Search

Genomic data for wild species of the genus Bubalus (Asian buffaloes) are still lacking while several whole genomes are currently available for domestic water buffaloes. To address this, we sequenced the genome of a wild endangered dwarf buffalo, the lowland anoa (Bubalus depressicornis), produced a draft genome assembly, and made comparison to published buffalo genomes.The lowland anoa genome assembly was 2.56 Gbp long and contained 103,135 contigs, the longest contig being 337.39 kbp long. N50 and L50 values were 38.73 kbp and 19.83 kbp, respectively, mean coverage was 44x and GC content was 41.74%. Two strategies were adopted to evaluate genome completeness: (i) determination of genomic features with de novo and homology-based predictions using annotations of chromosome-level genome assembly of the river buffalo, and (ii) employment of benchmarking against universal single-copy orthologs (BUSCO). Homology-based predictions identified 94.51% complete and 3.65% partial genomic features. De novo gene predictions identified 32,393 genes, representing 97.14% of the reference’s annotated genes, whilst BUSCO search against the mammalian orthologues database identified 71.1% complete, 11.7% fragmented and 17.2% missing orthologues, indicating a good level of completeness for downstream analyses. Repeat analyses indicated that the lowland anoa genome contains 42.12% of repetitive regions. The genome assembly of the lowland anoa is expected to contribute to comparative genome analyses among bovid species.

Recent studies have reported evidence suggesting that portions of contemporary human genomes introgressed from archaic hominin populations went to high frequencies due to positive selection. However, no study to date has specifically addressed the post-introgression population dynamics of these putative cases of adaptive introgression. Here, for the first time, we specifically define cases of immediate adaptive introgression (iAI), in which archaic haplotypes rose to high frequencies in humans as a result of a selective sweep that occurred shortly after the introgression event. We define these cases as distinct from instances of selection on standing introgressed variation (SI), in which an introgressed haplotype initially segregated neutrally and subsequently underwent positive selection. Using a geographically diverse dataset, we report novel cases of selection on introgressed variation in living humans and shortlisted among these cases those whose selective sweeps are more consistent with having been the product of iAI rather than SI. Many of these novel inferred iAI haplotypes have potential biological relevance, including three introgressed haplotypes that contain immune-related genes in West Siberians, South Asians, and West Eurasians. Overall, our results suggest that iAI may not represent the full picture of positive selection on archaically introgressed haplotypes in humans and that more work needs to be done to analyze the role of SI in the archaic introgression landscape of living humans.

Amongst 15 bird species, representative of 7 orders, recurrent breakages evocating the presence of fragile sites were detected in the chromosomes of the 5 species belonging to Passeriformes. These breaks appeared when 5-bromodeoxyuridine (BrdU) was added to the cell culture medium at a dose inefficient for inducing chromosome structure alterations in other birds and mammals. They involved, similarly in male and female, 3 loci on the Z chromosome of 3 Turdus species (Turdidae). Labeling by BrdU antibody confirmed the correlation between BrdU incorporation into DNA and breakage, especially around and in the sites of breakage. Thus, 3 BrdU-sensitive fragile sites were present in the Z chromosomes of these birds. Three fragile sites were also detected at different locations in the Z chromosomes of the European robin (Erithacus rubecula, Muscicapidae), suggesting that a structural rearrangement occurred during the evolution of Turdidae and Muscicapidae. Chromosome banding confirmed this interpretation. Finally, in the more distantly related species Parus major (Paridae), the almost acrocentric Z chromosome displayed a single BrdU-sensitive fragile site in its short arm, and the W appeared to be pulverized by BrdU incorporation. Although it cannot be excluded that the BrdU-sensitive fragile sites may be involved in rearrangements, their conservation in many species, and possibly all Passeriformes, provides evidence that they do not constitute a pejorative character during evolution.

