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Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.

Fragile sites are classified as common or rare depending on their occurrence in the populations. While rare sites are mainly associated with inherited diseases, common sites have been involved in somatic rearrangements found in the chromosomes of cancer cells. Here we study a mouse locus containing the ionotropic glutamate receptor delta 2 (grid2) gene in which spontaneous chromosome rearrangements occur frequently, giving rise to mutant animals in inbred populations. We identify and clone common fragile sites overlapping the mouse grid2 gene and its human ortholog GRID2, lying respectively at bands 6C1 and 4q22 in a 7-Mb-long region of synteny. These results show a third example of orthologous common sites conserved at the molecular level, and reveal an unexpected link between an inherited disease and an aphidicolin-sensitive region. Recurrent deletions of subregions of band 4q22 have been previously described in human hepatocellular carcinomas. This 15-Mb-long region appears precisely centered on the site described here, which strongly suggests that it also plays a specific role in hepatic carcinogenesis.

Genetic alterations implicated in malignant melanoma are still poorly understood. Malignant melanomas present highly variable histologic and cytologic patterns. The aim of the present study is to define genomic imbalances associated with the development of 2 histologic types of swine hereditary cutaneous melanoma. We have investigated I I swine tumors by comparative genomic hybridization (CGH), 4 superficial spreading melanomas (SSMs) and 7 nodular melanomas (NMs). Following laser capture microdissection and degenerate oligonucleoticle primed-polymerase chain reaction, we were able to isolate and then amplify DNA from the 2 histologic subtypes. Consensus regions of chromosome gains were identified on both histologic subtypes, on swine chromosomes 3p13-p17 (75% of the SSMs and 71% of the NMs), 12q (100% of the SSMs and 57% of the NMs) and 14q11-q21 (75% of the SSMs and 42% of NMs). Chromosomal loss was restricted to NM lesions and the swine 13q36-49 region was lost in 100% of the NMs. Interphase fluorescence in situ hybridization with a probe mapping to the 13q41-q42 region indicates loss of the corresponding region on NM lesions. Taking into account this CGH analysis and the comparative genomic data between swine and human genomes, we suggest that a role for the human chromosomes 3p11-qter and chromosome 21 losses should be investigated in human nodular melanoma progression. (C) 2004 Wiley-Liss, Inc.

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.

Le cancer de la prostate est en fréquence le deuxième cancer chez l’homme. Souvent, initialement hormono-dépendant, la perte de réponse aux traitements anti-androgéniques survient au cours de la progression tumorale, traduisant l’émergence d’un phénotype hormono-indépendant. Afin d’étudier les bases morphologiques, génétiques et moléculaires associées à la perte d’hormono-dépendance, nous avons établi un modèle de xénogreffe d’adénocarcinome prostatique humain, PAC120, avec ses deux variants : hormono-dépendant (HD) et hormono-indépendant (HID). La croissance de PAC120 HD peut être inhibée par castration chirurgicale ou par administration d’un antagoniste de la LHRH, le FE200486 (Ferring, San Diego, CA). L’évolution vers l’hormono-indépendance est associée à une différenciation mucoïde ou neuroendocrine, à la survenue d’altérations chromosomiques supplémentaires et à des variations d’expression de gènes. PAC120 est un nouveau modèle de cancer de prostate qui offre l’opportunité d’étudier les mécanismes d’échappement hormonal et d’évaluer l’efficacité de nouveaux agents thérapeutiques.

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.

Transgenic mice expressing the c-Myc oncogene driven by woodchuck hepatitis virus (WHV) regulatory sequences develop hepatocellular carcinoma with a high frequency. To investigate genetic lesions that cooperate with Myc in liver carcinogenesis, we conducted a genome-wide scan for loss of heterozygosity (LOH) and mutational analysis of β-catenin in 37 hepatocellular adenomas and carcinomas from C57BL/6 x castaneus F1 transgenic mice. In a subset of these tumors, chromosome imbalances were examined by comparative genomic hybridization (CGH). Allelotyping with 99 microsatellite markers spanning all autosomes revealed allelic imbalances at one or more chromosomes in 83.8% of cases. The overall fractional allelic loss was rather low, with a mean index of 0.066. However, significant LOH rates involved chromosomes 4 (21.6% of tumors), 14, 9 and 1 (11 to 16%). Interstitial LOH on chromosome 4 was mapped at band C4–C7 that contains the INK4a/ARF and INK4b loci, and on chromosome 14 at band B–D including the RB locus. In man, the homologous chromosomal regions 9p21, 13q14 and 8p21–23 are frequently deleted in liver cancer. LOH at chromosomes 1 and 14, and β-catenin mutations (12.5% of cases) were seen only in HCCs. All tumors examined were found to be aneuploid. CGH analysis of 10 representative cases revealed recurrent gains at chromosomes 16 and 19, but losses or deletions involving mostly chromosomes 4 and 14 generally prevailed over gains. Thus, Myc activation in the liver might select for inactivation of tumor suppressor genes on regions of chromosomes 4 and 14 in a context of low genomic instability. Myc transgenic mice provide a useful model for better defining crosstalks between oncogene and tumor suppressor pathways in liver tumorigenesis.

