Bernard Dutrillaux

A simple method for simultaneous R- or G-banding and fluorescence in situ hybridization of small single-copy genes

A significant improvement in fluorescence in situ hybridization, enabling the detection of single-copy genes as small as 500 bp directly on banded chromosomes, is presented. The induction of chromosome banding, which does not require additional handling or any system of amplification, is obtained simply by using an alkaline (pH 11) p-phenylenediamine anti-fade solution. As the banding produced is related to the timing of 5-bromodeoxyuridine incorporation, either R- or G-banding, constitutive heterochromatin staining, or chromosome asymmetry can be observed simultaneously with the fluorescent hybridized spots. Results of hybridization of small cDNA probes for the human genes for motilin, thymidylate synthetase, and lymphocyte activation-3 are provided as examples of the high-resolution mapping obtainable with this technique.

Characterization of chromosomal anomalies in human breast cancer: A comparison of 30 paradiploid cases with few chromosome changes

A comparison of chromosomal anomalies detected in 30 cases of breast cancer in females with near-diploid karyotypes is reported. The tumors, of which 20 were previously unpublished, were selected for the relatively low complexity of their karyotypes, among a sample of 118 cases. Almost all of the 151 structural rearrangements detected were unbalanced, and 67% of breakpoints were located in or had contact with heterochromatin. In cases with few anomalies, rearrangements of chromosomes 1 and/or 16 were very frequent, leading principally to a gain of 1q and loss of 16q. In cases with more anomalies (5-16), deletions involving 17p, 4p, 13, 6q, 8p, 9p, 11p, and 11q and gains of 1q and 8q were the most frequent. Homogeneously staining regions (HSR) were detected in 14 tumors, mostly on 8p (6/22) and chromosome 19 (3/22). No double minutes (dmin) were observed. We conclude that trisomy 1q and monosomy 16q are early chromosomal changes in breast cancer, whereas other deletions and gain of 8q are clearly secondary events.

Mutations in OGG1, a gene involved in the repair of oxidative DNA damage, are found in human lung and kidney tumours

The human OGG1 gene encodes a DNA glycosylase activity catalysing the excision of the mutagenic lesion 7,8-dihydro-8-oxoguanine from oxidatively damaged DNA. The OGG1 gene was localized to chromosome 3p25, a region showing frequent loss of heterozygosity (LOH) in lung and kidney tumours. In this study, we have analysed by RT–PCR the expression of OGG1 in 25 small cell lung cancers, in 15 kidney carcinomas and the 15 normal kidney counterparts. The results show that OGG1 messenger RNA can be detected in all tumours tested and that no significant difference was observed in the level of expression between normal and tumoral kidney tissues. Denaturing gradient gel electrophoresis (DGGE) was used to screen this series of human tumours for alterations in the OGG1 cDNA. The study revealed homozygous mutations in three tumours, two from lung and one from kidney. Sequencing analysis of the mutants identified a single base substitution in each of the three cases: two tranversions (GC to TA and TA to AT) and one transition (GC to AT). All three substitutions cause an amino acid change in the hOgg1 protein. For the mutant kidney tumour, the normal tissue counterpart shows a wild-type profile. These results suggest a role for OGG1 mutations in the course of the multistage process of carcinogenesis in lung or kidney.

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.

High frequencies of inversions and translocations of chromosomes 7 and 14 in ataxia telangiectasia

The R-banding of more than 1100 lymphocytes and fibroblasts from 11 patients with ataxia telangiectasia (AT) showed rearrangements of chromosomes 7 and/or 14 in about 7% of the cells. Among these rearrangements, pericentric inversion of chromosome 7 was the most frequent, and the paracentric inversion of chromosome 14 not very rare. These inversions are believed to be fairly specific of AT, and their absence in the previously reported literature may be due to technical problems. Inversions were also observed in the lymphocytes of presumed heterozygote carriers with a lower frequency, and may be of some help for the detection of healthy heterozygote carriers of the AT gene.

Breast cancer genetic evolution: I. Data from cytogenetics and DNA content.

