Expression Of Common Fragile Sites Research Articles

Variability in expression of common fragile sites: in search of a new criterion

Fragile sites are nonrandom, heritable sites on chromosomes that can be induced to form gaps, breaks, and rearrangements under specific conditions. There is currently no established criterion to define a common fragile site. We applied seven published criteria to our data from three groups of subjects: (1) three pairs of like-sexed twins, (2) four unaffected von Hippel-Lindau (VHL) family members, and (3) six patients affected with VHL disease. Substantial differences were present in the numbers of sites considered positive by these criteria. While some of this variability can be attributed to technical factors, our data illustrate the problems in comparing results from different studies to assess the significance of fragile sites. A recently published criterion is based upon the Poisson distribution. We found this criterion to be flawed in its presentation, and furthermore, the Poisson distribution did not provide an adequate approximation to our data. We propose here an alternative approach based upon the negative binomial distribution.

Possible association of rare autosomal folate sensitive fragile sites and idiopathic mental retardation: a blind controlled population study

The expression of folate sensitive fragile sites (FS) was assessed in cord blood lymphocyte cultures obtained from 790 newborns (NB) and in peripheral blood lymphocyte cultures from 326 institutionalized mentally retarded residents (MR). The mean rate of expression of common FS and the occurrence of rare FS was significantly higher in the MR population. Age, sex and history of chronic medication use did not appear to influence common FS expression in the MR population, 3/790 (0.38%) NB and 5/326 (1.53%) MR exhibited rare autosomal folate sensitive FS, a 4-fold difference in incidence (P = 0.009, Poisson test). Four of the five MR who expressed rare FS were considered to have idiopathic MR (4/179 or 2.2%). The occurrence of rare FS in 1/147 (0.68%) MR with known etiology is not significantly different from the frequency of occurrence in the NB population (P = 0.428, Poisson test). In this population, rare FS appear to be overrepresented in the idiopathic etiology MR group.

Common fragile sites in man and three closely related primate species

The expression of common fragile sites was studied in peripheral lymphocytes of man, gorilla, chimpanzee, and orangutan after induction with aphidicolin, methotrexate, or fluorodeoxyuridine. As far as the chromosomal localization is concerned, it appears that many of these sites have been highly conserved during primate evolution. However, differences were found in the relative expression of certain sites. In all four species, mapping of approximately 500 lesions disclosed the most breakage-prone common fragile sites, at which about 90% of all induced aberrations were localized. Comparison of chromosome regions involved in evolutionary changes to fragile sites in the four primate species revealed 30 sites that were located at or close to the same chromosomal band. However, no correlation was found between the relative expression of a certain common fragile site in vitro and a potential involvement of this chromosomal site in evolutionary changes.

The expression frequency of common fragile sites and genetic susceptibility to lung cancers

The chromosomal aberration rate (including gap and break) and expression frequency of common fragile sites were examined in peripheral blood lymphocytes cultured with TC199 medium from 96 patients with lung cancers, 40 of their first-degree relatives, and 45 normal control subjects. Both the chromosomal aberration rates and expression frequencies of common fragile sites observed in patients and their relatives were significantly higher than those in normal control subjects. About 60% of chromosomal aberrations were derived from the expression of common fragile sites either in the patients and their relatives or in the controls. The expression of fra(3)(p14) was most frequently observed, and the mean frequencies of its expression in patients and their relatives were significantly higher than in control subjects. It is suggested that common fragile sites might be unstable factors of the human genome, and their expression might be affected by some genetic factors, and they might play an important role in the genetic susceptibility to lung cancers. The significantly high expression of fra(3)(p14) in patients and their relatives may be related to the generation of the breakpoint at band 3p14 found in lung cancers.

Expression of Fragile Site 8q22 in Peripheral Blood Lymphocytes Taken from Patients with Acute Leukemia M2 having t(8;21)(q22;q22)

The relation between the expression of common fragile sites and chromosomal breakpoints in neoplastic cells in the same patients has not been well studied. In the present study, the frequency and distribution of aphidicolin-induced breaks on chromosomes 8 and 21 in peripheral blood lymphocytes (PBL) taken from three patients with acute myeloid leukemia, French-American-British classification type M2, having chromosomal translocation t(8;21)(q22;q22) (M2t) were studied prior to any initial treatment. Seven patients with other types of acute leukemia without t(8;21) and 13 healthy people were also studied. In PBL from patients with M2t, the numbers of chromosomal breaks were 1, 7 and 3/216 metaphasees at bands 8q21, 8q22 and 8q24, respectively; the frequencies at 8q22 and 8q24 being significantly higher than those for patients with the other leukemias and for the healthy subjects (p less than 0.001 and p less than 0.01, respectively). These results suggest that the fragility on chromosome 8q21-24 is related to a predisposition to this particular type of leukemia.

