Chromosomal fragile sites and relationship between genetic predisposition to small cell lung cancer.

Fragile sites are non-staining gaps and breaks on mammalian chromosomes. Several investigators have pointed out that these sites may act as factors that predispose to specific chromosomal rearrangements that are present in some cancer cases. The expression of common fragile sites induced by aphidicolin (Apc) was evaluated on prometaphase chromosomes obtained from the peripheral blood lymphocytes of 15 patients with lung cancer, 20 of their clinically healthy family members, and 20 age-matched normal controls. As a result of cytogenetic evaluation carried out by the High Resolution Banding (HRB) technique, 1q21, 2q33, 3p14, 7q32, 13q13, 16q23, 17q21, and 22q12 are defined as fragile sites in patients and relatives. The rate of total fragile sites and 2q33, 3p14, and 16q23 are statistically significant in both patients and relatives when compared with the control group. Therefore, our results showed that common fragile sites might be unstable factors in the human genome and they can be used as suitable markers for genetic predisposition to lung cancer.     

The expression frequency of common fragile sites and genetic predisposition to colon cancer

The expression frequency of common fragile sites induced by aphidicolin (Apc), bromodeoxyuridine (BrdU), and caffeine was evaluated on prometaphase chromosomes obtained from the peripheral blood lymphocytes of 32 patients with colon cancer, 30 of their clinically healthy family members and 30 age-matched normal controls. The proportion of damaged cells (P < 0.001), the mean number of chromosomal aberrations and the expression frequencies of fragile sites were significantly higher in the patient and relative groups compared to the control group. Our findings show an increased genetic instability in patients with colon cancer and their first-degree relatives. In addition, common fragile sites can be used as a suitable marker for determining genetic predisposition to cancer.     

Fragile Sites in Human and Macaca Fascicularis Chromosomes are Breakpoints in Chromosome Evolution

We have analysed the expression of aphidicolin-induced common fragile sites at two different aphidicolin concentrations (0.1 µmol/L and 0.2 µmol/L) in three female and one male crab-eating macaques (Macaca fascicularis, Cercopithecidae, Catarrhini). A total of 3948 metaphases were analysed: 1754 in cultures exposed to 0.1 µmol/L aphidicolin, 1261 in cultures exposed to 0.2 µmol/L aphidicolin and 933 in controls. The number of breaks and gaps detected ranged from 439 in cultures exposed to 0.1 µmol/L aphidicolin to 2061 in cultures exposed to 0.2 µmol/L aphidicolin. The use of a multinomial FSM statistical model allowed us to identify 95 fragile sites in the chromosomes of M. fascicularis, of which only 16 are expressed in all four specimens. A comparative study between the chromosomes of M. fascicularis and man has demonstrated that 38 human common fragile sites (50%) are found in the equivalent location in M. fascicularis. The analysis of the rearrangements that have taken place during chromosome evolution has revealed that the breakpoints involved in these rearrangements correspond significantly (p < 0.025) to the location of M. fascicularis fragile sites.     

Common fragile sites: their prevalence in subjects with constitutional and acquired chromosomal instability

Chromosomal fragile sites that are inducible by methotrexate and aphidicolin are frequent in the human population. To assess the frequency and distribution of these common fragile sites, we performed a cytogenetic survey on lymphocytes from subjects known to be particularly prone to breakage because of constitutional chromosomal instability, the possession of a rare fragile site, or Fanconi anemia. Furthermore, a group of cancer patients was included in this study in view of possible acquired chromosomal instability. Lymphocyte chromosomes from several healthy donors were analyzed under identical conditions. We found that methotrexate- and aphidicolin-induced fragile sites are widespread in the general population, showing a similar breakpoint distribution. Ten fragile sites (3p14, 16q23, 2q32, 6q25, 4p16, 4q31, 14q24, 1p31, 20p12, 7q21) were observed in at least 40% of the individuals among the different groups. Our data point out a significantly increased breakage induced by aphidicolin in lymphocytes from cancer patients and, to a lesser extent, from rare fragile sites carriers. These results suggest that common fragile sites are enhanced in some constitutional and acquired conditions.     

FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.      

Synergism between aphidicolin and adenoviruses in the induction of breaks at fragile sites on human chromosomes.