SummaryA general scheme of chromosome alterations occurring during tumor progression is proposed from the cytogenetic study of 113 breast carcinomas. For 76 of these tumors, chromosome numbers and rate of chromosome rearrangements were correlated with DNA content studied by flow cytometry. A series of 536 cases was used as control for flow cytometry. The following evolution can be proposed: 1. occurrence of unbalanced rearrangements decreasing chromosome number and DNA content; 2. correlatively to the rate of chromosome rearrangements, formation of endoreduplications leading to hyperploid sidelines; 3. persistence of the near diploid cells and decrease of chromosome number to about 35 and of DNA index to .85; 4. more frequently, elimination of the near diploid cells and complete passage to hyperploidy; 5. further losses of chromosomes in the hyperploid tumors, whose karyotypes can decrease to about 55 chromosomes and a DNA index of 1.35; 6. eventually, occurrence of a second endoreduplication, leading to an apparent near tetraploidy. The rate of rearranged chromosomes may reach 80% in both near diploid tumors with 35–40 and hyperploid tumors with 55–65 chromosomes which can be regarded as those with the highest degree of tumor progression. It is shown that the increase of chromosome number and DNA index above diploidy is very limited, and that all tumors with more than 50 chromosomes and 1.35 DNA content passed through endoreduplication. This results in many possible losses of heterozygosity in these cases.

Cytogenetics of colorectal adenocarcinomas

The occurrence of nonrandom chromosomal anomalies in colorectal adenocarcinomas could be demonstrated from the cytogenetic study of 100 cases. The most frequent changes are a rearrangement of chromosome 17, leading to the loss of its short arm and a loss of one chromosome 18. Three types of tumors with abnormal karyotypes can be defined. First are the monosomic-type near-diploid tumors (MD), characterized by a monosomy of both 17p and chromosome 18 mostly associated with other recurrent monosomies. In two of three cases, one or several minor derived polyploid subclones are also observed. Second are the monosomic-type polyploid tumors (MP), which have a pattern of chromosome imbalance very similar to that of MD tumors. They derive from MD tumors by endoreduplication followed by complete disappearance of the original MD clone. Third are the trisomic-type tumors (TT), which lose either 17p or chromosome 18 or none, most of the anomalies being gains of entire chromosomes. These TT tumors never undergo endoreduplication. In addition, seven tumors with normal karyotypes were found and may constitute another category (NT). A nonrandom distribution of these tumor types in relation to tumor site was observed, since in the distal colon, TT and NT tumors are underrepresented and endoreduplications are significantly more frequent. The level of chromosomal mutagenesis is two- to threefold higher in MD and MP than in TT tumors. More than 95% of the rearrangements are unbalanced, and most of them result from breakpoints located in juxtacentromeric heterochromatin. A good correlation is found between our results and the available molecular data on allelic losses. The involvement of recessive tumor suppressor genes in colorectal tumorigenesis and the possible relationship between chromosomal imbalances and deviations in metabolic pathways is described.

The Peripheral Benzodiazepine Receptors: A Review

Peripheral benzodiazepine receptors (PBRs) have been identified in various peripheral tissues as well as in glial cells in the brain. This review describes the tissue and subcellular distribution of the PBR in mammalian tissues and analyzes its many putative endogenous ligands. It deals with the pharmacological, structural and molecular characterization of the PBR, the proteins associated with the receptor (VDAC, ANC, PRAX-1) and their roles in cell growth and differentiation, cancer, steroid biosynthesis, and other physiological roles.

Establishment of Human Colon Cancer Cell Lines from Fresh Tumors versus Xenografts: Comparison of Success Rate and Cell Line Features

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.

Specific chromosome instability induced by heavy ions: a step towards transformation of human fibroblasts?

Cultures of human skin fibroblasts were exposed to heavy ions: neon (E = 10.74 MeV/u) and argon (E = 10.52 MeV/u) at fluences of 10(6), 2 x 10(6) and 4 x 10(6) and lead (E = 9.5 MeV/u) at a fluence of 2 x 10(6) particles/cm2. Cultures were further prolonged for up to 25 passages and karyotyping was performed at various times. Radiation-induced chromosome anomalies progressively decreased, became quite rare at passages 5-7 and increased at later passages. Around passages 20-25, most anomalies occurring were dicentrics, involving telomeric regions of 13p and q arms principally and to a lesser degree those of 1p, 16p and 16q arms. These non-random rearrangements paralleled the appearance of clones with unbalanced karyotypes. In particular, two independent proliferating clones were characterized by a monosomy 13. It is concluded that most chromosome lesions directly induced by heavy ions are hardly compatible with cell survival and thus disappear after a few cell generations. However, surviving cells acquire a de novo chromosome instability leading to the formation of clones with unbalanced karyotypes at late passages.