Polymorphic HSRs in chromosome 1 of the two semispecies Mus musculus musculus and M. m. domesticus have a common origin in an ancestral population

HSRs (homogeneously staining regions) are the cytological correlates of DNA amplification. In the house mouse, Mus musculus, many populations are polymorphic for the presence or absence of HSRs on chromosome 1. In the semispecies M. m. domesticus the amplified DNA is present within one HSR, whereas in M. m. musculus chromosomes 1 with two HSRs are found. Hybridization of HSR-specific probes to Southern blots of HSR-carrying genomic DNAs from different localities and semispecies revealed similar complex band patterns. the remaining variation is restricted to sequences with a low degree of amplification. Variation is higher between semispecies than within one semispecies. It is assumed that HSRs are derived from one original amplification event and that unequal recombination is the mechanism underlying the length variation of HSRs present today in both semispecies. Evidence from G-banding and in situ hybridization shows that the two HSRs of M. m. musculus originated from a single HSR by means of a paracentric inversion, where one break-point was located within the single HSR and the second outside the HSR. As a consequence of the paracentric inversion the two HSRs of M. m. musculus are permanently linked together. Since exchange of genes between the two semispecies is restricted to a narrow hybrid zone the amplification that gave rise to the HSR most probably occurred prior to the divergence into the semispecies M. m. domesticus and M. m. musculus about 1 million years ago.by D. Schweizer     

Chromosome anomalies associated with amplification of the adenosine deaminase gene (ADA) in rat hepatoma cells

Two independently selected series of rat hepatoma cell lines resistant to the drug deoxycoformycin (dCF) were analyzed karyotypically. Several forms of homogeneously staining regions (HSRs) were present on metaphase chromosomes of these cells. In some instances HSRs comprised nearly an entire chromosome, which are among the largest chromosomes in the karyotype. Stable resistance to dCF is acquired in rat cells by overproduction of the enzyme adenosine deaminase (ADA) as a result of amplification of ADA gene sequences. We have localized the amplified ADA gene sequences to HSRs on metaphase chromosomes from both series of dCF-resistant cell lines by in situ hybridization. Based upon the number of ADA gene sequences present and the lengths of the HSRs, we have estimated the size of the amplified unit to range from 450 to 1,000 kb.     

Recurrent HSR in the centromeric region of chromosome 8 in breast cancer

In a sample of 44 human breast cancers, the presence of HSRs was detected in 15 cases (36%). The occurrence of HSRs seems correlated poorly with tumor progression, but the number of HSRs per cell certainly does. Chromosome 8 was the most frequently involved, and the HSRs were always located near its centromere, suggesting that an amplification of gene(s) located in this region has occurred. Among these, LHRH is a candidate, but further studies are needed to confirm or reject this hypothesis.     

Intrachromosomal Gene Amplification Triggered by Hairpin-Capped Breaks Requires Homologous Recombination and is Independent of Nonhomologous End-Joining

Gene amplification is one of the major mechanisms of acquisition of drug resistance and activation of oncogenes in tumors. In mammalian cells, amplified chromosomal regions are manifested cytogenetically as extrachromosomal double minutes (DMs) and chromosomal homogeneously staining regions (HSRs). We recently demonstrated using yeast model system that hairpin-capped double strand breaks (DSBs) generated at the location of human Alu-quasipalindromes can trigger both types of gene amplification. Specifically, the dicentric chromosomes arising from replication of hairpin-capped molecules can be precursors for intrachromosomal amplicons. The formation of HSRs can be accounted for either by breakage-fusion-bridge (BFB) cycle which necessitates nonhomologous end-joining pathway (NHEJ) or by the repair event involving homologous recombination (HR). In this study, we report that intrachromosomal gene amplification mediated by hairpin-capped DSBs is independent of NHEJ machinery, however requires the functions of Rad52 and Rad51 proteins. Based on our observations, we propose a HR-dependent mechanism to explain how the breakage of dicentric chromosomes can lead to the formation of HSRs.     

Changes in the genotype and phenotype determining the resistance of Djungarian hamster somatic cells to adriablastin

The development of adriablastin resistance in Djungarian hamster DM-15 cells is accompanied by the appearance of small chromatin bodies (SCB) and long homogeneously staining regions (HSRs) in the chromosomes--the structures that contained amplified genes. The pattern of karyotypic alterations (the appearance of additional chromosome 4, and emergence of SCB, formation of the HSRs in one of three of chromosome 4, transposition of the HSRs from chromosome 4 to other chromosomes) during the development of adriablastin resistance is identical to that found in these cells before, namely during the development of colchicine resistance. Adriablastin- and colchicine-resistant cells have similar changes in plasma membrane permeability for 3H-colchicine, 3H-actinomycin D, 3H-puromycin, 3H-cytochalasin B, and 3H-vinblastine. Apparently, adriablastin resistance has the same mechanism as colchicine resistance, being connected with gene amplification and decreased plasma membrane permeability for these drugs.     

