Induction of sister chromatid exchanges at common fragile sites.

Experiments were performed to gain further insight into chromosome structure and behavior at common fragile sites by testing the hypothesis that gaps at these sites predispose to intrachromosomal recombination as measured by sister chromatid exchanges (SCEs). Human lymphocytes were concurrently treated with aphidicolin, for determination of fragile site expression, and with 5-bromodeoxy-uridine, for SCE analysis. Aphidicolin induced chromosome gaps nonrandomly, with the great majority of gaps occurring at common fragile sites. On average, 66% of gaps were accompanied by an SCE at the site of the lesion. Analysis of two specific common fragile sites at 3p14 and 16q23 showed the same pattern; that is, on average 70% of gaps at these sites were accompanied by an SCE. These results show that common fragile sites are hot spots not only for chromosomal lesions such as gaps but also for SCE formation.     

Population cytogenetics of aphidicolin-induced fragile sites

Summary Chromosome fragile sites are inducible by aphidicolin in cultured human lymphocytes. To assess the frequency and distribution of these common fragile sites in the general population, a cytogenetic survey was performed on 126 subjects, 59 males and 67 females, whose age ranged from 1 day to 72 years. Common fragile sites, induced by aphidicolin, were widespread and showed a remarkably different sensitivity among individuals; age influenced the overall frequency of fragile sites. Moreover, both age and sex seemed to modulate the expression of specific fragile sites. In our population, the most common fragile sites were: 3p14, 16q23, Xp22, 6q26, 1p31, 4q31, 1p22, 7q22, 2q33, 3q27, 2q31, 7q32, 14q24, 10q22, 5q31, 2q37, 6p21.     

Autosomal fragile sites

It is possible to distribute the 17 autosomic fragile sites presently known in three categories according to their sensitivity: BrdU-sensitive sites (10q25, 16q22, 17p12), distamycin A-sensitive sites (16q22, 17p12) and folate- and thymidilate-sensitive sites (2q11-q14, 3p14, 6p23, 7p11, 8q22, 9p21, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, 16q23, 17p12, 20p11). Four fundamental problems are discussed, first the relation between the presence of a fragile site and the phenotype, secondly the incidence of autosomic sites, third the origin of fragility (particularity of DNA structure, defect of the DNA/proteins binding and abnormal arrangement of chromatin, abnormality of the metaphasic scaffold) and fourth the localization of fragile sites.     

Population cytogenetics of rare fragile sites in Japan

Summary A population cytogenetic study of three groups of rare fragile sites defined in Human Gene Mapping 8 (HGM8, Berger et al. 1985) has been conducted using peripheral blood lymphocytes of healthy Japanese subjects. We have examined 1,022 blood donors for folate-sensitive and bromodeoxyuridine (BrdU)-requiring, and 845 for distamycin A-inducible fragile sites. Out of 17 rare autosomal fragile sites defined in HGM8, the following six were identified in Japan; folate-sensitive fra(2)(q11), fra(11)(q13) and fra(11)(q23), distamycin A-inducible fra(16)(q22) and fra(17)(p12), and BrdU-requiring fra(10)(q25). The incidences of distamycin A-inducible fra(16)(q22) (1.42%) and fra(17)(p12) (3.08%) were considerably higher than those of the other sites in Japan. Furthermore, a folate-sensitive fra(17)(p12) and a distamycin A-inducible fra(8)(q24.1) have been newly found in the present study. Their incidences were 0.10% (1/1,022) and 0.71% (6/845), respectively. Since the expression of this fra(17)(p12) was induced by fluorodeoxyuridine, supressed by thymidine, but not induced by distamycin A, it can be classified as a folate-sensitive site. The expression of the new distamycin A-inducible fra(8)(q24.1) was also enhanced by treatment with Hoechst 33258, berenil and 4,6-diamidino-2-phenylindole (DAPI). This fragile site fulfils all four classical criteria suggested by Sutherland (1979) and also new criteria for a rare fragile site defined in HGM8 (Berger et al. 1985).     

Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA.

We determined previously that the selectable marker pSV2neo is preferentially inserted into chromosomal fragile sites in human x hamster hybrid cells in the presence of an agent (aphidicolin) that induces fragile-site expression. In contrast, cells transfected without fragile-site induction showed an essentially random integration pattern. To determine whether the integration of marker DNA at fragile sites affects the frequency of fragile-site expression, the parental hybrid and three transfectants (two with pSV2neo integrated at the fragile site at 3p14.2 [FRA3B] and specific hamster fragile sites [chromosome 1, bands q26-31, or mar2, bands q11-13] and one with pSV2neo integrated at sites that are not fragile sites) were treated with aphidicolin. After 24 h the two cell lines with plasmid integration at FRA3B showed structural rearrangements at that site; these rearrangements accounted for 43%-67% of the total deletions and translocations observed. Structural rearrangements were not observed in the parental cell line. After 5 d aphidicolin treatment, the observed excess in frequency of structural rearrangements at FRA3B in the cell lines with pSV2neo integration at 3p14 over that in the two lines without FRA3B integration was less dramatic, but nonetheless significant. Fluorescent in situ hybridization (FISH) analysis of these cells, using a biotin-labeled pSV2neo probe, showed results consistent with the gross rearrangements detected cytogenetically in the lines with FRA3B integration; however, the pSV2neo sequences were frequently deleted concomitantly with translocations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of aphidicolin, FUdR and caffeine‐induced fragile sites in lymphocytes of healthy Turkish individuals

The expression of common fragile sites (c‐fra) and frequency of chromosomal aberrations were studied in peripheral lymphocytes of 50 healthy Turkish individuals (26 males and 24 females from 1 to 87 years of age) after induction with aphidicolin (APC), 5′‐fluorodeoxyuridine (FUdR), and caffeine. A correlation was seen between age and the frequency of chromosomal aberrations in APC and caffeine treated cultures, but there were no significant differences in the frequencies of chromosomal aberrations between males and females in any of the treatments. The mean frequency of aberrations induced by FUdR was significantly higher than that induced by APC and caffeine. A chromosome aberration is defined as a fragile site when present in 1% of the cells analyzed from each culture and in at least 50% of the individuals studied. Using these criteria, 12 c‐fra were observed in the three treatments: 1p21, 1q21, 2p11‐q11, 3p14, 4q31, 6q26, 7q22, 7q32, 8q24, 11q23, 16q23, and Xp22. Sites 3p14, 16q23, and Xp22 were the most frequently observed c‐fra, with only the frequency of Xp22 being significantly increased in females in APC treated cultures. The results of these studies are important as a base against which the effects of other clastogenic and environmental agents, as well as genetic background, can be compared. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA polymerase α inhibition by aphidicolin induces gaps and breaks at common fragile sites in human chromosomes

Summary Aphidicolin, a specific inhibitor of DNA polymerase , is known to induce chromosomal aberrations. At concentrations that did not greatly affect mitotic index, aphidicolin induced a striking number of chromosome gaps and breaks distributed in a highly nonrandom manner in cultured human lymphocytes. Specific chromosome bands, especially 2q31, 3p14, 6q26, 7q32, 16q23, and Xp22 were preferentially damaged in lymphocytes from each of 12 subjects studied. Total and site-specific damage was dose dependent and greatly increased when folic acid was removed from the medium. The sites most sensitive to aphidicolin damage include the hot spots seen under conditions of thymidylate stress and in studies of spontaneous chromosomal damage. The fragile X site, which can also be induced by thymidylate stress, was not induced by aphidicolin in lymphocytes, suggesting a separate mechanism for its induction. Aphidicolin represents a novel tool for detection of hot spots on human chromosomes through the mechanism of DNA polymerase inhibition. The hot spots induced by aphidicolin represent a new class of fragile sites which we term common fragile sites.     

Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture.

The expression of heritable fragile sites on human chromosomes has been shown to be dependent upon composition of the tissue medium for sites at 2q11, 10q23, 11q13, 16p124, 20p11 and Xq27 or 28 but not for the site at 16q22. Expression of the fragile sites is inhibited by folic acid, thymidine, folinic acid, and probably bromodeoxyuridine, and induced by methrotrexate. In addition, there is a correlation between frequency of expression of the sites and pH of the culture medium for the sites on 2q, 10q and Xq. Possible reasons for these findings are discussed, and a definition and classification of fragile sites is proposed.     

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site

Summary Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(pl2), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did nto induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra (16)(pl2) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.     

Fragile X expression is decreased by 5-azacytidine and S-adenosylhomocysteine

A lymphoblastoid cell line derived from a patient with fragile X chromosome exhibited fragility only when 5-fluoro-2'-deoxyuridine (FUdR) was added to the culture medium. Addition of methionine with FUdR greatly increased the frequency of fragile X. Addition of 5-azacytidine (an inhibitor of methylation at the level of DNA) or S-adenosylhomocysteine (an inhibitor of the synthesis of the methyl group donor S-adenosylmethionine) reversed the effect of methionine as well as that of FUdR. It is proposed that DNA methylation is in some way involved in the mechanism of fragile X expression.     

Telomeric associations involving Yq12 in a lymphoblastoid cell line

A number of apparent telomeric associations/fusions (TAs) involving Yq12 were observed in a lymphoblastoid cell line (GM6892A) established from a fragile-X-negative, phenotypically normal male with the fragile X gene mutation. The TAs were seen when these cells were grown for an extended period of time in medium 199, which is deficient in thymidine and folic acid. Because the low thymidine and folic acid condition of medium 199 is known to induce chromosome and chromatid gaps and breaks at folate-sensitive fragile sites, other fragile site-induction regimes were examined to determine if the TAs seen in GM6892A were due to a fragile site in the Yq12 band. No TAs were seen with any of the other regimes that were tried.     

Synergistic effect of DAPI and thymidylate stress conditions on the induction of common fragile sites

When supplied to human leukocytes grown in complete medium (RPMI 1640), DAPI, a nonintercalating compound specific for the AT bases of DNA, induces the appearance of three common fragile sites (CFRA) mapped at 1q42, 2q31, and 7p22. The same treatment with DAPI in a medium deficient in folic acid and thymidine (199 M) considerably increases the expression of these sites and induces the appearance of a further 16 CFRA sites at 1q24, 2p25, 4p16, 4q25, 5p15.3, 6p21.3, 6p25, 6q13, 9p24, 16p13.3, 16q23, 17q21, 18q23, 20q13.1, 21q21, and Xq28. The results point to the existence of a synergism between DAPI and thymidylate-stress culture conditions in inducing site-specific chromosome damage. The results also agree with the hypothesis that DAPI-induced CFRA sites are DNA late-replicating chromosomal areas rich in AT bases.     

Expression and identification of folate-sensitive fragile sites in British Suffolk sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>)

An investigation to understand the dynamics and biological significance of fragile site expression, and identification of 5-fluorodeoxyuridine (FUdR) induced chromosomal gaps/breaks, were carried out in an experimental flock of 45 Suffolk sheep. The statistical comparison revealed, highly significant variation in the frequency of chromosomal fragile site expression between control and FUdR cultures. Mean (± S.D.) values for cells with gaps and breaks, or aberrant cell count (AC), and the number of aberrations (NoA) per animal were 2.02 ± 0.34, 2.42 ± 0.48, 13.26 ± 0.85 and 21.87 ± 1.88 (P < 0.01) in control and FUdR cultures, respectively. The comparison of age revealed nonsignificant variation between control and FUdR cultures. The G-band analysis of fragile site data revealed gaps in 29 autosomal and two X-chromosomal bands in the control cultures, whereas FUdR treated cultures scored 78 unstable bands in autosomes of which 56 were significantly fragile. X-chromosomes expressed breaks and gaps in six G-negative bands and five of them (Xq13, Xq15, Xq17, Xq24 and Xq26) were significantly fragile. The distribution comparison of autosomal fragile sites between sex groups did not reveal any significant variation. Female X-chromosomes were significantly more fragile than the male X-chromosomes. The distribution comparison for age groups (lambs versus adults) revealed significantly higher number of fragile bands in adults. Comparison of published data on reciprocal translocations in sheep with the fragile-site data obtained in this study indicated that the break sites of both phenomena were correlated. Similarities were also found between fragile sites and breakpoints of evolutionary significance in family Bovidae.     

