Patients with different lung cancers show normal expression of fra(3)(p14.2) in aphidicolin-treated lymphocyte cultures

Among common fragile sites, fra(3)(p14.2) is the most expressed either spontaneously or after treatment with aphidicolin (APC) in lymphocyte cultures. Because recurrent chromosomal abnormalities involving the short arm of chromosome 3 in tumor tissue are present in various malignancies, including lung cancer, the induction of fra(3)(p14.2) elicited by APC was investigated with the aim of detecting possible interindividual polymorphism in its expression that might be relevant to predisposition toward cancer-related events. Thirty-four patients affected with various lung cancers (14 squamous cell carcinomas, 13 adenocarcinomas, and seven small cell carcinomas) and 14 controls (patients undergoing routine routine follow-up after coronary by-pass) were included in this study. The frequency of fra(3)(p14.2) expression was not significantly different among the patients grouped either by disease or by sex and age. It was estimated that fra(3)(p14.2) accounts for about 20% of total breakage in APC-treated lymphocyte cultures from the general population.     

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.     

Expressivity of a common fragile site,fra(3)(p14.2), in patients with cancer and other diseases

Summary A population survey of a common folate-sensitive fragile site, fra(3)(p14.2), has been carried out on PHA-stimulated peripheral lymphocytes of patients with cancer and other diseases, under both culture conditions of folate deprivation and aphidicolin treatment. Overall findings regarding variability of expressivity due to age and sex were very similar to those obtained in a healthy population. The expression of fra(3)(p14.2) by folate deficient condition appeared hardly influenced by such exogenous factors as tobacco smoking habit, past histories of radiotherapy and chemotherapy, while it was associated with the unfavorable prognosis of cancers. Furthermore, proportion of those with higher expression was slightly but significantly larger in both lung and breast cancer patients. These findings suggest that some factors relevant to the expression of fra(3)(p14.2) may be associated with development and progression of certain kinds of cancer.     

Translocation t(3;8)(p14.2;q24.1) in renal cell carcinoma affects expression of the common fragile site at 3p14(FRA3B) in lymphocytes

The common fragile site at 3p14(FRA3B) is cytogenetically close to the positions of translocation and deletion breakpoints frequently observed in renal cell carcinoma (RCC) and small cell carcinoma of the lung. Possible involvement of this fragile site in the familial RCC t(3;8)(p14.2;q24.1) was investigated. Expression of FRA3B, induced by treatment of lymphocytes with aphidicolin, is altered by the translocation. These results suggest that the fragile site is very close to, if not coincident with, the translocation breakpoint.     

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.     

Investigation of genetic susceptibility to non-small cell lung cancer by fragile site expression

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.     

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.     

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.     

Fragile sites and genitourinary tumors

We tested for fragile sites in lymphocytes from nine patients with genitourinary tumors to determine if a correlation existed between their cancer chromosome breakpoints and fragile sites. Induction was done for rare fragile sites in all known classes by exposure of cells to fluorodeoxyuridine and bromodeoxyuridine (BrdU). No rare fragile sites were found. Induction was also done for common fragile sites in all known classes using aphidicolin (Apc), 5-azacytidine, and BrdU. Although 56 common fragile sites were detected, only a single site corresponded in location to a genitourinary tumor chromosome breakpoint. That was the common fragile site in band 3p14. No overall correlation was found between fragile sites and chromosome rearrangements in carcinoma of the kidney, ureter, bladder, and testis. The sole known candidate for a possible biologic role is the 3p14 common fragile site in renal cell carcinoma.     

The effect of hydroxyurea on the expression of the common fragile site at 3p14.

Hydroxyurea (HU) is an inhibitor of DNA synthesis, which can inhibit the enzyme ribonucleotide reductase, reduce the syntheses of all four deoxyribonucleoside diphosphates (dNDP), and disturb the balance of the dNTP pool. We have studied the effect of HU on the common fragile site at 3p14 (FRA3B) and have found that G2 treatment with HU increased not only the frequency of chromosomal aberration but also the expression of FRA3B in both complete and folate deficient media. There is a synergistic effect between HU and growth in folate deficient medium on the induction of FRA3B. Our results suggest that the inhibition of DNA repair, including the inhibition in G2 phase, plays an important role in the expression of FRA3B, supporting other authors' data on the effect of other DNA repair inhibitors, such as aphidicolin, caffeine, 1-beta-D-arabinofuranosylcytosine, and 5-fluorodeoxyuridine, on the expression of FRA3B.     