Amongst Cercopithecidae, the species of the Cercopithecini tribe underwent a very active chromosome evolution, principally by fissions, which increased their chromosome number up to 72. In contrast, all the species of Papionini have fairly similar karyotypes with 42 chromosomes. In animals, nucleolus organizer regions (NORs) are generally considered as instable structures, which frequently vary in size, number, and location at both infra- and interspecific levels. Although in Cercopithecinae the NORs, involved in breaks, exchanges, and translocations, behave like fragile sites in somatic cells, their number and location appear to be very stable between species. Fluorescence in situ hybridization of a 28S rDNA probe on metaphase chromosomes displayed a unique interstitial location in either an acrocentric pair (in 12 species of Cercopithecini) or a metacentric pair (in 6 species of Papionini). A non-exhaustive survey of literature data on NOR location in other primates shows that numerical variations of the NORs principally depend on their location: most multiple NORs are in terminal positions, while almost all unique NORs are in interstitial positions. We propose that this correlation is the consequence of the selection against gametic imbalances involving the chromosomal material distal to the NORs, which is effective when they are interstitially, but not terminally, located. Thus, the consequences of the interstitial NOR instability for reproduction are essentially limited to their size variations, as observed in Cercopithecidae.

Lactase is the enzyme that breaks down the milk sugar lactose, and in most mammals, including most humans, lactase activity is down-regulated after the weaning period is completed. However, in about 35% of adults worldwide, lactase continues to be expressed throughout adulthood, a feature termed lactase persistence (LP). Genetic evidence indicates that LP is a recent human adaptation, and its current geographic distribution correlates with the relative historical importance of dairying in different human populations. Investigating archaeological evidence for fresh milk consumption has proved crucial in building an account of the joint evolution of LP and dairying. A powerful technique for investigating food processing, including milk processing, in ancient populations is lipid residue analysis on archaeological pottery. We review here the archaeological and genetic evidence available that have contributed to a better understanding of the gene-culture co-evolution of LP and dairying.

Previous morphological and molecular analyses failed to resolve the phylogenetic position of the critically endangered saola (Pseudoryx nghetinhensis) with respect to its placement in Bovina (cattle, bison, and yak) or Bubalina (Asian and African buffaloes). In the present study, G- and C-banding, Ag-staining and FISH with 28S and telomeric probes was undertaken for 17 bovid species. An analysis of these data allowed us to identify 49 structural rearrangements that included autosomes, gonosomes and 17 different NOR sites. The combined data set was subjected to a cladistic analysis aimed at: (i) providing new insights on phylogenetic relationships of the saola and other species within the subfamily Bovinae, and (ii) testing the suitability of different classes of chromosomal characters for phylogenetic reconstruction of the family Bovidae. The study revealed that nucleolar organizing regions (NORs) are phylogenetically informative. It was shown that at least one, or sometimes two of these characters punctuate divergences that include nodes that are the most basal in the tree, to those that are the most recent. In this context, the shared presence of three NORs in saola and species of Syncerus and Bubalus strongly suggests the saola’s placement within the subtribe Bubalina. This contrasts with Robertsonian rearrangements which are informative only at the generic level. These findings suggest that NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae that may also have applicability at higher taxonomic levels, possibly even for Pecora.

Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interface of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G 2 but not in G 1 phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G 2 phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle.

In a series of 260 cytologically abnormal breast cancers studied in our laboratory, we observed a single case of near-haploid karyotype: 27,X, +der(1;16)(q10;p10), +7, +14, +15. A review of published cases of near-haploid malignancies suggests that near haploidy belongs to a process of chromosome evolution distinct from that of most epithelial cancers in which hypodiploidy is strongly associated with the occurrence of unbalanced structural rearrangements. In near-haploid tumors, chromosome loss is independent from chromosome rearrangements and may not be associated with an adverse prognosis.

We investigated the possibilities of using scanning ion analytical microscopy (SIAM) to detect bromine in human metaphase chromosomes. The experiments were performed after incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), into the DNA or by in situ hybridization of a BrdU-labelled probe for the subcentromeric repeated DNA sequences. The possibilities offered by this microanalytical method were compared with immunofluorescent staining techniques. Well-defined maps of bands containing bromide were obtained with metaphase chromosomes that had incorporated BrdU during the late S-phase. Their patterns were similar to the labelling obtained by immunofluorescence. In addition, SIAM reveals the presence of bromine within constitutive heterochromatic regions in which BrdU is poorly detected by immunofluorescence. The comparison of the 12C14N, 31P and 81Br maps of controls and fluorescence plus Giemsa (FPG) metaphase chromosomes shows the loss of bromide from DNA during this treatment. SIAM emerges as a new powerful microanalytical technology for investigating chromosome structure further.

