Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA.

Human neuroblastoma cells often carry amplified DNA encompassing the gene N-myc. Amplified N-myc has been found localized in "double minutes" in direct tumor cell preparations. In contrast, later passages carried amplified N-myc almost exclusively within a single homogeneously staining chromosomal region located at a chromosomal site different from the normal location of N-myc. We used pulsed field gel electrophoresis to define the structural arrangement of the amplified DNA. Long-range mapping was facilitated by the presence of several sites for rare cutting restriction endonucleases in the 5' region of N-myc. Amplified DNAs of different neuroblastoma cell lines were heterogeneous in size and had undergone recombination at various distances from N-myc. N-myc occupied a central position within the amplified DNA, and in no case was the coding region affected by recombination. Among neuroblastoma cells, varying proportions of amplified DNA (in some instances close to 100%) consisted of multiple tandem arrays of DNA segments ranging in size from 100 to 700 kilobase pairs. Tumor cells with low degrees of amplification revealed regions of amplified DNA in excess of 1,500 kilobase pairs without apparent rearrangement. Our observations, in concert with the cytogenetic findings, suggest a model of gene amplification which involves unscheduled DNA replication, recombination, and formation of extrachromosomal DNA followed by integration into a chromosome and subsequent in situ multiplication. The central position which N-myc occupies within the amplified sequences and the lack of recombination within the coding region of N-mc indicate that N-myc rather than other genetic information provides the selective advantage for retention of the amplified DNA.     

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.     

Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis

We characterized N-myc gene amplification in three human neuroblastoma cell lines (IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rare-cutting enzymes completely digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case. We estimated that the minimal distance between neighboring N-myc genes is at least several hundred kilobases. Our data suggest that amplification units contain several DNA fragments derived from ditterent loci, but that they are homogeneous.     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Characterization of N-myc amplification units in human neuroblastoma cells.

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.     

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.     

Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.     

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.     

Generation of high-density DNA markers from yeast artificial chromosome DNA by single unique primer-polymerase chain reaction

We have developed a method for the whole sequence amplification of yeast artificial chromosome (YAC) DNA excised from preparative pulsed-field gel electrophoresis using single unique primer-polymerase chain reaction procedures. We used seven contiguous YAC clones, which span 2 Mbp of the Huntington disease gene region on 4p16.3, to amplify the YAC DNAs. The average size of the amplified DNA was ∼300 bp long, and 12 DNA markers located on the YAC clones positively hybridized with these amplified products, implying that the sequences of the YAC clones were comprehensively amplified by our procedures. These amplified YAC DNAs greatly facilitate the characterization of YAC clones, leading to the detailed analysis of the defined chromosomal region.     

Excision of N-myc from chromosome 2 in human neuroblastoma cells containing amplified N-myc sequences.

Amplification of one of three growth-stimulating myc genes is a common method by which many tumor types gain a proliferative advantage. In metastatic human neuroblastoma, the amplification of the N-myc locus, located on chromosome 2, is a dominant feature of this usually fatal pediatric cancer. Of the many models proposed to explain this amplification, all incorporate as the initial step either disproportionate overreplication of the chromosomal site or recombination across a loop structure. The original locus is retained within the chromosome in the overreplication models but is excised in the recombination models. To test these models, we have used somatic cell hybrids to separate and analyze the chromosomes 2 from a neuroblastoma cell line containing in vivo amplified N-myc. Our results demonstrate that N-myc is excised from one of the chromosomes, suggesting that deletion is a requisite part of gene amplification in a naturally occurring system.     

Amplified cellular oncogenes in neoplasms of the human central nervous system.

Significant advances have recently been made in a number of areas concerning central nervous system (CNS) neoplasia. Particularly salient are the following: (1) gene amplification is related to increasing grade of human glioma malignancy and occurs in approximately 40% of the most common and most malignant variety of glioma, glioblastoma multiforme (GBM), (2) by far the most commonly amplified gene in glioblastomas is the epidermal growth factor receptor (EGFR) gene, which is amplified in about one third of GBMs, (3) a small percentage of GBMs amplify N-myc or the novel sequence gli, (4) the EGFR gene is rearranged in at least half of gliomas in which it is amplified, and (5) EGFR gene rearrangement results in external domain deletions that yield truncated EGF receptors. Antibodies specific for the mutant EGF receptor fusion junction have been successfully produced and provide stimulating new potential avenues for tumor imaging and therapy. For pediatric CNS neoplasms, only medulloblastoma has been investigated in adequate numbers; a small percentage exhibit amplification of either the N-myc or c-myc genes.     