Infection of human embryonic kidney cells with adenoviruses results in the induction of gaps and breaks in cell chromosomes. With adenovirus type 12, cytogenetic damage is known to occur primarily at fragile sites on chromosomes 1 and 17. We have mapped adenovirus type 5-induced breaks and have observed that, although they occur on all chromosomes, they are localized primarily on bands where fragile sites have been mapped. The susceptibility of fragile sites to adenovirus led us to investigate their expression upon combined treatments with virus and aphidicolin, a frequently used inducer of fragile sites. Under these experimental conditions, the frequency of damage at all sites was found to increase significantly, and the magnitude of such increases indicated a synergistic effect between drug and virus.     

Common fragile sites in chromosomes of bone marrow cells and peripheral blood lymphocytes from healthy persons and leukemia patients

Frequency and distribution of aphidicolin-induced fragile sites (c-fra) on chromosomes of both peripheral blood lymphocytes (PBL) and bone marrow (BM) from 15 leukemia patients were studied in comparison with 22 PBL and six BM samples from healthy volunteers. In normal controls, the most frequent c-fra was 3p14 in PBL, but it was 4q21-25 in BM. The second most frequent site was 16q23 in PBL, but it was 7q11.2 in BM. These differences in fragile sites between PBL and BM may be related to distinct functions of cells in different tissues. The total number of breaks in PBL and BM showed a significant difference among individuals, but the sites were generally common. The frequency of breaks in PBL from leukemia patients was higher than in controls when the leukemic cells had any karyotypic abnormalities. In leukemia without karyotypic abnormalities and acute myeloid leukemia (AML) with (15:17), the frequency of breaks fell within normal or slightly above normal ranges. Breaks at 3p14 (22.0% of total breaks), 16q23 (7.3%), 7q32 (4.3%), Xp22 (3.7%), and 6q26 (2.9%) were frequent in PBL from seven AML patients. Breaks at 4q21-25 (2.1%), 7q22 (2.2%), 7q32, and Xp22 were more frequently induced than in controls, and 1p32 (0.1%), 3p14, 6q26, and 16q23 were less often expressed than in controls. On the other hand, PBL from acute lymphoblastic leukemia patients showed a higher frequency of breaks only at 1p22 (3.4%) and the frequency of breaks at 3p14 (30.2%) decreased (p less than 0.05). The PBL from AML patients with t(8;21) (q22;q22) showed breaks at 8q22 and 8q24, and the frequencies were significantly higher than those of other types of leukemia or in controls (p less than 0.001). The results of this study suggest that fragility of chromosomes may be related to the chromosomal rearrangement in or predisposition to leukemia.     

Allele-specific late replication and fragility of the most active common fragile site, FRA3B

FRA3B at 3p14.2 is the most active of the common fragile sites in the human genome and is expressed when cells are exposed to the DNA replication inhibitor, aphidicolin. Several lines of evidence suggest that fragile sites are regions of late replication. To elucidate the relationship between the timing of replication across the FRA3B region and its corresponding fragility, we labeled cells with 5-bromo-2'- deoxyuridine (BrdU) and adopted an immunofluorescent procedure to visualize late replicating DNA (BrdU-substituted DNA) in metaphase chromosomes. We also chose 21 markers along the FRA3B region and analyzed the timing of replication using BrdU-labeled DNA from different stages of the cell cycle sorted by flow cytometry. Our results show that there are two distinct alleles that replicate at different stages in the cell cycle and that breaks/gaps preferentially occurred on the chromosome 3 with the late replication allele. These results provide direct evidence that allele-specific late replication is involved in the fragility of the most active common fragile site, FRA3B.      

Fragile sites and neuroblastoma: fragile site at 1p13.1 and other points on lymphocyte chromosomes from patients and family members

Fragile sites on lymphocyte chromosomes of 20 patients with neuroblastoma of different clinical stages and histologic differentiation were studied. A hitherto unknown break in the band p13.1 of chromosome #1 was found in a frequency of 3%-15% in nine cases. The same phenomenon was observed in three members of a neuroblastoma family. In another instance, this fragile site was also seen in the mother of a patient. Fragile sites were expressed when cells were cultured in folate-deprived medium and could be slightly enhanced in frequency by aphidicolin. Additional aphidicolin induced possible fragile sites hitherto unknown or not yet accepted by the HGM 8 were detected.     

Enhanced expression of 1p32 and 1p22 fragile sites in lymphocytes in cutaneous malignant melanomas.