A modified comparative genomic hybridization (mCGH) technique was used to identify and map amplified DNA sequences in six homogeneously staining regions (hsr) from three primary breast carcinomas. Five different chromosomal regions and bands were identified as sites of amplification: 8p1, 17q21.1, 17q23 (two cases), 19q13.3, and 20q13.3. The mCGH site located on 17q21.1 was demonstrated to correspond to a 50-100-fold amplification of ERBB2. Further in situ hybridization experiments were used to confirm the mCGH results and to characterize the organization of the amplified sequences within the hsr. In five of six instances, two or more chromosomal regions were found amplified in the same hsr. In the tumor with the less modified karyotype, the two hsr comprised DNA sequences from three different chromosomes and showed different patterns of amplification. In the tumor with the most rearranged karyotype, the hsr-carrying chromosomes were formed by the translocation and amplification of sequences from three or four different chromosomal sites. This illustrates the complexity of the amplification process in breast cancers.

After immortalization of human normal mammary epithelial cells by replication-defective SV40 genome integration, 2 cultures were developed independently. Both had the same integration site, in band 9q21, but rapidly diverged karyotypically. After a few passages, one, designated SC2T2, exhibited near-diploid (a) and the other, designated SL2T2, near-tetraploid (b) karyotypes. The simplest formulas were 44, X, -X, der(3;22) (q10;q10), der(4)t(4;9)(q34;q12), +8, +9, add(13)(pl), der(19) t(8; 19)(q21;p 13.3), add(22)(p 1) for karyotype (a) and 93, XXXX, add(1)(q12), add(11)(q13), +20 for karyotype (b). A number of alterations were further acquired with passages. Both cell cultures were tumorigenic, but their efficiency of grafting in nude mice largely differed: it was low for SL2T2 and high for SC2T2 cultures. All cultures of the xenografted tumors, obtained from either SL2T2 or SC2T2, exhibited the same clonal anomalies as those characterizing karyotype (a). It was concluded that only cells with karyotype (a) were tumorigenic, and that the difference in the tumorigenic potential of cultures SC2T2 and SL2T2 was related to their richness in cells with this karyotype. The comparison of the various karyotypes, together with data obtained in other cell types transformed by SV40, suggests that the acquisition of tumorigenicity in S2T2 mammary epithelial cells may be related to the loss of chromosome 3p arm. © 1995 Wiley-Liss, Inc.

Two-color fluorescent in situ hybridizations using probes for alphoid (alpha) and classical satellite (CS) DNAs from chromosomes 1 and 16 were performed to characterize i(1q), der(1;16), and complex rearrangements observed in breast cancer cells from fresh tumors and established cell lines. Six of seven i(1q) occurred after breakage in the alpha 1 containing region and one of seven was dicentric, with breakage in 1p11.2. The five der(1;16)(q10;p10) studied appeared to result from a variety of breakpoints involving alpha 1, alpha 16, CS1, and CS16 DNAs. All had conserved alpha 16 DNA, suggesting a segregation of the der(1;16) leading to a loss of 16q and a gain of 1q in most cases. One complex rearrangement of chromosome 1 also appeared to involve chromosome 16, suggesting that a der(1;16) occurred first, followed by another rearrangement. Both the apparent preferential involvement of constitutive heterochromatin harboring alpha and CS DNAs and the variety of breakpoints spanning along heterochromatin suggest that the important consequence of the rearrangement is not the breakage per se but the resulting imbalance.