Synergistic effect of hydroxyurea and excessive thymidine on the expression of the common fragile sites at 3p14 and 16q23

There is a synergistic effect between hydroxyurea (HU) and high concentrations of thymidine (TdR) on the induction of the common fragile sites at 3p14 and 16q23. The frequencies of expression of these fragile sites were significantly higher than in the controls when excessive TdR (0.5 mM) and HU (4 mM) were added into lymphocyte cultures in MEM medium 24 h and 2 h before harvest. Two conditions for the maximum expression of common fragile sites, such as fra(3)(p14), fra(16)(q23) are discussed. The TdR-HU fra(3)(p14)-inducing system might be modified effectively and consistently to improve the prenatal diagnosis of the fragile X syndrome.

Effects of folate in culture medium on common fragile sites in lymphocyte chromosomes from normal and leukemic children.

The expression of common fragile sites at 69 bands was evaluated in 20 normal children and in 15 children with newly diagnosed acute leukemia using medium with folate (FA+) and without folate (FA-). As expected, the FA- medium significantly increased expression of aberrations in all study groups but the differences were larger for normal children than leukemic children. The major effect of the FA- medium was a generalized increase in aberration frequency over all sites rather than site-specific increases. A tendency toward clumping of aberrations within cells was exhibited in both media. Aberrations were seen at 81% (FA+) and 83% (FA-) of the 69 bands, with 4 sites - 3p14, 6p21, 9q13, and 17q23 - recorded in most of the study individuals. In addition, 12 sites not previously recorded as common or rare sites had significant levels of expression in at least one study group.

Multiple common fragile sites are expressed in the genome of the laboratory rat.

Splenic lymphocytes from Sprague Dawley and Fischer 344 rats were exposed to two chemicals known to induce common fragile site expression in man: fluorodeoxyuridine (in conjunction with the enhancing effects of caffeine) and aphidicolin. Of 39 sites that were significantly damaged in excess, 12 meet the criteria for fragility proposed in this investigation. Rat fragile sites appear to differ from those in man in that no common hierarchical frequency of expression is evident from the two methods of induction. In addition, a comparison of published cancer-specific chromosome breakpoints from a variety of rat tumors reveals little or no apparent concordance with the identified fragile sites. The rat is an animal model in which multiple common fragile sites can be induced and, as such, will be valuable for testing hypotheses concerning the biological basis of chromosomal fragility.

Study of human chromosome V. The effects of thymidine concentration and timing on the expression of uridine-induced constitutive fragile sites.

The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 microM up to 200 microM) and the "rescuing" effect of thymidine can be achieved if the cells were treated prior to 2-3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.

The effect of caffeine on fragile X expression

Caffeine has been reported to enhance the expression of the fragile X [fra(X)] and common fragile sites in peripheral blood lymphocyte cultures (PBLC) treated with 5-fluorodeoxyuridine (FUdR). One of the effects of caffeine on replicating cells is inhibition of DNA repair suggesting that fragile sites may be regions of DNA with a high rate of misreplication under the conditions of thymidylate stress induced by FUdR. We have studied the effect of caffeine on the expression of the fra(X) and common folate-dependent fragile sites in PBLC from two fra(X) expressing individuals and in five lymphoblastoid cell lines (LCL) established from individuals in families in which the fra(X) is segregating. Caffeine did not enhance the expression of the fra(X) in the PBLC or in the three LCL from fra(X) expressing individuals nor did it elicit fra(X) expression in LCL from a non-expressing obligate-carrier female and a transmitting male. However, in all cultures there was a marked increase of common fragile site expression due to caffeine treatment. These data suggest that the mechanism of expression of the common fragile sites and the fra(X) may be quite different.

Caffeine enhances fragile (X) expression in somatic cell hybrids.

Yunis and Soreng recently demonstrated enhanced expression of common fragile sites as and of fra(X) when 2.2 mM caffeine is added to FUdR treated lymphocyte cultures 6 hours before harvest. We failed to replicate this finding for fra(X) expression in lymphocytes. However, we do find a consistent increase in expression levels in somatic cell hybrids between a Chinese hamster cell line and 3 unrelated individuals with the fra(X) mutation when caffeine is present for the last 2 hours before harvest. This was particularly true for "low-expressing" cell lines, in which a 4-6 fold increase was observed. Using a thymidylate synthase deficient hybrid which could be blocked in the S phase by thymidine deprivation, we found that caffeine significantly reduced the recovery time from thymidine release to mitosis. This produced the highest level of fra(X) expression (48%) only one hour after release from thymidine deprivation. These results show that caffeine does enhance fra(X) expression in at least some cell types. The effect is probably indirect, inhibiting the mitotic delay usually associated with DNA damage.