Regular pattern of karyotypic alterations accompanying gene amplification in Djungarian hamster cells: study of colchicine,adriablastin, and methotrexate resistance

Chromosomal analysis of 26 Djungarian hamster cell lines obtained from 11 independent clones and possessing different levels of resistance to colchicine or adriablastin as a consequence of gene amplification revealed regular patterns in the karyotypic changes that accompanied the development of drug resistance. Usually the sequence of karyotypic changes was as follows: first an additional chromosome 4 appeared; then single unpaired small chromatin bodies (SCBs) arose; later in the middle part of the long arm of one of three chromosomes 4 long homogeneously staining regions (HSRs) and double minute chromosomes (DMs) were formed; and finally in the most resistant variants large clusters of SCBs appeared. The emergence of the clusters of the SCBs correlated well with the occurrence of autonomously replicating, amplified DNA sequences. In contrast to DNA of the HSRs the DNA of the SCBs could replicate outside the S-phase of the cell cycle. When kept in a non-selective medium, the cells gradually lost their resistance to colchicine: 1%–4% of the cells lost the capacity to form colonies in the selective medium independently of the pattern of location in them of amplified genes (in chromosomal HSRs, SCBs, or DMs). Loss of drug resistance was accompanied by disappearance of the chromosomal HSRs, SCBs, and DMs. Chromosomal analysis of the set of methotrexate-resistant Djungarian hamster cell lines indicated the following karyotypic evolution: first the additional material on the distal part of one of two chromosomes 3 appeared; then the light HSRs were formed on the distal part of one of two chromosomes 4; later clusters of SCBs and HSRs arose on the distal part of the short arm of chromosome 3. Probably the amplification of different genes is characterized by specific patterns of karyotypic alterations.     

Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.     

Cytological, flow cytometric, and molecular analysis of the rapid evolution of mammalian chromosomes containing highly amplified DNA sequences

Transfection of a mouse dihydrofolate reductase (DHFR) cDNA contained in a plasmid "expression vector" into DHFR deficient Chinese hamster cells, followed by progressive selection of cells in increasing concentrations of methotrexate (MTX), leads to marked amplification of the exogenous DHFR sequences in the recipient hamster cells. This gene amplification is evident at the cytological level, in the form of homogeneously staining chromosomal regions (HSRs), at a gene expression level, in the form of fluorescein-methotrexate binding, and at the DNA level. Flow sorting, based on variable fluorescein-MTX binding, or direct cellular cloning, followed by chromosome analysis, revealed intercellular heterogeneity of HSRs in size and distribution. This suggested that there was a rapid evolution of HSRs in MTX-resistant transfectants. Chromosomal analysis of HSR evolution in situ, by examining individual colonies presumably derived from one or a few cells, underscored this impression of chromosome structural fluidity. Rates of HSR change in excess of 0.01 per cell division, increased by low doses of the recombinogen, mitomycin C, were detected. The Chinese hamster DHFR transfectants described should be amenable to detailed, coordinate cytological and molecular characterization. Such an analysis should contribute to an understanding of processes such as homologous recombination in mediating HSR evolution in mammalian chromosomes.     

Occurrence and evolution of homogeneously staining regions may be due to breakage-fusion-bridge cycles following telomere loss

Chromosomes with homogeneously staining regions (HSR) were analysed in a subclone of the H4 rat hepatoma cell line, where they represent amplification of the ribosomal RNA (rRNA) genes. Detailed G-band analysis of the subclone revealed that an HSR on the short arm of chromosome 3 became unstable and changed its position within the chromosome. The evolution of this marker chromosome was associated with the terminal deletion of the normal long arm of the HSR-bearing chromosome 3 and may have involved ring formation as a result of fusion between the HSR on the short arm and the broken end of the long arm. Evidence was obtained for breakage at different sites within the ring, producing chromosomes with HSRs located terminally on either the long arms or both arms. The terminally located HSR underwent elongation in some cells presumably as a result of a breakage-fusion-bridge cycle characteristic of instability due to telomeric loss. It is suggested that terminally located HSRs may generally occur this way.     

