Characteristic chromosomal fragility of human embryonic cells exposed in vitro to aphidicolin.

The frequency and distribution of aphidicolin (APC)-induced common fragile sites (cfs) were analyzed in human embryonic cells of different origins. Embryonic lung fibroblasts (MRC-5), amniocytes (AMINO) and embryonic retina cells (HERO790) are as sensitive to the APC-induced clastogenic effect as peripheral lymphocytes, whereas embryonic kidney cells (HEK) seem more resistant to the induction of chromosomal gaps and breaks by the drug. Analysis of the distribution of fragile sites confirmed that the expression of specific APC-induced cfs varies in different cells and that the embryonic cell strains show a greater similarity among themselves than to lymphocytes. In addition, HEK, MRC-5, HERO790 and AMINO cells show specific APC induction of the cfs at the 1p31.2 chromosomal band, which seems to be a distinctive feature of the embryonic stage of cells.

Subregional localization of 14 yeast artificial chromosomes to human chromosome region 1p by fluorescence in situ hybridization.

We have sublocalized to the region between 1p22 and 1p33 a total of 14 yeast artificial chromosomes previously assigned to a broader area of human chromosome 1p. Our purpose was to map DNA sequences that could be used for the molecular characterization of the two common fragile sites present in bands 1p31.2 and 1p32, the expression of which is increased in patients with neuroblastomas.

Hypersensitivity of lymphoblastoid lines derived from ataxia telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16).

Mammalian DNA topoisomerase II represents the cellular target of many antitumor drugs, such as epipodophyllotoxin VP-16 (etoposide). The mechanism by which VP-16 exerts its cytotoxic and antineoplastic actions has not yet been firmly established, although the unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors suggest the involvement of DNA double-strand breaks. In the present study we analyzed the chromosomal sensitivity of lymphoblastoid cell lines derived from ataxia telangiectasia (AT) patients to low concentrations of the drug. Our results indicate that AT derived cells are hypersensitive to the clastogenic activity of VP-16 either when the drug is present for the whole duration of the cell cycle or specifically in the G2 phase, confirming that the induction of DNA double strand breaks, to which AT cells seem typically sensitive, could have an important role in the biological activity of VP-16.

Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident.

The present study concerns the monitoring of children from the Byelorussian, Ukrainian and Russian republics exposed to the fall-out of the Chernobyl accident. Cytogenetic analyses have been performed on 41 children coming from different areas and exhibiting varying amounts of 137Cs internal contamination, as evaluated by whole-body counter (WBC) analysis. On a total of 28,670 metaphases scored, radiation-induced chromosome damage is still present, although at a very low frequency. Due to the very low fraction of dicentrics, because of the time elapsed from the accident and the relatively low doses of exposure, radiobiological dosimetry is not possible for these children. However, considering that the WBC data indicate that the children are still exposed to 137Cs contamination, the observed occurrence of stable chromosome rearrangements and breaks may represent the persisting effect of continuous low doses of radiation. The present study also indicates that the parallel use of internal contamination dosimetry and cytogenetics could be usefully employed to monitor individual exposure to radiation and to define further management measures.

Increased chromosome fragility in lymphocytes of short normal children treated with recombinant human growth hormone.

A few years ago it was reported that some growth-hormone-deficient children had developed leukemia following therapy with human growth hormone. This raised concern that this therapy may stimulate tumor development. Since it is known that the tendency to develop cancer is closely related to chromosome breakage, we decided to investigate whether recombinant human growth hormone (rhGH) therapy can increase chromosome fragility. Ten short normal children were studied during their first year of treatment. Lymphocytes were collected at 0, 6 and 12 months of rhGH therapy, and we assessed the rate of spontaneous chromosome aberrations, the frequency of sister chromatid exchanges, the proliferative rate indices, the expression of common fragile sites induced by aphidicolin, and the sensitivity towards the radiomimetic action of bleomycin. At 6 months of therapy, there was a significant increase in bleomycin-induced chromosome aberrations, which remained unchanged after 1 year of treatment. An increase in spontaneous chromosome rearrangements at 6 and 12 months of therapy was also observed. These findings are further supported by data obtained from the analysis of 16 short normal children already on rhGH therapy.

Incidence of chromosome abnormalities and clinical significance of karyotype in de novo acute myeloid leukemia.

