

Molecular profiling of common fragile sites in human fibroblasts

Common fragile sites have been mapped primarily in lymphocytes, but recent analyses show that the setting of these sites relies on cell type–dependent replication programs. Using a new approach, we molecularly mapped common fragile sites in human fibroblasts and showed that commitment to fragility depends on similar replication features in fibroblasts and lymphocytes, although different loci are committed in each cell type. Notably, the common fragile sites that we identified overlapped heretofore unexplained deletion clusters observed in tumors.

Localization of chromatid breaks in Fanconi's anemia, using three consecutive stains

SummaryThe location of 339 break points was analyzed in three patients with Fanconis anemia, using three consecutive stains: ordinary Giemsa, Q-banding, and R-banding. Almost all the breaks seem to take place in the Q bands, using R-banding, and in the R bands, using Q-banding. A very important artifact, varying according to the method used, is thus demonstrated. In fact, the breaks take place in the interbands, between R and Q bands.The breaks were also localized in relation to sister chromatid exchanges (SCEs), seen after BUDR treatment. There is a clear excess of breaks at places of SCE (29%). This may indicate a possible correlation between breaks and SCEs.

Complex evolution of sex chromosomes in Gerbillidae (Rodentia).

The sex chromosomes of 11 species of Gerbillidae (Meriones tristrami, M. crassus, M. libycus, M. persicus, M. unguiculatus, Gerbillus hesperinus, G. nigeriae, G. gerbillus, G. campestris, Gerbillurus tytonis, and Taterillus gracilis) are reported. A very complex evolution of the X chromosome and, to a lesser degree, of the Y chromosome was observed, including several inversions, translocations with autosomes, and an increase in heterochromatin. The role of constitutive heterochromatin, isolating autosome and gonosome segments in translocations (thus preventing any position effect), is discussed. The sequence of rearrangements affecting the sex chromosomes is considered with a view toward establishing the phylogenetic relationship of the species studied. The tendency to accumulate the same rate type of rearrangement, namely, gonosome-autosome translocations, is another demonstration that chromosomal evolution is not random in a given group of species.

Recurrent homogeneously staining regions in 8p1 in breast cancer and lack of amplification of POLB, LHRH, and PLAT genes.

In a cytogenetic study of 125 primary and untreated breast cancers, 107 were selected for the quality of their metaphases permitting detection of amplifications:homogeneously staining regions (HSRs), abnormally banded region (ABRs), and double minutes (dmins). HSRs and ABRs were detected in 62 cases (58%), but no cases of dmins were observed. The localizations of HSRs and ABRs were not random because they were observed in the 8p1 position in 14 cases. The possible amplifications of five sequences, MOS (8q1), LHRH (8p21.1), POLB (8p11.2), PLAT (8p12), and D8Z2 (8c) were investigated in three tumors with HSR on the short arm of chromosome 8. Because these sequences were not amplified, two interpretations can be proposed: 1) there is a frequent amplification of a sequence from the 8p1 region, located between the investigated sequences; and 2) the amplifications do not occur in 8p1, but HSRs or ABRs of undetermined origin have a strong tendency to be translocated onto 8p. Because cases with HSR(8p) have less complex karyotypes than with HSRs in other locations, the first interpretation is the most likely: HSRs may be formed in 8p and further translocated on other chromosomes in the course of tumor progression.

Radiation sensitivity of bloom's syndrome lymphocytes during S and G2 phases

In order to study chromosome sensitivity of Blooms syndrome (BS) cells in relation to the replication stage, gamma-ray irradiation was performed immediately before adding bromodeoxyuridine (BrdU) to lymphocyte cultures of one BS patient and of one control. It was found that BS cells are much more sensitive to the irradiation than control cells at the end of S and at G2 phases. The rate of induction of chromosome breaks is significantly increased and that of chromatid breaks and exchanges is also increased, though to a lesser degree. Our results also favor the existence of a cell subpopulation in BS characterized by a slow cycle, a high spontaneous chromosome aberration rate, and a high radiation sensitivity.