Frequency and distribution of 5-fluorodeoxyuridine (5-FdU) plus caffeine-induced fragile sites on chromosomes of peripheral blood lymphocytes (PBL) from 10 patients with cutaneous melanoma were studied in comparison with 10 PBL samples from normal donors of corresponding sex and age. The total number of breaks showed a significant difference among individuals in both groups, however, the average frequencies of 5-FdU plus caffeine-induced, as well as spontaneous damages in PBL from melanoma patients, were higher than those from healthy volunteers. The analysis of the breakpoint distribution showed a statistically significant increase in the expression of several fragile sites. The highest enhancement was observed at 1p32 and 1p22 sites (p less than 0.001). Earlier, the increase in the expression of 1p32 fragile sites was reported for neuroblastoma patients. We believe that enhanced expression of fragile sites in 1p may play a yet-unknown pathogenetic role in the development of some neuroectodermal tumors.     

Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction

The FRA3B at 3p14.2 is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress. The molecular basis for chromosome fragility at FRA3B is unknown. In contrast to the rare fragile sites, including FRAXA, no repeat motifs, such as trinucleotide repeats, have been identified within FRA3B. Several lines of evidence suggest that fragile sites are regions of DNA whose replication is unusually sensitive to interference. We have used fluorescence in situ hybridization to determine the relative timing of replication of FRA3B sequences. Our studies revealed that FRA3B sequences are late replicating. Exposure to aphidicolin, an inhibitor of both DNA polymerase alpha and delta, results in a reproducible delay in the timing of replication, and some cells enter G2without having completed replication of FRA3B sequences. Our results support a model in which common fragile sites are sequences that initiate replication late in S phase or are slow to replicate, and the chromosomal breaks and gaps observed in metaphase cells are due to unreplicated DNA.      

The role of late/slow replication of the FRA16D in common fragile site induction

The FRA16D, at 16q23, spans the WWOX gene and is one of the most highly expressed common fragile sites observed when DNA replication is perturbed by aphidicolin. Several lines of evidence suggest that fragile sites are regions of DNA that are unusually sensitive to interference during replication. We have determined that the FRA16D alleles replicate in a synchronous fashion and that replication of these sequences occurs primarily in late S phase extending into G2 phase. Exposure to aphidicolin, an inhibitor of DNA polymerase alpha, results in a modest increase in cells with replication of FRA16D sequences in early S phase. This may represent initiation of replication in early S phase coupled with slow replication progression, or, alternatively, these cells may have passed through mitosis, entered the G1-S phase of the next cell cycle, and initiated replication/repair. Our results support a model in which common fragile sites are sequences that may initiate replication in early-mid S phase but are slow to complete replication, and the chromosomal breaks and gaps observed in metaphase cells result from unreplicated DNA.     

骨肉瘤患者染色体不稳定性和脆性部位表达的研究

对48例骨肉瘤患者和36例正常人外周血淋巴细胞染色体不稳定性和脆性部位表达进行了研究,发现患者染色体裂隙、断裂点、异常细胞率和脆性部位表达率均显著高于正常对照组,且脆性部位3p~(14),7q~(32).11q~(13),13q~(13)在半数以上病例中表达。这些位点与某些癌基因同位或毗邻。染色体不稳定性增加和脆性部位的过度表达可解释骨肉瘤患者有较高的肿瘤遗传易感性。     

Distribution of autosomal fragile sites in specimens cultured for prenatal fragile X diagnosis

We reviewed the distribution of autosomal fragile sites (FS) and spontaneous chromosome breaks or gaps (CB) at chromosome locations other than those recognized as FS from 100 amniotic fluid samples (AF), 19 chorionic villus samples (CVS), and 5 percutaneous umbilical blood samples (PUBS) referred for fragile X [fra(X)] analysis. We present data on the degree of expression of autosomal fragility in AF, CVS, and PUBS samples, and the relationship between degree of expression and induction system. The most common observed FS were: 3p14, 9p32, and 6q26 in AF; 9q32, 3q27, and 8q22 in CVS; and 3p14, Xq22, and 16q23 in PUBS cases. Distribution of FS and CB, when compared by induction system, was not found to be identical. Our data also indicate that the presence of any particular FS cannot be used as an indicator for the effectiveness of the fra(X) induction system in prenatal samples.     