We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor α. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf–Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.

A family of negative regulators of JAK signaling pathway referred to as suppressor of cytokines signaling (SOCS) or cytokine-inducible SH2 protein (CIS) has been recently identified. In order to find additional members of this family, we have used a consensus amino acid sequence contained in the well-conserved central SH2 domain to search DNA databases. We isolated cDNA coding for the human homologue of SOCS-5, referred to as CIS6. Northern blot analysis revealed CIS6 mRNA expression in various tissues such as heart, muscle, spleen, and thymus and in all myeloma cell lines examined. The gene was assigned to human chromosome bands 2p21 and 3p22 by in situ hybridization. CIS6 is structurally related to other members of the CIS family and therefore could act as a negative regulator of signal transduction.

The metabolism of polymers of ADP-ribose (ADP-Rib), a rapid cellular response to DNA damage, involves the concerted action of poly-ADPR polymerase (PARP) and poly-ADPR glycohydrolase (PARG). We have recently described the isolation and characterization of bovine cDNA encoding poly(ADP-ribose) glycohydrolase (PARG) Lin et al. (1997). Surprisingly, this cDNA codes for a protein of 111 kDa, nearly twice the size of the isolated protein from bovine thymus. More recently, we have cloned the human and mouse PARG cDNAs (manuscript in preparation) and analysis of the protein sequences shows that mammalian PARGs are highly conserved proteins constituted by two main domains, a catalytic domain of 65 kDa at the C-terminus and a domain of regulation of 45 kDa located at the N-terminus. Southern hybridization experiments indicated that PARG is encoded by a single copy gene. Thus, mapping of the human and mouse PARG genes may provide important insights to facilitate the study of many, presently unanswered questions pertaining to the biological significance of PARG and the pathway in which it is involved. Chromosomes were prepared from human peripheral blood lymphocyte cultures after BrdU incorporation during the last 7 h before harvesting. Mouse chromosomes were prepared from normal mouse fibroblast cultures. A 2.7-kb fragment of human PARG in vector Lafmid BA (EST H17106) and a 2.8-kb fragment of murine PARG in vector pTZ18R were labeled by nick-translation with biotin-11-dUTP (Sigma, France) and used as probes for FISH. A standard hybridization was performed as described previously (Apiou et al., 1996). The mouse PARG probe was used at a concentration of 15 ng/µl in the presence of 100 fold excess of mouse Cot-1 DNA. The human PARG probe was used at a concentration of 20 ng/µl in 15 µl of hybridization buffer for each slide. For human chromosomes, direct banding of BrdU-substituted chromosomes was obtained by incubation in an alkaline solution of p-phenylenediamine, (PPD11) (Lemieux et al., 1992) and stained with propidium iodide. Mouse chromosomes were stained with DAPI and identified with computer generated reverse DAPI banding. Immunochemical detection of hybridization was performed using goat antibiotin antibodies (Vector laboratories, Burlingame, CA) and rabbit FITC conjugated anti-goat antibodies (Biosys, Compiègne, France). Metaphases were observed under a fluorescent microscope (DMRB, Leica, Germany). Images were captured using a cooled photometrics CCD camera and Quips-smart capture software (Vysis).Probe name: hPARG2.7 human, mPARG2.8 mouseProbe type: cDNAInsert size: 2.7 kb human; 2.8 kb mouseVector: pTZ18R, and Lafmid BAGene reference: GeneBank AF005043 human and AF079557 mouseLocation:10qNumber of cells examined: 25Number of cells with specific signals: 20Most precise assignment:10q11.23Location of background signals (sites with >2 signals): none observedLocation:14Number of cells examined:25Number of cells with specific signals:19Most precise assignment:14BLocation of background signals (sites with >2 signals): none observed (fig. 1)Supported by Fondation pour la Recherche Médicale and by NIH Grant CA43894.

