A South American origin of the genus Dynastes (Coleoptera: Scarabaeidae: Dynastinae) demonstrated by chromosomal analyses.

The karyotypes of 5 species of Dynastes, D. hercules, D. tityus, D. granti, D. satanas, and D. neptunus, and 2 subspecies of D. hercules are compared with those of 6 other selected Dynastinae species. In the 3 former species, there are 18 chromosomes, including neo-sex chromosomes formed by the fusion of an acrocentric autosome with the X and Y chromosomes. In all other species, including D. neptunus and D. satanas, free X and Y chromosomes are observed in 20,Xyp karyotypes. The acrocentric autosome presumably involved in the fusion is present in D. neptunus and D. satanas (pair No. 8). It replaces a submetacentric observed in the other Dynastinae species. Thus, the karyotypes of D. neptunus and D. satanas derive from that of ancestral Dynastinae by an inversion in chromosome 8, and those of D. hercules, D. granti and D. tityus derive from that of D. neptunus by the translocation of this inverted chromosome to sex chromosomes. Because the Dynastes species with the most derived karyotype occur in North and Central America and Lesser Antilles, while D. neptunus and D. satanas are limited to the northern part of the South American Andes, we suggest a South American origin of the genus Dynastes.

Chromosome analysis of 82 species of Scarabaeoidea (Coleoptera), with special focus on NOR localization.

The aim of this study was the identification of the ancestral location of the nucleolus organizer region (NOR) in the Scarabaeoidea superfamily, and its evolutive trends in the karyotypes. For this purpose, the mitotic and meiotic chromosomes at pachynema of 82 species belonging to 4 families and 8 subfamilies, including 49 species without any published data, were examined after Giemsa staining, C-banding and NOR staining. It could be perceived that most karyotypes are composed of 18 nonacrocentric autosomes, an acrocentric X and a punctiform Y. NORs are frequently located on the X independent of its morphology. In contrast, autosomal NORs are frequently on the rare acrocentric short arms. Thus, it could be shown that the ancestral karyotype was very probably composed of 18 metacentric/submetacentric autosomes, an NOR carrier acrocentric X and a punctiform Y. The NOR translocation on autosomes parallels the passage to their acrocentric morphology. It is proposed that the frequent location of the NOR on the X of beetles, and possibly other insects, is made possible by their mode of dosage compensation of the X chromosome, consisting in the overexpression of the unique X of the males.

Cyclocephala (Coleoptera: Scarabaeidae: Dynastinae) evolution in Lesser West Indies indicates a Northward colonization by C. tridentata.

A dual cytogenetic and molecular analysis was performed in four species of Cyclocepala (Coleoptera: Scarabaeidae: Dynastinae) from Lesser Antilles (Martinique, Dominica and Guadeloupe). Two species/sub-species, C. mafaffa grandis and C. insulicola, are endemic to Guadeloupe. They have their own non-polymorphic karyotype and a fairly homogeneous haplotype of the COI gene. C. melanocephala rubiginosa has a distinct karyotype. Its COI haplotype is homogeneous in Guadeloupe and heterogeneous in Martinique. Finally, C. tridentata has highly different karyotypes and haplotypes in the three islands. In Martinique, its karyotype, composed of metacentrics, is monomorphic while its haplotype is fairly heterogeneous. Both are close to those of other Cyclocephala and Dynastinae species, thus fairly ancestral. In Guadeloupe, its karyotype is highly polymorphic, with many acrocentrics, and its haplotype fairly homogeneous. Both are highly derived. In Dominica, both the karyotype and the haplotype represent intermediate stages between those of Martinique and Guadeloupe. We conclude that several independent colonization episodes have occurred, which excludes that C. insulicola is a vicariant form of C. tridentata in Guadeloupe. Both chromosome and COI gene polymorphisms clearly indicate a recent colonization with a northward direction for C. tridentata.

Evolution of European cockchafers (Melolonthinae: Scarabaeidae: Coleoptera): a morphological, molecular and chromosomal study of intra- and inter-specific variations.

In cockchafers of the genus Melolontha, there is a marked intraspecific polymorphism for morphological characters, making some specimens of one species resemble another. A cytogenetic and molecular (mitochondrial COI gene sequence) study of typical and atypical forms of M. melolontha and M. hippocastani, captured at the same period and area, was performed. Karyotypes and haplotypes clearly characterize each taxon, placing atypical specimens in one or the other species unambiguously. This formally discards the role of hybridization in phenotypic resemblance, as usually proposed. Karyotypes and haplotypes were compared to those of M. pectoralis and Phyllophaga pleei, a more distantly related Melolonthinae, and some Dynastinae species, to reconstruct their ancestral karyotype. The karyotype of M. melolontha is the most derivative and that of P. pleei the most conserved among the Melolonthinae studied, which fits with the phylogeny established by COI gene analysis. Both karyotypes and COI haplotypes demonstrate the proximity of M. pectoralis and M. melolontha. The karyotype of M. melolontha is polymorphic, without relationship with morphological variations. Finally, the existence of similar morphological variations in different Melolontha species and chromosomal polymorphism in M. melolontha is discussed in relation with a network (reticulated) mode of speciation.