Common fragile site expression and genetic predisposition to breast cancer

The expression of common fragile sites induced by aphidicolin and caffeine was evaluated on prometaphase obtained from the peripheral blood lymphocytes of 35 women with breast cancer, their 35 clinically healthy female family members, and 20 sex- and age-matched normal controls. As a result of the cytogenetic and statistical evaluation, the number of damaged cells, chromosomal aberrations, and expression frequencies of fragile sites detected in patients with breast cancer and their first-degree relatives were found to be significantly higher than those in the control group. Our findings indicate an increased genetic instability in women with breast carcinomas and their relatives. Therefore, fragile sites may be used as a reliable marker for defining genetic susceptibility to cancer in general.     

Cytogenetic analysis of spermatozoa in the father of a child with a de- novo reciprocal translocation t(7;9) (q22;p23)

Analysis of sperm chromosomes was carried out in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23) by G-banding and chromosome painting. Sperm metaphases were obtained using the zona- free hamster oocyte-human sperm fusion technique. A total of 138 complements were sequentially analysed by G-banding and fluorescence in- situ hybridization (FISH). The frequency of spermatozoa with structural chromosome abnormalities (5.1%) and the estimated conservative aneuploidy (1.4%) were within the range obtained in our control donors (6.9 and 4%). The sex ratio (45.3% X versus 54.7% Y) was not significantly different from the theoretical 1:1. A total of 309 sperm complements was analysed by FISH, 138 sequentially analysed by G- banding-FISH and another 171 analysed by FISH only. The frequencies of structural chromosome abnormalities for chromosomes 7 and 9 (0.6 and 0% respectively) were not significantly different from those obtained in our control donors (0.6 and 0.8%). No spermatozoa with the t(7;9) (q22;p23) were observed, showing no evidence for a germ-cell mosaicism. A statistically significant, positive association between sperm breakpoints and fragile sites (P = 0.0225) was observed. However, the coincidence between fragile sites and sperm breaks (80%) was not significantly different from that obtained in our control donors (79.2%). These results suggest that in this case the risk of structural chromosome abnormalities in further offspring is not increased, although an association between fragile sites and sperm chromosome breaks in the father does exist.      

Fragile site induction by aphidicolin may be increased in parents of neuroblastoma patients

We recently demonstrated an increased expression of fragile sites, induced by aphidicolin, in lymphocytes of neuroblastoma patients. We have now extended our studies to parents of affected children with neuroblastoma to verify if this characteristic may be genetically transmitted. We have examined 20 families. In most of them, the hypersensitivity to aphidicolin was found in the affected child and in at least one parent. Moreover, some of the parents showed an increase in the expression of the fragile sites 1p32, 1p13, or both that are preferentially expressed in neuroblastoma patients. The possible relations between the hypersensitivity to aphidicolin and the inheritance of predisposition to neuroblastoma must be clarified.     

Fragile sites, cancer chromosome breakpoints, and oncogenes all cluster in light G bands

Fragile sites tend to be bands where breaks occur in cancer chromosome rearrangements that can involve oncogenes. The locations of fragile sites, cancer breakpoints, and oncogenes were therefore charted. All were predominantly in light G bands. Specifically, 78 of 89 (88%) fragile sites were in light G bands including the large group of common fragile sites inducible with aphidicolin (p less than 0.001). Of 61 cancer breakpoints, 50 (82%) were in light bands including translocation breakpoints (p less than 0.001). Thirteen of 14 (93%) oncogenes localized to light bands. The sharing of chromosome bands can stem from a biologically meaningful relationship, as between cancer breakpoints and oncogenes. Joint occupancy of chromosome bands can also reflect independent reasons to be in the same sector of the genome. Thus, fragile sites may well be in light bands because they are associated with active genes. This clearly does not rule out a biologic relationship between specific fragile sites and specific cancer breakpoints.     

Animal model: Chromosomal fragile site expression in dogs: I. Breed specific differences

Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P < 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folatesensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folatesensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes.     

Chromosome instability in lymphocytes from two patients affected by three sequential primary cancers: the role of fragile sites

The chromosomal aberration rate and the expression of fragile sites induced by aphidicolin were evaluated in metaphase chromosomes obtained from peripheral blood lymphocytes of two untreated patients with multiple primary cancers. Spontaneous aberrations of chromosome number and structure and chromosome fragility were compared with controls with the use of the same methods. Chromosomal aberration rates and expression frequencies of fragile sites were significantly higher in the patients than in normal control subjects. In the patients, all but one structural chromosome aberration involved at least one fragile site. Our results suggest that fragile sites may be unstable regions of the human genome, which might play an important role in the genetic instability associated with cancer predisposition.