Chromosome breakage and recombination at fragile sites.

Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3p14.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3p14.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

Fragile sites induced by FUdR,caffeine, and aphidicolin

Summary The frequencies of common fragile sites (c-fra) induced in peripheral blood lymphocytes by fluorodeoxyuridine (FUdR), aphidicolin, or caffeine, in eight healthy controls were studied. There was a significantly higher frequency of breaks (P<0.05) in the latter two treatments than the former. Also, significant variation in total number of breaks was observed among the eight individuals within the three treatments. The relative frequency of a fragile site in relation to the total number of fragile sites in an individual rather than its expression in total cells was considered important. Use of a frequency of 4% or more of total fragile sites was proposed to eliminate apparent random breaks that were observed. Using these criteria, a total of 31 c-fra were observed in the three treatments. The distribution of the fragile sites was different in FUdR-treated cells as opposed to caffeine- and aphidicolin-treated cells. Sites 3p14 and 16q23 and Xp22 were the three most frequently observed c-fra. The higher frequency of expression of some fragile sites in normal controls, as observed here, suggests that any relationship between fragile sites and neoplastic transformation has to be carefully evaluated. A classification based on frequency in the population, rather than mode of induction, is suggested.     

Heritable fragile sites on human chromosomes

Summary A kindred is described in which six members have a fragile site at 12q13. This fragile site was found to be suppressed by folic acid and thymidine in lymphocyte culture. An updated classification of known fragile sites is presented.     

Segregation analysis of rare autosomal fragile sites

Summary Segregation analyses were performed on pedigrees with rare autosomal fragile sites. The results of the analysis of pedigrees with folate sensitive fragile sites, including 2q1, 6p23, 7p11, 8q22, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, and 20p11, suggested that expression of the gene depended on the carrier parent: it was only 50% penetrant when transmitted by a carrier father, but fully penetrant when transmitted by a carrier mother. Pedigrees with the bromodeoxyuridine (BrdU) fragile site, fra(10)(q25), showed the same trend but the results were not statistically significant. In addition, 38 of the 44 probands with folate sensitive or BrdU-sensitive fragile sites received the gene from their carrier mother and only six received it from their father. In contrast, the analysis of pedigrees with the distamycin A-inducible site, fra(16)(q22), gave the results expected for a simple codominant trait with complete penetrance. Probands with this fragile site received the gene equally from mothers or fathers. The genetic implications of these results are discussed.     

Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes

Human papillomaviruses (HPV) 16 and 18 are closely linked with human genital cancer. In most cervical carcinomas, viral sequences are integrated into the host genome. HeLa, a cervical carcinoma cell line, has multiple copies of integrated HPV 18 DNA. In this study, in situ chromosome hybridization was used to assign the integration sites of HPV 18 DNA sequences on HeLa cell chromosomes. Four sites of hybridization were identified at 8q23----q24, 9q31----q34, p11----p13 on an abnormal chromosome 5, and q12----q13 on an abnormal 22. Three of these sites correspond with the locations of MYC, ABL, and SIS protooncogenes, and are at or in close proximity to fragile sites. The chromosomal localization of HPV 18 DNA may be useful in assessing the role of viral integration in the development of this malignancy.     

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.