Livia Bertoni

Fluorescence in situ hybridization with a synthetic (T2AG3)n polynucleotide detects several intrachromosomal telomere-like repeats on human chromosomes

(T2AG3) repeats comprise the telomeres of human chromosomes and also are present at interstitial locations. Using a long synthetic (T2AG3)n probe, we have localized telomere-like repeats at several internal sites on human chromosomes.

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Molecular organization of internal telomeric sequences in Chinese hamster chromosomes

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.

Intrachromosomal telomere-like DNA sequences in Chinese hamster.

The simple repetitive sequence (TTAGGG)n constitutes the telomeres of vertebrate chromosomes. In several species, telomeric repeats have been observed at intrachromosomal sites (Meyne et al. 1990). It has been proposed that interstitial (TrAGGG)n sequences may be prone to fragility and recombination and that breakage within such repetitions may produce telomere-like structures that stabilize the ends of rearranged chromosomes (Ijdo et al. 1991; Meyne et al. 1990). This hypothesis is supported by the cytogenetic correlation between induced breaks and intrachromosomal telomeric sites in Chinese hamster cell lines (Alvarez et al. 1993; Balajee et al. 1994). Furthermore, it has been shown that the recombination events that accompanied CAD gene amplification frequently involved (TTAGGG) repetitions (Bertoni et al. 1994) and that the introduction of human telomeres into rodent or human cells could induce chromosome fragmentation (Farr et al. 1991; Barnett et al. 1993). In Chinese hamster the precise localization of (TTAGGG)n sequences has not been analyzed. In this paper we report the chromosomal localization of these sequences in primary diploid fibroblasts, and we show that they map at telomeric, centromeric and interstitial sites. The interstitial sequences, which have not been described before in Chinese hamster, contain a relatively small number of repeats and may derive from telomere fusion or centromere inactivation of ancestral chromosomes. Moreover, we have observed that spontaneous breaks, occurring during the senescence crisis of primary fibroblasts, are located preferentially within chromosome bands containing (TTAGGG)n repeats. We then mapped the (TTAGGG)n sequences in a CHO-K1 derived cell line and showed that the chromosomes can be identified without banding. New information about the origin of some marker chromosomes has been obtained. Metaphase chromosomes from a Chinese hamster (CricetuIus griseus) primary fibroblast cell line (CHL) were hybridized in situ with a (TTAGGG)n probe and G-banded after hybridization according to the methods previously described (Bertoni et al. 1994). The probe was a mixture of biotinylated synthetic (TTAGGG)n fragments with a size range between 1 and 20 kb. The localization of hybridization signals has been analyzed on 37 metaphases, which were obtained from cells passaged 8 times. In Fig. la two examples from each chromosome pair are shown. The karyotype of CHL cells is identical to the normal Chinese hamster karyotype, which has been described (Ray and Mohandas 1976; Simi et al. 1988). Briefly, 11 chromosome pairs are present: 9 metacentric or submetacentric (1, 2, 3, 4, 8, 9, 10, X, and Y) and 3 acrocentric pairs (5, 6, and 7). All chromosomes show hybridization signals at both ends, indicating that long stretches of (TTAGGG)n repetitions are present at the telomeres. A subterminal localization of the telomeric signal, which is observed in some CHL chromosomes of Fig. la, has been previously described by others and seems to be due to chromosome condensation (Moyzis et al. 1988).

Establishment and characterization of a new cell line from primary human breast carcinoma

SummaryA new cell line (BRC-230) was established from surgical material of primary ductal infiltrating breast carcinoma. The epithelial nature of this cell line was confirmed by ultrastructural analysis and demonstrated the retention of structural properties characteristic of the original tumor. The BRC-230 cell line induced tumor in athymic Cr1:nu/nu(CD-1)BR nude mice, it possessed an abnormal karyotype with a modal chromosome number between 60–61 with eight recurrent marker chromosomes, and it presented a doubling time of 30.5 hr. Scatchard analysis demonstrated that both primary tumor and BRC-230 cells were estrogen and progesterone receptor negative.Immunoenzymatic and radioimmunoassays showed a production of marker antigens (CEA, TPA, CA125, CA15-3, CA19-9) which was similar in the patients serum and BRC-230 cells. Thein vitro drug sensitivity assay of the cell line and of the parental tumor tissue showed overlapping results to all tested antiblastic drugs. BRC-230 cells were resistant to 4-Idroperoxy-cyclophosphamide, Idarubicinol, Mitoxantrone, Etoposide, 4′Epidoxorubicin, and Doxorubicin, showing a multiple drug resistance phenotype. Amplification or rearrangement of Her-2neu, Ha-ras, and C-myc genes was observed neither in the original tumor nor in BRC-230 cells; the mdr-1 gene was also present in a single copy.We conclude from these studies that the BRC-230 cell line maintains the same characteristics as the original tumor and may provide us with a good model to studyin vitro the biology of drug resistance of breast cancer.