Rodents represent the largest order of living mammals. It comprises 5 sub-orders, among which Sciuromorpha (Sciuridae, Gliridae and Aplodontiidae) are assumed to occupy a basal position in rodent evolution. Banded karyotypes of some representatives of the Sciuridae family have been compared to each other, and comparisons with man were performed using chromosome paintings. Sciuridae karyotypes have conserved several eutherian ancestral syntenies. Like Sciuridae, Gliridae possess some chromosomes easily comparable with those of Primates. Comparisons of Gliridae and Sciuridae chromosomes with those of the presumed eutherian ancestor provide information about their chromosomal evolution and their position among Rodentia. Although both Sciuridae and Gliridae karyotypes are relatively conserved, they display many differences, indicating their early divergence. The reconstruction of their chromosomal evolution allowed us to propose the composition of their presumed ancestral karyotypes, with 2n = 48 and 2n = 38 for Gliridae and Sciuridae, respectively. Since rodent emergence, a single rearrangement is common to these 2 families. It formed a chromosome with fragments homologous to human chromosomes 4-8p-4-12-22, not detected in other rodents, and thus characteristic for the Sciuromorpha. This allowed us to reassess the chromosomal signatures of Rodentia. Finally, we show that the speed of chromosomal evolution in Gliridae is intermediate between that of Sciuridae (low) and Muridae (high).

A 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.

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.

We have previously shown that assessment of chromosome alteration rate by cytogenetics is well correlated with breast cancer prognosis factors. As karyotypes are usually difficult to obtain from solid tumors using conventional methods, a new approach is proposed. Metaphase-like chromosomes were directly obtained following chromosome condensation using calyculin A (okadaic acid) from cytologic specimens of breast cancers sampled by fine needle. Chromosome counts and rearrangement rates were established in a series of 45 tumors, as early as 24-48 h after sampling. A high rate of rearranged chromosomes was found to correlate with high histological grade, TNM stage and S-phase fraction, loss of estrogen receptor expression and DNA aneuploidy. The indication of genome alteration provided by this method constitutes a simple, potent and early potential prognostic factor in breast cancer directly applied on cytological specimens.

In a sample of 329 patients with a solid tumor (colon and breast adenocarcinoma, cervical carcinoma, and meningioma), four balanced constitutional translocations were observed. Two were t(13q14q), and two were reciprocal translocations. Comparison with surveys of newborns showed a significant excess of translocations in our sample.

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus–transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic ,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2–rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3–rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.

The global DNA methylation status was investigated on a series of 59 breast cancers by Southern blotting, using methylation sensitive restriction enzymes. By comparison to control DNA, almost all tumor DNAs were found globally hypomethylated. However, the demethylation was variable from tumor to tumor. Compared to other biological parameters, the methylation did not correlate with chromosome alterations, steroid hormone receptor status, or histopathological grading. Tumors which appeared to be the most evolved for other parameters were only mildly hypomethylated, whereas tumors with strongly hypomethylated DNA corresponded to those with slight alterations of the other parameters. Thus, DNA hypomethylation is a consistent characteristic of breast cancer, but its variations may not correlate with tumor progression of most breast cancers.

Gene amplification can be defined as an increase in the copy number of a restricted region of the genome. Originally discovered as a mechanism of drug resistance in cultured cell lines, it also occurs during tumourigenesis, more frequently in solid tumours than in hemopoietic malignancies (for review, Alitalo and Schwab, 1986; Schwab and Amler, 1990). Two types of chromosomal structures, the homogeneously staining regions (hsr) and double minute chromosomes (dmin), are currently regarded as hallmarks of gene amplification, hsr consist of chromosomal regions that fail to exhibit bands. They can vary in size and do not usually reside at the native locus of the amplified gene (Wahl, 1989). dmin are small paired acentric elements that replicate autonomously once per cell cycle and segregate randomly between daughter cells at mitosis (Wahl, 1989). Thus, unequal segregation of these structure provides means to increase the copy number of certain DNA sequences during a single cell cycle. Submicroscopic circular molecules, called episomes, were found in cells with amplified genes and might represent precursors of dmin (Carroll et al., 1988; Van Hoff et al., 1988; Wahl, 1989).