Y-chromosome disomy and trisomy in scarabaeid and cerambycid beetles.

In a series of about 500 specimens, including 420 males, of karyotyped Polyphaga beetles, 5 males with chromosome Y aneuploidy were detected. One male of each Dicronorrhina derbyana oberthuri (Scarabaeidae), Agapanthia violacea and Morimus funereus (Cerambycidae) were XYY, and 2 probably related and sterile males of Marmylida marginella (Scarabaeidae) were XYYY. These and literature data suggest that Y chromosome aneuploidies are much more frequent in polyphagan beetles than any other group of animals with an XY/XX sex determinism. The origin of this particularity probably lies in the unique mode of sex chromosome association at meiosis I: it is not synaptic but realized through nucleolar proteins forming the well-known parachute-like structure (Xy(p)). This has 2 possible consequences. The first one is the regular association of several sex chromosomes at metaphase I and segregation at anaphase I. It allows, for instance, XYY (Xyy(p)) males to procreate XYY sons. The second consequence is the occasional remain of nucleolar proteins embedding sex chromosomes in spermatocytes II. We propose that it could impede the correct segregation of Y chromatids after centromere split at anaphase II, and contribute to form YY gametes by XY males and YYY gametes by XYY males. The tendency for increasing the number of Ys would not be strongly limited at the XY level, but only at the XYY level by male infertility at higher Y ploidies.

Chromosome polymorphism and Fanconi-like instability in the scarabaeid beetle Macraspis tristis from Guadeloupe.

The karyotype of Macraspis tristis Laporte is described. It is composed of 18 chromosomes. C-band positive heterochromatin is very abundant and is located at centromeric regions and, for some chromosomes, at telomeric regions. There is a high inter-individual chromosomal polymorphism for the presence and extension of telomeric heterochromatin. In one of the 8 specimens studied, 2 populations were observed in testicular cells. Besides groups of normal metaphases, other groups displayed multiple chromatid and chromosome alterations such as breaks, exchanges (radials), deletions and translocations, resembling those described in Fanconi anemia. The insect had a normal phenotype, but its gametogenesis did not reach the spermatocyte II stage, and was quite poor in spermatocytes I. The clonal character of the chromosome instability was obvious owing to the incomplete cytodiaeresis of germ cells which remain associated by cytoplasmic bridges. This may be the first example of chromosome instability observed in animals from nature.

Sex chromosome rearrangements in Polyphaga beetles.

The presence of a parachute sex chromosome bivalent (Xyp) at metaphase I of male meiosis is a well-known characteristic of Coleoptera, present in almost all families of this order and assumed to represent their ancestral sex chromosome formula. Sex chromosomes appear to be manifold more frequently involved in inter-chromosomal rearrangements than the average of the nine autosomal pairs usually forming their karyotype. This leads to various formulae such as neo-sex, multiple sex and perhaps unique sex chromosomes. These rearrangements alter the intimate association between sex chromosomes and nucleolar proteins, which are usual components of the Xyp. Different situations, selected in a series of 125 mitotic and meiotic cytogenetic studies of Polyphaga beetle species, are reported and discussed, with the aim to improve our knowledge on the mechanisms of sex chromosome rearrangements, the relationships with nucleoli and the consequences on dosage compensation and chromosome segregation.

Nucleolus and chromosome relationships at pachynema in four Scarabaeoidea (Coleoptera) species with various combinations of NOR and sex chromosomes.