Two extended arrays of a satellite DNA sequence at the centromere and at the short-arm telomere of Chinese hamster chromosome 5

We have cloned a Chinese hamster chromosome-specific repeated sequence (SatCH5). This satellite is composed of a 33-bp unit organized in two extended tandem arrays. It is localized at the centromere and at the short-arm subtelomere of chromosome 5. Altogether, SatCH5 covers about 1–2 Mb per diploid genome and is not present in other species, including the Syrian hamster and mouse. Since it is known in the Chinese hamster and numerous other vertebrate species that telomeric (TTAGGG)n repeats are localized at the centromeres of several chromosomes, we studied the localization of SatCH5 relative to (TTAGGG)n sequences. Using two-color fluorescence in situ hybridization on stretched chromosomes and on DNA fibers, we have shown that at the centromere of chromosome 5 SatCH5 and the (TTAGGG)n arrays are contiguous. SatCH5 is the first chromosome-specific repetitive sequence located at both the pericentromeric and subtelomeric regions of the same chromosome.

Different genome organization in two new cell lines established from human gastric carcinoma

Two gastric cancer cell lines, AKG and GK2, were established from a pleural and an ascitic effusion, respectively. GK2 cells have a pseudodiploid karyotype with an add(6)(q27) chromosome in all metaphases examined. The karyotype of AKG cells is highly rearranged: FISH analysis with painting probes has shown that DNA sequences derived from single chromosomes are scattered on several (as many as eight) markers. In this cell line, the C-MYC and the K-RAS oncogenes are amplified. The organization and the copy number of the C-MYC-amplified units are different from the K-RAS units, suggesting that the two oncogenes were amplified independently. The presence of a few marker chromosomes carrying both C-MYC and K-RAS could be due to translocation events that followed the amplification.

Amplification of the pericentromeric region of chromosome 1 in a newly established colon carcinoma cell line

The LRWZ cell line was established from an ascitic effusion of a colon adenocarcinoma. We studied the karyotype of LRWZ cells using G-banding and chromosome painting. The cell line is near triploid and is characterized by several chromosome rearrangements and pronounced intermetaphase variation. Chromosome painting probes revealed numerous labeled regions on different chromosomes, indicating that several translocations occurred during the evolution of the cell population. The 10 recurrent marker chromosomes identified (M1-M10) were derived from complex rearrangements involving up to three different chromosomes. M2 is a particularly interesting marker that originated from the amplification of the pericentromeric region of chromosome 1 and has a peculiar organization comprising five copies of the region included between 1p21 and 1q21 and is surprisingly stable: it is present in all the metaphases analyzed, has telomeric DNA at both termini, and contains one active and four inactivated centromeres. To provide insights into the molecular mechanisms that generated M2, we performed fluorescence in situ hybridization experiments using a panel of probes mapping near the centromere of chromosome 1 and three probes for different satellite sequences; the formation of chromosome M2 required the intervention of several rearrangements including unequal exchange, chromatid breakage followed by fusion of the sister chromatids, and loss of centromeric heterochromatin.

Assignment of the Equus caballus interleukin 8 gene (IL8) to chromosome 3q14.2→q14.3 by in situ hybridization

Human interleukin 8 (IL-8) is an anti-infl ammatory cytokine. A signifi cant increase of the IL-8 concentration has been found in the plasma of marathon runners (Suzuki et al., 2000) and a similar upregulation of this interleukin was recently detected in lymphocytes of horses immediately after intense exercise (Cappelli, Verini-Supplizi, Capomaccio and Silvestrelli unpublished results). Equus caballus (ECA) IL8 full length cDNA, isolated from peripheral blood lymphocytes of an endurance horse, was sequenced (GenBank accession number AY184956). The mRNA sequence is 1089 nt in length and contains a 306 nt region that putatively codes for a 101 amino acid protein. In the framework of studying the modulation of gene expression profi les in racing horses, we report here the chromosomal location of ECA IL8 . This result adds a new type I marker to the FISH map of the horse.

Late onset of CAD gene amplification in unamplified PALA resistant Chinese hamster mutants

In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has always been found associated with amplification of the CAD gene (carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase). We describe two PALA resistant Chinese hamster mutant cell lines in which amplification of the CAD gene was not present. The PALA resistant phenotype was stable when the cells were grown in non-selective medium. However, after prolonged growth in the presence of the same drug concentration used for selection, cells with increased CAD gene copy number and higher levels of resistance overrode the original population. In these cell populations, a heterogeneous organization of the CAD genes was revealed by fluorescence in situ hybridization on mitotic chromosomes indicating that the additional copies of the gene were generated in several ways, such as non-disjunction and breakage-fusion-bridge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberrations in the cells grown in the presence of the drug, could have favored the late onset of the amplified mutants. It is tempting to speculate that, during the expansion of tumor populations, different drug resistance mechanisms, including gene amplification, could occur in succession and lead to the generation of cells highly resistant to chemotherapeutic agents.