The cytogenetic dose response following in vivo localized irradiation is difficult to establish because of the occurrence of clones defined by chromosome alterations, with various proliferative rates. The biological meaning of these clones is not well understood. Two sets of experiments were performed to follow their behavior. R-banded karyotypes were established on human fibroblasts irradiated either before or after initiation of the cultures. Clones were observed in cultures developed after irradiation of biopsies, whereas irradiated cultures exhibited karyotypes with multiple non-clonal rearrangements. This difference suggests that most radiation-induced chromosome anomalies do not confer a selective advantage on the carrier cells in vitro. The appearance of clonal anomalies following biopsy irradiation would rather be a consequence of a strong selection at the time of the growth of the cells out of the explants, which would give rise to the progeny of a limited number of progenitor cells.

A modified comparative genomic hybridization (mCGH) technique was applied to a series of 17 primary breast carcinomas in which cytogenetic study (CG) demonstrated the presence of homogeneously staining region(s), suggesting the occurrence of DNA amplification. mCGH demonstrated recurrent amplifications of the whole chromosome arms 8q (9 times) and 1q (7 times) and of DNA loci in the following bands: 11q13 (6 times), 9p13 and 17q21.1 (4 times), 1q21.1 and 16p11.2 (3 times), and 8q22, 8q24.1, 10q22, 15q26, 17q23, and 20q13.3 (twice). Amplification of whole chromosome arms is likely to have resulted from unbalanced translocations or isochromosomes, whereas amplifications of smaller chromosomal segments probably arose through real DNA amplification processes. In all tumors but one, more than one amplified locus was detected. The fact that many chromosomal sites were involved suggests that the process of amplification is complex and that many genes are potential targets.

Data on aneuploidy from a prospective study on a large number of lymphocyte metaphases (over 1000 in 72-h and 100 in 48-h cultures) per individual from eight healthy donors of various ages are reported. Chromosome losses were dependent on culture time, being significantly more frequent in 72-h than in 48-h cultures. All donors exhibited various degrees of aneuploidy which increased with age in women. This increase resulted essentially from X chromosome losses, as previously reported. Although the rate of aneuploidy limited to autosomes was similar in newborns and in adults, the distributions of the missing autosomes were different. In the two newborns studied, autosome aneuploidy was random. In the adults, a significant inverse correlation with autosome lengths was observed. The inverse correlation between chromosome lengths and losses may be explained by selective pressure against monosomic cells in the adults.

In a cytogenetic study of 125 primary and untreated breast cancers, 107 were selected for the quality of their metaphases permitting detection of amplifications: homogeneously staining regions (HSRs), abnormally banded region (ABRs), and double minutes (dmins). HSRs and ABRs were detected in 62 cases (58%), but no cases of dmins were observed. The localizations of HSRs and ABRs were not random because they were observed in the 8p1 position in 14 cases. The possible amplifications of five sequences. MOS (8q1), LHRH (8p21.1), POLB (8p11.2). PLAT (8p12), and D8Z2 (8c) were investigated in three tumors with HSR on the short arm of chromosome 8. Because these sequences were not amplified, two interpretations can be proposed: 1) there is a frequent amplification of a sequence from the 8p1 region, located between the investigated sequences; and 2) the amplifications do not occur in 8p1, but HSRs or ABRs of undetermined origin have a strong tendency to be translocated onto 8p. Because cases with HSR(8p) have less complex karyotypes than with HSRs in other locations, the first interpretation is the most likely: HSRs may be formed in 8p and further translocated on other chromosomes in the course of tumor progression.