Heritable fragility at 11q13 and 12q13

The chromosomes of two mentally retarded probands were investigated because they were suspected of having the fragile X syndrome. However two other fragilities were detected. In one patient a fra(11)(q13) was found and in the other a fra(12)(q13). Family studies revealed that both fragile sites were real heritable ones. Besides these two heritable fragile sites, the common fragile site at 3p14 was frequently observed. The effects of BUdR, FUdR and methotrexate on the frequency of the three fragilities were studied. The two heritable fragile sites differed from the common fragile site at 3p14 with respect to their inducibility by FUdR and methotrexate.     

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.     

Fra(X)(q27.2), the common fragile site, observed in only one of 760 cases studied for the fragile X syndrome.

Cell cultures from 760 whole blood, amniotic fluid, chorionic villus sample, and peripheral umbilical blood sample specimens were exposed to multiple fra(X)(q27.3) induction systems (none had aphidicolin). Fifty-three exhibited the rare fragile site, fra(X)(q27.3) or FRAXA, none of which demonstrated the common fragile site or FRAXD at band Xq27.2. Only one cell in one of the negative whole blood FUdR-treated cultures from a mentally retarded male showed FRAXD. Therefore, it appears that FRAXD occurs very rarely in cultures treated to induce FRAXA since only one positive cell was observed in over 88,000 analyzed. It appears that very low frequencies of fra(X)(q27) can be accounted for only in part by the presence of the common fragile site since only one of 9 cases, each with one fra(X)(q27) positive cell, exhibited FRAXD and the others were FRAXA. After confirmation of FRAXA with direct DNA testing in a large number of low frequency cases, it should be possible to rely on the detection of very low frequencies of fra(X)(q27.3), e.g., 1% with at least 2 positive cells.     

Autosomal folate sensitive fragile sites in normal and mentally retarded individuals in Greece

The frequencies of autosomal folate sensitive fragile sites were compared in populations of mentally retarded fra(X) negative (N = 220) and normal children (N = 76) in Greece. In addition, the frequency of autosomal fragile sites was studied in 20 known fra(X) children in order to test if the fra(X) syndrome is associated with general chromosome instability. The frequencies of both common and rare autosomal fragile sites did not differ significantly between the mentally retarded and the normal children, although the rate of expression was considerably higher in the retarded group. Autosomal fragile sites were not increased in the fra(X) patients. Fra(3)(p14) was by far the most frequent one in all groups. The frequency of fra(6)(q26) was found to be considerably higher among the mentally retarded children, this difference being almost statistically significant. Further cytogenetic studies of normal and retarded individuals are required in order to elucidate this point further.     

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 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.     

Loss of common 3p14 fragile site expression in renal cell carcinoma with deletion breakpoint at 3p14

The common fragile site in human chromosome band 3p14 is a constant cytogenetic marker present on every normal chromosome #3. Therefore, we selected a renal cell carcinoma with a deletion breakpoint in 3p14 for analysis of the 3p14 fragile site. Aphidicolin was used to induce the expression of the 3p14 fragile site. The fragile sites expressed in the renal carcinoma cells generally mirrored those expressed in lymphocytes. The normal chromosome #3 in the renal carcinoma cells expressed the common 3p14 fragile site. The partially deleted #3 did not. The deletion breakpoint, therefore, cannot be beyond the 3p14 fragile site. The common fragile site in 3p14 must be at or very near the deletion breakpoint in 3p14 in renal cell carcinoma. These results are consistent with this fragile site causing this cancer chromosome deletion.     

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.      

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.     

Reduced expression of aphidicolin-induced common fragile sites in peripheral lymphocyte chromosomes of patients with B-cell chronic lymphocytic leukemia

The occurrence and frequency of aphidicolin-induced common chromosomal fragile sites were examined in 12 B-cell chronic lymphocytic leukemia (B-CLL) patients (ten males and two females) and three normal individuals. The mononuclear cells separated by Ficoll-Hypaque gradient were cultured in vitro for 96 hours stimulated by pokeweed mitogen (PWM) in combination with T-leukemia cell conditioned medium or 10% B-cell growth factor. For the final 24 hours the cells were treated with aphidicolin (0.07 microgram/ml). Results indicate that there was a significant reduction in the overall mean frequency of common fragile sites in CLL patients with a wide individual variation. Fragile sites were found to be localized either on a single chromatid or both chromatids, but rarely involved homologous chromosomes. No definite relationship between the frequency of fragile sites and the staging of CLL disease was observed. A significant reduction and variability in the frequency of fragile sites suggest the heterogenous nature of B-CLL and probably a different mechanism of induction of fragile sites in CLL cells compared to controls.