Human alpha-endosulfine is an endogenous regulator of the beta-cell K(ATP) channels. The recombinant alpha-endosulfine inhibits sulfonylurea binding to beta-cell membranes, reduces cloned K(ATP) channel currents, and stimulates insulin secretion from beta-cells. These properties led us to study the human ENSA gene that encodes alpha-endosulfine. Here, we describe the isolation, the partial characterization, and the chromosomal localization of the ENSA gene. The ENSA gene appears to be a 1.8-kb-long sequence that contains the transcription initiation site located 528 bp upstream of the initiation codon. The ENSA gene is intronless, and a single copy gene seems to be present in the genome. Finally, the ENSA gene co-localizes on human chromosome 14 (14q24.3-q31) with a locus for susceptibility to type 1 diabetes called IDDM11; thus, the ENSA gene represents an IDDM11 candidate.

The human OZF gene (ZNF146), located in chromosome band 19q13.1, is amplified and overexpressed in pancreatic carcinomas. It encodes a protein consisting solely of ten Krüppel zinc finger motifs. We report here the isolation and the characterization of the murine OZF cDNA (Zfp146). Comparison of the deduced amino acid sequences between murine, human and bovine cDNAs revealed a strong identity (95%). A closely related gene, Zfp260, was also isolated and characterized. It encodes a putative protein consisting of three vestigial zinc finger motifs followed by ten Krüppel zinc fingers sharing 79% identity and no gap insertion with the Zfp146 zinc fingers. In vitro transcription/translation of both genes led to synthesis of proteins of the predicted size. Co-expression was observed at the mRNA level in eight adult mouse tissues. Two-color FISH revealed co-localization of both genes on mouse chromosome 7 (band B1–B3). The co-expression and co-localization of Zfp146 and Zfp260 together with the close similarity of their zinc finger domains, suggests that both participate in the same regulatory pathway.

Purine nucleotide metabolism was studied in two human cutaneous melanoma cell lines IPC182 and IGR221. IPC182 cells do not differentiate, while IGR221 cells differentiate spontaneously at confluency, with intense melanin production. The activities of 11 enzymes involved in the de novo or salvage synthesis or the catabolic pathway of purine nucleotides were measured at different times (from day 3 to day 18), after subculture, during exponential growth and the stationary phase, with or without differentiation. The results demonstrated remarkable differences in the enzyme activity levels and/or the evolution from exponential growth to the stationary phase for each cell line, as well as between the two cell lines. In the non-differentiating IPC182 cells, the activity of enzymes involved in purine nucleotide synthesis decreased when the growth rate slowed down and remained at a low level with a concomitant increase in catabolic activities. In the differentiating IGR221 cells, the activity of enzymes involved in purine nucleotide salvage synthesis increased during the proliferative phase and was maintained at a high level when the cells reached confluency and differentiated; catabolic activities were always lower than in the IPC182 cells. This suggests that extra purine nucleotides, synthesized preferentially by the salvage pathway, could be required for the differentiation of human melanoma cells. Since the two cell lines were cultured in the absence of any differentiation-inducing agents, these results indicate that various metabolic modifications are associated with the natural processes of cell proliferation and differentiation. This research could help to identify some of the enzymes involved in purine metabolism as the targets for the induction of differentiation.

To characterize the biological role of Kin17 protein, a mammalian nuclear protein which participates in the response to UV and ionizing radiation and binds to curved DNA, EBV-derived vectors carrying (Mm)Kin17 cDNA were constructed and transfected in tumorigenic cells harboring different p53 profiles (HeLa, H1299, and HCT116) and in immortalized HEK 293 cells. (Mm)Kin17 protein expression induced a tremendous decrease in cell proliferation of the three tumorigenic cell lines 2 weeks after transfection. Transfection of HEK 293 cells with an pEBVCMV(Mm)Kin17 plasmid gave rise to numerous (Mm)Kin17-expressing cells which constantly disappeared with time, preventing the establishment of (Mm)Kin17-expressing cells. Several independent clones were isolated from HEK 293 cells carrying a pEBVMT(Mm)Kin17 vector. The two clones described here (B223.1 and B223.2) exhibited different (Mm)Kin17 protein levels and displayed a gradual decrease in their proliferative capacities. In B223.1 cells, the basal expression of (Mm)Kin17 greatly reduced plating efficiency and cell growth. B223.1 cell morphology was altered, with numerous round-shaped cells whose spreading on the culture support was hampered. We observed giant multinucleated cells or cells containing micronuclei-like structures and/or multilobed nuclei. To conclude, (Mm)Kin17 overexpression reduced the proliferation of tumorigenic cells independently of their p53 status and modified cell growth and cell morphology of established HEK 293 cells producing (Mm)Kin17 protein. It is likely that (Mm)Kin17 may interfere with DNA replication.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleusin vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.