Nucleolus organizer regions (NORs) and nucleolus locations were studied after silver staining in spermatocytes at pachynema from four beetle species selected for their various combinations of sex chromosomes. Their karyotypic formulae were: 18,neoXY (Dorcus parallelipipedus); 25,X (Passalus unicornis) and 20,Xyp (Cetonia aurata and Protaecia (Potosia) opaca). NORs were located in the short arms of a unique acrocentric autosome pair in the first three and in intercalary position in a sub-metacentric autosome pair in the last species. Silver staining gave remarkably more consistent results in pachytene than in mitotic spreads, enabling the detection of both NORs and nucleoli, and also better results in embryo than in spermatogonial metaphases. At pachynema the NORs were elongated, roughly in proportion to the number of nucleoli, which always remained associated with NOR. Nucleoli were not recurrently associated with sex chromosomes, except in P. unicornis, at late pachynema. In C. aurata and P. opaca the sex body was recurrently associated with acrocentric short arms and metacentric telomeres, respectively. Even in these simple situations, with NORs located in a single autosome pair, the number of nucleoli and their relationships with sex chromosomes varied strongly from species to species. These variations appear to be largely determined by the chromosome rearrangements which have occurred during evolution, which makes extrapolations and generalizations quite hazardous. In D. parallelipipedus pachytene cells a quasi-systematic and transient fusion between the terminal heterochromatin of two sub-metacentrics was detected. Other chromosome bivalents could also be occasionally associated, but not the NOR carrier one. A strong enhancement of DAPI or quinacrine mustard staining was observed at the fusion point.

X-Y-autosome translocation, chromosome compaction, NOR expression and heterochromatin insulation in the Scarabaeid beetle Dynastes hercules hercules.

The karyotype of the giant beetle Dynastes hercules hercules is composed of only 16 autosomes and large sex chromosomes. Meiotic studies in the males showed that a large part of the sex chromosomes undergo synapsis at pachynema similarly to autosomes, demonstrating that both derived from an autosome-gonosome translocation. Therefore, karyotype formula is 18,neoXY. The heterochromatisation of the neoX short arm at pachynema indicates that it corresponds to the ancestral X. It carries the nucleolar organizer region (NOR) in its proximal part, which is undercondensed, especially in male mitotic and meiotic cells. In female mitotic cells, both NOR staining and undercondensation were more difficult to observe in the neoX short arms. In somatic interphase nuclei, NOR expression strongly varies with the sex. Two separated compact groups of silver dots were observed in female nuclei, while a single dispersed and large group of silver deposit exists in the males. Both the lower condensation and the higher NOR expression of the single neoX of the males, compared to each of the two neoXs of the females, is interpreted to be a consequence of dosage compensation, a mechanism not yet described in Coleoptera. In mammals as well as in Coleoptera, the carriers of gonosome-autosome translocations not exhibiting deleterious phenotypes show constitutive heterochromatin at the autosome-gonosome junction. Thus, heterochromatin may play an important universal role by clearly separating chromosome segments with different regulations of gene expression, such as inactivation or dosage compensation of the X chromosome on the one side and a conventional autosomal structure on the other side.

High chromosomal polymorphism and heterozygosity in Cyclocephala tridentata from Guadeloupe: chromosome comparison with some other species of Dynastinae (Coleoptera: Scarabaeidae).

Very distinct karyotypes have been observed in two Cyclocephala species from Guadeloupe, considered as very close and possibly vicariant: C. insulicola with only metacentric and C. tridentata tridentata with many acrocentric autosomes. This prompted us to study the karyotype of a few other neotropical Dynastinae belonging to four of the eight existing tribes, to find out which one of these two species had the most divergent chromosomes from their ancestral condition. In the four additional species studied, i.e., Cyclocephalamaffafa, Strategus syphax, Ligyrus cuniculus and Megasoma actaeon, a karyotype composed of 20 chromosomes, including 18 meta- or submetacentric autosomes was found, as it was in C. insulicola. Thus, the karyotype of C. t. tridentata, in which most of the 18 autosomes were acrocentric, is apomorphic. In addition, it was highly polymorphic, with six different karyotypes observed among the ten specimens studied. All had one to four heterozygous chromosome pairs formed by one acrocentric and one submetacentric carrying a large juxta-centromeric heterochromatic component. This heterozygosity did not seem to impair either meiotic synapsis or chiasma formation and chromosome segregation. Such high rates of chromosome heterozygosity and polymorphism are infrequent and never described in beetles. This suggests that C. t. tridentata undergoes an active process of chromosome evolution. A possible relationship with insularity and/or pesticide exposure is briefly discussed.

Meiotic behaviour of a new complex X-Y-autosome translocation and amplified heterochromatin in Jumnos ruckeri (Saunders) (Coleoptera, Scarabaeidae, Cetoniinae).