Amplification of Hot DNA segments in Escherichia coli

In Escherichia coli, a replication fork blocking event at a DNA replication terminus (Ter) enhances homologous recombination at the nearby sister chromosomal region, converting the region into a recombination hotspot, Hot, site. Using a RNaseH negative (rnhA-) mutant, we identified eight kinds of Hot DNAs (HotA-H). Among these, enhanced recombination of three kinds of Hot DNAs (HotA-C) was dependent on fork blocking events at Ter sites. In the present study, we examined whether HotA DNAs are amplified when circular DNA (HotA plus a drug-resistance DNA) is inserted into the homologous region on the chromosome of a rnhA- mutant. The resulting HotA DNA transformants were analysed using pulsed-field gel electrophoresis, fluorescence in situ hybridization and DNA microarray technique. The following results were obtained: (i) HotA DNA is amplified by about 40-fold on average; (ii) whereas 90% of the cells contain about 6-10 copies of HotA DNA, the remaining 10% of cells have as many as several hundred HotA copies; and (iii) amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs are amplified to the same level as HotA. Furthermore, HotL DNA, which is activated by blocking the clockwise oriC-starting replication fork at the artificially inserted TerL site in the fork-blocked strain with a rnhA+ background, is also amplified, but is not amplified in the non-blocked strain. From these data, we propose a model that can explain production of three distinct forms of Hot DNA molecules by the following three recombination pathways: (i) unequal intersister recombination; (ii) intrasister recombination, followed by rolling-circle replication; and (iii) intrasister recombination, producing circular DNA molecules.     

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.     

Amplification of the N-myc oncogene in an adenocarcinoma of the lung

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.     

Human proto-oncogene N-myc encodes nuclear proteins that bind DNA.

N-myc is a gene whose amplification has been implicated in the genesis of several malignant human tumors. We have identified two proteins with molecular weights of 65,000 and 67,000 encoded by N-myc. The abundance of these proteins in tumor cells was consonant with the extent of amplification of N-myc. The two proteins apparently arose from the same mRNA, were phosphorylated, were exceptionally unstable, were located in the nucleus of cells, and bound to both single- and double-stranded DNA. These properties suggest that the products of N-myc and of the related proto-oncogene c-myc may have similar biochemical functions and that N-myc may be a regulatory gene. Our findings sustain the view that inordinate expression of N-myc may contribute to the genesis of several different human tumors.     

Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Two-colour in situ hybridization with probes for two co-amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase-long inverted repeats. Their organization is remarkably well accounted for if breakage-fusion-bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.     

Multiplex PCR with the blunt hairpin primers for next generation sequencing

The multiplex PCR is one of the important methods to enrich the target DNAs for next generation sequencing. The non-specific amplification and interaction between the primers are the pivotal challenges of multiplex PCR. Here, we introduce the novel blunt hairpin primers for effective reducing the primer dimers and mispriming events. We also used a pair of auxiliary primers to enhance PCR efficiency. We simultaneously amplified 89 target regions from 44 samples and sequenced all amplicons on ion torrent PGM platform. Among all the filtrated amplicons (3438 different amplicons), 99.7, 97.6, 90.1 and 72.8% had sequencing depths fell within 200, 100, 50 and 25-fold range. The sequencing depth variations among all the samples were less than 27-fold. We also amplified multiplex regions with blunt hairpin, stick hairpin and normal linear primers, and the blunt hairpin primers could significantly reduce the amount of primer dimers and unspecific products.These results show that multiplex PCR with the blunt hairpin primers is a flexible, specific and economical target-region captured approach for the next generation sequencing.     

Molecular cytogenetic characterization of the 11q13 amplicon in head and neck squamous cell carcinoma

Amplification of 11q13 DNA sequences and overexpression of CCND1 are common findings in head and neck squamous cell carcinoma (HNSCC), identified in about 30% of the cases. However, little is known about initiation of the amplification and the organization of the amplicon. In order to study the structure of the amplicon in more detail and to learn more about the mechanisms involved in its initiation, prometaphase, metaphase, and anaphase fluorescence in situ hybridization (FISH) with 40 BAC clones spanning a 16-Mb region in chromosome bands 11q12.2 to 11q13.5 was performed in nine HNSCC cell lines with homogeneously staining regions. FISH analysis showed that the size of the amplicon varied among the nine cell lines, the smallest being 2.12 Mb and the largest 8.97 Mb. The smallest overlapping region of amplification was approximately 1.61 Mb, covering the region from BAC 729E14 to BAC 102B19. This region contained several genes previously shown to be amplified and overexpressed in HNSCC, including CCDN1, CTTN, SHANK2, and ORAOV1. The cell lines were also used to study the internal structure of the amplicon. Various patterns of amplified DNA sequences within the amplicon were found among the nine cell lines. Even within the same cell line, different amplicon structures could be found in different cell populations, indicating that the mechanisms involved in the development of the amplicons in HNSCC were more complex than previously assumed. The frequent finding of inverted repeats within the amplicons, however, suggests that breakage-fusion-bridge cycles are important in the initiation, but the fact that such repeats constituted only small parts of the amplicons indicate that they are further rearranged during tumor progression.