5-bromodeoxyuridine incorporation was studied in metaphase chromosomes from 24 breast tumour specimens, including 23 adenocarcinomas. In these 23 cases, a slow cell cycle was observed, with a long (8 h) G2 phase. This slowing of the cell cycle, which was poorly related to the degree of polyploidy, was significantly related to the number of chromosome anomalies: the cell cycle was particularly slow when many rearranged chromosome were observed. These in vitro findings during the first cell cycle cannot easily be transposed to the in vivo situation. By analogy with Fanconi anaemia, in which both chromosome lesions and a long G2 phase are detected, a DNA repair defect and/or high DNA mutagenesis might exist in breast cancer cells.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Cytogenetic studies on fresh human breast cancers revealed that homogeneously staining regions (HSRs), which are assumed to represent DNA amplification, are observed in almost half of the cases. To search for a possible relationship between HSRs and proto-oncogene amplification, 16 proto-oncogenes, including ERBB2, were studied by Southern blot analysis in four tumors with two or three HSRs, and in three tumors without HSRs. Only four proto-oncogenes were found to be amplified in at least one tumor each: HST and INT2 (x3), MYC (x2-3), and FES (x greater than 10). The large sizes of the HSRs, which each corresponded to several percent of the haploid genome, were hardly compatible with the low rate of amplification, except for FES and then only if a large adjacent segment was co-amplified. This incomplete correlation was demonstrated by in situ hybridization, using biotinylated probes, which showed fluorescent spots on only one HSR for FES in one tumor and for INT2 in another one. Our results indicate that most of the large amplifications corresponding to HSRs do not involve the proto-oncogenes usually studied in breast cancer. The large amplification of FES, detected in one tumor, may be coincidental.

A biotinylated probe (L23-21) specific for the 1q12 band of human karyotype was used to detect the 1q segment in interphase nuclei of breast and colon carcinomas. This probe was selected because trisomy or polysomy 1q is the most frequent chromosomal change observed in solid tumors. This method enables cancerous cells, including near-diploid ones carrying an unbalanced rearrangement of 1q, to be easily identified.

The effect of low-dose (0-0.5 Gy) gamma-radiations was studied on R-banded chromosomes from lymphocytes of healthy donors of various ages. In cells from newborns, an increase of chromosome damage roughly proportional to the dose was found. In lymphocytes from young adults chromosomal aberrations were not detected at doses of 0.05 and 0.1 Gy, and in lymphocytes from old adults chromosomal aberrations were not detected at doses of 0.05 and 0.1 Gy, and in lymphocytes from old adults not even at 0.2 Gy. The difficulty in detecting aberrations in lymphocytes from adults is largely due to a considerable background of chromosomal anomalies which should be borne in mind in dosimetry studies. The rate of induction largely depends on the types of rearrangements. One-break terminal deletions are efficiently induced at 0.1 and 0.2 Gy and are the best indicators of exposure at these doses. At 0.5 Gy, the frequencies of 2-break lesions, i.e., dicentrics and reciprocal translocations, increase, whereas that of deletions decreases.

Chromosome lesions detected in lymphocytes from 14 patients previously treated with melphalan, a bifunctional alkylating agent, have been analyzed on R-banded preparations. In comparison to controls, there was no significant increase of chromatid-type lesions, but chromosome-type lesions were quite frequent, affecting 21.5% of metaphases, on the average. Reciprocal translocations represent 54%, unbalanced translocations 15%, deletions 19% and inversions 6% of all rearrangements. Most of these would not have been detected without the use of chromosome banding. The distributions of affected chromosomes and chromosome bands were not random. Almost all imbalances resulting from rearrangements lead to losses but not to gains. The distribution of the abnormal chromosomes has been compared to that observed in controls and in in vitro experiments, and to the characteristic pattern of malignant cells from patients affected by secondary acute leukemia (ANLL).

Twenty-five human breast cancers, surgically resected, were studied by cytogenetic analysis and DNA flow cytometry (FCM). The establishment of karyotypes showed that multiple cell populations probably were derived from a single ancestor clone, because common marker chromosomes always could be demonstrated. Differences of up to 30% were observed when the estimates of DNA content by the two methods were compared. A general tendency toward the acquisition of large marker chromosomes should be at the origin of this discordance, as the proportion of markers for each case correlated significantly with the magnitude of the difference. Parallel use of the two methods revealed the existence of tumors with DNA diploid FCM profiles and highly abnormal hypodiploid karyotypes (35-40 chromosomes), which may explain the limited value of DNA ploidy as an independent prognostic factor in breast cancer.