Species belonging to the Cetoniinae subfamily studied so far possess 20 chromosomes, including a small X and a punctiform Y: 20,Xyp in the males. In a series of species from the Goliathini tribe under study we found a very unusual karyotype, with 12 autosomes and large sex chromosomes (14,neoXY) in Jumnos ruckieri from Thailand. Applying various techniques including pachytene bivalent spreading, we showed that 40% (mitotic and meiotic prophases) to 60% (metaphases) of the karyotype length was composed of heterochromatin. Both sex chromosomes were NOR carriers. At pachynema they underwent a complete synapsis of their distal regions, indicating their autosomal origin. At contrast, their very uneven central regions remained separated, but associated with nucleolus material. This association persisted until diakinesis, forming a pseudo-chiasma between the neoX and the neoY, which were always in end-to-end association. Compared to free autosomes the autosomal parts of the neo-sex chromosomes had a significant lack of interstitial chiasmata, indicating a possible lack of recombination at their proximal regions. As in the cases of X-autosome translocations in mammals, autosomal and gonosomal parts of the neo-sex chromosomes were insulated by heterochromatin, which may be a necessary condition to avoid deleterious position effects, whatever the mechanisms of gene dosage compensation.

Use of meiotic pachytene stage of spermatocytes for karyotypic studies in insects.

Coleopterans represent by far the largest animal group, with more than 300,000 identified species. Only little progress in their chromosome analysis has been accomplished during recent decades, compared with that made in vertebrate cytogenetics. Both the small size of their genome and the difficulty of obtaining mitotic cells with nice chromosomes have limited the application of conventional techniques, such as chromosome banding. A method for obtaining chromosome banding on well-spread bivalents from the pachytene stage of the meiotic prophase, the most frequent stage in young imagines, is described. It makes possible the identification of all bivalents and the establishment of the karyotype with greater ease and accuracy than with mitotic cells. In addition, it gives some insight into chromosome organization at a stage when autosomes are assumed to undergo an intense transcriptional activity. The results of the technique, which was successfully applied to many species, are described here in two of them, Cetonia aurata and Adesmia montana as examples.

Correlation between genetic alterations and growth of human malignant glioma xenografted in nude mice.

In order to develop preclinical models of malignant astrocytomas and oligodendrogliomas, a series of 54 resected gliomas (37 from oligodendroglial lineage and 17 from astrocytic lineage) were xenografted subcutaneously into nude mice. Molecular alterations commonly observed in gliomas subtypes, including LOH 1p and 1q, LOH 19q, LOH 10p and 10q, LOH 9p, TP53 and PTEN mutations, EGFR amplification, CDKN2A homozygous deletion and telomerase reactivation were systematically screened in the original and xenografted tumours. In all, 23 gliomas grew in nude mice. The most anaplastic tumours were selected as shown by pathological and molecular studies of the original tumour as well as shorter survival in patients whose tumours were successfully grafted. Comparison between the two growth profiles showed that 10q LOH and EGFR amplification gave a tumorigenic advantage. With a few exceptions, the genetic pattern was remarkably stable before and after growth in nude mice. These results suggest that subcutaneous xenografts are useful and reproducible models to analyse the molecular profile of malignant astrocytoma and oligodendroglioma. This represents the first step to improve our understanding of the correlations between molecular alterations and response to standard or experimental therapies.

[A new model of human prostate cancer, the PAC120 xenograft]. / Un nouveau modèle de cancer humain de prostate, PAC120.

Prostate cancer is the second cause of cancer death in men. Often, initialy hormono-independent, escape from anti-androgen therapy is a key event of tumoral progression showing an hormone-independent phenotype. To study morphological, genetic and molecular bases associated with the hormono-dependence escape, a new model of human adenocarcinoma prostate xenograft, PAC120, was established with its hormono-dependent and independent variants. Its growth was strongly inhibited by surgical castration or by administration of the new gonadotrophin-releasing hormone antagonist, FE 200486 (Ferring, San Diego, CA). Evolution to hormono-independence was frequently associated with a mucoid differentiation or a neuroendocrine-like pattern, with the apparition of new chromosomic alterations and variations of human gene expressions. PAC120 xenograft is a new model of hormone-dependent prostate cancer, opening the opportunity to study the hormone dependence escape mechanism and to evaluate the efficacity of new therapeutics.

Clinical and experimental progression of a new model of human prostate cancer and therapeutic approach.