Cytogenetic studies with high-resolution banding were performed on specimens from 132 consecutive patients with de novo acute myeloid leukemia (AML). All patients were treated according to therapeutic protocols in the same institution. Clonal abnormalities were detected in 97 of the 124 patients in whom an adequate number of mitoses was obtained (78.2%). Neither sex, FAB classification, WBC, or the extent of bone marrow infiltrate affected the rate of chromosomal aberrations, whereas patients younger than 40 years had a greater proportion of normal karyotypes (p = 0.047). Two different chromosomal classifications were evaluated: the presence of normal and abnormal metaphases (NN-AN-AA classification), and a classification in cytogenetic categories, the latter being based on the frequency of cytogenetic abnormalities. Both classifications were found to correlate significantly with the clinical outcome. They also showed independent prognostic significance when age, sex, and FAB morphology were considered in a multivariate analysis. Two abnormalities were closely associated with specific clinical-pathologic subsets of AML. All the 15 patients with t(15;17) had acute promyelocytic leukemia; this translocation was not found in any other subset of AML. Eight of the nine patients presenting rearrangements at 11q23 belonged to a FAB subset with monocytic differentiation (M4 and M5). Our data suggest that cytogenetic findings should influence the therapeutic approach to AML. In particular, young patients with karyotypes associated with poor responses may be considered for more eradicating treatments, including allogenic bone marrow transplantation.

Sensitivity to bleomycin and arabinoside cytosine in lymphocytes of patients affected by neuroblastoma and in those of their parents.

Chromosomal instability has been described in patients affected by various tumors. We previously reported a high sensitivity to fragile sites induction by aphidicolin in lymphocytes from patients affected by neuroblastoma and in those from their parents. In the search for the most suitable clastogenic agent to enhance the possible differences between healthy controls and patients affected by tumors, we have now tested two other drugs: bleomycin, a radiomimetic agent already used in vitro on chromosomes of patients affected by other tumors and arabinoside cytosine, an inhibitor of DNA polymerases alfa and beta. We observed a high sensitivity to bleomycin both in patients and in their parents, but to arabinoside cytosine only in NB patients. Moreover, the two drugs induced more fragile sites in 1p in patients and in their parents than in healthy controls. This phenomenon, which we already observed after treatment with aphidicolin, might be related to the frequent deletions and loss of heterozigosity in 1p in neuroblastoma cells.

Enhanced chromosomal fragility in neuroblastoma: correlation with poor prognosis.

In a previous study we demonstrated spontaneous fragility and hypersensitivity to fragile site induction by aphidicolin in lymphocytes from some neuroblastoma patients and their parents. Here we report data based on a total of 40 patients and 37 families. Possible correlations between higher sensitivity to aphidicolin and a variety of personal and clinical characteristics were verified. Patients with a poor prognosis generally proved to be more susceptible to fragile site induction.

Population cytogenetics of aphidicolin-induced fragile sites.

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.

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.

Structural chromosomal rearrangements in HpaII-treated human lymphocytes.

Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.

Fragile sites and chromosome instability: the distribution of breaks induced by cis-diamine-dichloro-platinum (II) in Fanconi anemia lymphocyte cultures.

The distribution of chromosome breaks induced by the antitumor drug cis-diamine-dichloroplatinum (II) in lymphocyte cultures from Fanconi anemia patients was analyzed. Breakpoints occurred nonrandomly over an arbitrarily defined human karyotype of 319 bands. These bands were classified according to either their banding pattern or their fragile site status (whether or not a fragile site of a given type is located at a chromosomal band). A significant involvement of G-light and fragile site bands was detected. The preferential occurrence of breaks at fragile site bands was limited to common fragile site bands (essentially of the aphidicolin-type).

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.

Investigations on the PGM (phosphoglucomutase) polymorphism by isoelectric focusing in Drosophila melanogaster.

Pgm allele frequencies of 383 individuals were determined in a sample of Drosophila melanogaster from three laboratory Sardinian populations, using the techniques of standard electrophoresis, heat denaturation, and isoelectric focusing. The analysis of the progeny obtained from informative crosses showed that the isoelectric focusing patterns segregate in a Mendelian way. The Pgm1.00 and Pgm0.70 electrophoretic alleles displayed different isoelectric points, whereas the Pgm1.00,tr and Pgm1.00,ts isoelectrophoretic alleles could not be differentiated when tested by isoelectric focusing. Moreover, the Pgm0.70,ts allele was split into two classes, with isoelectric points of pH 6.4 and pH 6.6.

Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.

Despite the fact that the horse is one of the more common domesticated animals, there are few reports dealing with the properties of its blood, and no comprehensive study has been performed on the reactivity of horse haemoglobin towards organic and inorganic ions. Here we report data on the effects of the organic phosphates D-glycerate-2,3-bisphosphate (2,3-DPG) and InsP6, and of chloride on the properties of horse haemoglobin. Thus the effect of saturating concentrations of 2,3-DPG on the oxygen affinity of horse haemoglobin is about 60% lower than with human adult haemoglobin under the same experimental conditions. The same applies also to InsP6, whose effect on oxygen binding to horse haemoglobin is decreased by about 55% compared with human adult haemoglobin. On the whole, horse haemoglobin appears to be much less sensitive to organic phosphates than previously believed. These results are discussed in the light of the primary structure of the molecule.

Cytogenetic effects of near ultraviolet radiation in normal and systemic lupus erythematosus lymphocytes.

We have conducted a study on the spontaneous and UV-A-induced frequency of chromosomal breaks and sister-chromatid exchanges (SCE) in purified lymphocytes from normal donors and from systemic lupus erythematosus (SLE) patients who were in clinical remission at the time of the study. Our results show that although SLE lymphocytes exhibit a higher frequency of spontaneous SCEs than controls, the rate of chromosomal breakage is comparable in the 2 groups. In both controls and patients, irradiation with UV-A (320-400 nm) increases the SCE values but does not significantly affect the frequency of chromosomal aberrations.

Chromosome fragile sites in Down syndrome patients.

We report on the fragile site expression in lymphocytes from 16 Down syndrome (DS) men aged 24 to 52 years. Among rare fragile sites, 16q22 has been reported to be induced or enhanced by alpha-interferon. Since DS cells have three copies of the receptor for alpha-interferon, we hypothesized a possible enhancement of 16q22 expression. This fragile site has also been related to a specific rearrangement in M4 acute nonlymphocytic leukemia. In view of the high incidence of acute leukemia in DS subjects, we studied the expression of 16q22 in lymphocyte cultures treated with 5-bromodeoxyuridine or alpha-interferon. We also studied whether the repair deficiency of DS cells could affect the expression of aphidicolin-induced fragile sites. The level of chromosomal aberrations was compared with that found in aphidicolin-treated cultures from 12 normal subjects of the same age. We found neither spontaneous or BrdU-induced fragility at 16q22 nor induction by alpha-interferon. Chromosomal breakage rate was increased in alpha-interferon-treated cultures in comparison with control cultures of the same subjects. Aphidicolin-induced fragile sites expression in DS patients did not differ significantly from that found in the lymphocyte cultures from control subjects.

The segregation distortion (SD) phenomenon in wild populations of Drosophila melanogaster: interaction between chromosomes 3 and SD chromosomes 2.

The Segregation Distortion (SD) phenomenon is a typical case of non-Mendelian segregation in Drosophila melanogaster, due to the dysfunction of sperm bearing a non-SD homologous chromosome. In nature, several factors involved in the expression of the SD phenomenon have been described; among these, a genetic modifier carried by chromosome 3, which enhances the distortion effect of the SD chromosomes. The analysis of natural Sardinian populations, carried out in order to evaluate the presence of chromosome 3 bearing these enhancer factors, has enabled us to ascertain that (a) also in these populations chromosomes 3 with enhancer factors are present, although with frequencies lower than those previously reported in other publications; (b) among these enhancer chromosomes 3, some increase the k of certain chromosomes 2 from values of chromosomes considered non-distorting (k less than or equal to 0.66) to value typical of SD chromosomes. The data obtained also allow us to put forward some considerations regarding the dynamics of the SD phenomenon in Sardinian populations, where the frequency of SD chromosomes is fairly elevated.

The distribution of MspI-induced breaks in human lymphocyte chromosomes and its relationship to common fragile sites.

The restriction endonuclease MspI (cleavage site C/CGG) induces chromosomal breaks in human lymphocytes. The breakpoints are distributed non-randomly along the chromosomes and the pattern of MspI-induced breakage depends on the recovery time (20 h or 6 h). Chromosomal bands preferentially involved in breakage are likely to coincide with bands where common fragile sites are located.