Amplification of portions of the genome in mammalian somatic cells resistant to colchicine. II. Marker chromosomes with long homogeneously staining regions in colchicine-resistant cells of the Djungarian hamster

The series of sublines 170-750 times more resistant to colchicine were obtained from 10 independent clones of Djungarian hamster cells possessing 16-22-fold resistance to the drug. From each clone, several sublines with different levels of colchicine-resistance were developed. The drug resistance was unstable. 2,7-4,0% of cells per population doubling lost resistance to selective dosages of colchicine. The loss of resistance was stepwise. The chromosomes stained by trypsin G-banding technique were studied in 17 sublines. 15 sublines derived from 9 independent clones contained chromosomes with long homogeneously staining regions (HSRs). These were, as a rule, primarily localized in the long arm of chromosome 4. During cultivation, HSRs were transferred from chromosome 4 into other chromosomes. Evidently, transposition of HSRs was due to translocations of different chromosomes of HSRs in the chromosome 4 and to subsequent breakages of the resulting dicentrics within HSRs. A great number of different chromosomal rearrangements was also found in the cells containing HSRs. Possibly, formation of HSR leads to destabilization of the karyotype and to the variability of the genome. The length of HSRs varied in different cells of each subline. The levels of colchicine-resistance in different sublines did not correlate with the average length of HSRs in their cells. The lack of connection between the lengths of HSRs and the levels of drug resistance as well as the existence of highly resistant sublines with gene amplification, but without HSRs, suggest that amplified genes are localized in Djungarian hamster colchicine-resistant cells both in chromosomes and extrachromosomally.     

Monoclonal antibodies: Longitudinal prescribing information analysis of hypersensitivity reactions

Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy’s life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3–4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use.     

Adenosine deaminase gene amplification in deoxycoformycin-resistant mammalian cells

Deoxycoformycin (dCF)-resistant mutants of rat hepatoma, mouse LMTK-, and Chinese hamster ovary (CHO) cells have been isolated and shown to overproduce adenosine deaminase (ADA). The overproduction of ADA was found to be due to ADA-gene amplification in rat and mouse cells but not in CHO cells. Deoxycoformycin-resistant rat hepatoma cells have large HSRs (homogeneously staining regions), mouse cells carry DMs (Double minutes), and CHO cells do not appear to have any gross chromosomal anomalies. When dCF-resistant rat hepatoma and mouse cells are selected by increasing the concentration of the inhibitor in small increments, there is a good correlation between the increase in ADA gene copy number and the increase in the level of expression of ADA, suggesting that all of the amplified genes are equally active in the expression of ADA.     

Monoclonal antibodies: Longitudinal prescribing information analysis of hypersensitivity reactions

Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy’s life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3–4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use.     

Sunitinib Suppress Neuroblastoma Growth through Degradation of MYCN and Inhibition of Angiogenesis

Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common and deadly extracranial tumor of childhood. The majority of high-risk neuroblastoma exhibit amplification of the MYCN proto-oncogene and increased neoangiogenesis. Both MYCN protein stabilization and angiogenesis are regulated by signaling through receptor tyrosine kinases (RTKs). Therefore, inhibitors of RTKs have a potential as a treatment option for high-risk neuroblastoma. We used receptor tyrosine kinase antibody arrays to profile the activity of membrane-bound RTKs in neuroblastoma and found the multi-RTK inhibitor sunitinib to tailor the activation of RTKs in neuroblastoma cells. Sunitinib inhibited several RTKs and demonstrated potent antitumor activity on neuroblastoma cells, through induction of apoptosis and cell cycle arrest. Treatment with sunitinib decreased MYCN protein levels by inhibition of PI3K/AKT signaling and GSK3β. This effect correlates with a decrease in VEGF secretion in neuroblastoma cells with MYCN amplification. Sunitinib significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude mice and demonstrated an anti-angiogenic effect in vivo with a reduction of tumor vasculature and a decrease of MYCN expression. These results suggest that sunitinib should be tested as a treatment option for high risk neuroblastoma patients.     

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.     

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification.     

Molecular cloning of a cDNA for Chinese hamster ovary asparagine synthetase

In previous reports we have described the isolation and characterization of a number of Chinese hamster ovary (CHO) cell mutants resistant to the amino acid analogue albizziin (Alb). Multistep mutants were derived which showed a high degree of drug resistance and expressed increased levels of asparagine synthetase (AS) levels up to 300-fold over that of the parental cell line. Karyotypic analysis of these mutants revealed homogeneously staining regions (HSRs) usually indicative of gene amplification. In the present work, we provide further proof for gene amplification by showing that the mutants greatly overproduce functional AS mRNA, as evidenced by in vitro translation of purified mRNA and immunoprecipitation of AS.

By using these overproducing mutants as sources of mRNA coupled with velocity centrifugation, we have been able to greatly enrich for AS sequences in our mRNA preparations to the point where they represent 1–5% of the total message. This facilitated cloning and selection of the cDNA sequences complementary to the gene. Utilizing these cloned cDNAs, we have demonstrated a correlation between gene copy number and enzyme expression in the parent and Alb-resistant mutants, thus providing direct evidence that drug resistance is due to gene amplification.     

In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.