We report the clinical evolution of a prostate cancer, metastasizing to lungs and bones, recurring locally, and escaping from anti-androgen therapy. Key event of biological progression of the patient's tumor was the coincidence of allelic imbalance accumulation and of bone metastases occurrence. The recurrent tumor was established as the transplantable xenograft PAC120 in nude mice, where it grew locally. PAC120 displayed the same immunophenotype of the original tumor (positive for keratin, vimentin, prostatic acid phosphatase, and Leu-7) and expressed human HOXB9, HOXA4, HER-2/neu, and prostate-specific antigen genes, as detected by reverse transcriptase-polymerase chain reaction. It formed lung micrometastases detected by mRNA expression of human genes. Cytogenetic analysis demonstrated numerous alterations reflecting the tumor evolution. PAC120 was still hormone-dependent; its growth was strongly inhibited by the new gonadotropin-releasing hormone antagonist FE 200486 but weakly by gonadotropin-releasing hormone superagonist D-Trp(6)-luteinizing-hormone releasing hormone (decapeptyl). Tumor growth inhibition induced by anti-hormone therapy was linked to the hormone deprivation degree, more important and more stable with FE 200486 than with D-Trp(6)-luteinizing-hormone releasing hormone. Surgical castration of mice led to tumor regressions but did not prevent late recurrences. Transition to hormone-independent tumors was frequently associated with a mucoid differentiation or with a neuroendocrine-like pattern. Independent variations of mRNA expression of HER-2/neu and prostate-specific antigen were observed in hormone-independent tumors whereas HOXB9 gene expression was constant. In conclusion, PAC120 xenograft, a new model of hormone-dependent prostate cancer retained the progression potential of the original tumor, opening the opportunity to study the hormone dependence escape mechanism.

Genome instability in secondary solid tumors developing after radiotherapy of bilateral retinoblastoma.

Genome alterations of seven secondary tumors (five osteosarcomas, one malignant peripheral sheath nerve tumor, one leiomyosarcoma) occurring in the field of irradiation of patients treated for bilateral retinoblastoma have been studied. These patients were predisposed to develop radiation-induced tumors because of the presence of a germ line mutation in the retinoblastoma gene (RB1). Tumor cells were characterized by a high chromosome instability whereas microsatellites and minisatellites were found to be stable. In all tumors, the normal RB1 allele was lost with the corresponding chromosome 13, whereas the germ line mutated allele was retained. The two alleles of TP53 were inactivated, one by deletion of the short arm of chromosome 17, the other by mutation. As compared with non-radiation-induced tumors, the observed panel of TP53 mutations was uncommon with sites not recurrently found otherwise and a high rate of deletions (3/7). In these predisposed patients, the loss of the single normal allele of RB1 is rather due to the radiation-induced chromosome instability than a direct effect of ionizing radiation.

Cytogenetic study of eight new cases of radiation-induced solid tumors.

Radiation-induced tumors were selected according to the criteria defined by Cahan (1948) for sarcomas. Cell cultures and/or xenografts in nude mice were performed with biopsies obtained from second primary tumors. Karyotypes of eight tumors were established after R-banding. After comparison with literature data on 15 other cases, two distinct cytogenetic patterns could be distinguished. One was characterized by polyclonal karyotypes, of which a large proportion were simple and carriers of balanced translocations. Another one was characterized by monoclonal chromosome alterations observed in highly aneuploid and complex karyotypes, in which many deletions were observed. These two different patterns could be related to the modality of metaphase harvesting. Polyclonal karyotypes were preferentially observed after long-term cultures, and monoclonal karyotypes after short-term cultures or xenografts. The following scheme of radiation oncogenesis is proposed: a) induction of recessive gene mutations including that of tumor suppressor genes; b) accumulation of genomic alterations in the irradiated tissue with aging, including deletions or mutations of normal alleles from mutated tumor suppressor genes; and c) loss of tumor suppressor gene function and initiation of a multistage tumor development and progression. Polyclonal abnormalities are assumed to exist in noncancerous cells which acquired radiation-induced chromosome aberrations.

Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

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.

GAC1, a new member of the leucine-rich repeat superfamily on chromosome band 1q32.1, is amplified and overexpressed in malignant gliomas.

We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Amplification and overexpression of GAC1 was demonstrated in two of eight tumors where amplifications were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.

Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization.

Using chromosome painting, a study of chromosomal abnormalities has been performed in two prostatic carcinoma cell lines, PC-3 and DU145. In PC-3, this analysis revealed a highly rearranged hypotriploid karyotype with 54 to 61 chromosomes and numerous rearrangements of chromosomes 1, 3, 5, 8, 10, and 14. At passage 73, DU145 had a hypotriploid karyotype with few rearrangements of chromosomes 1, 3, 5, 12, 13, and 20, whereas at passage 153, this cell line showed a near-tetraploid karyotype with a great number of rearrangements involving chromosomes 3, 6, 8, 10, 12, and 17. A single rearrangement was shared by the 2 cell lines, an i(5)(p10). A comparative genomic hybridization study demonstrated a noticeable amplification of bands 10q22.3-q23 and 14q22-q24 in the PC-3 cell line. No amplification signal was detected for DU145.