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.     

Unusual chromosome architecture and behaviour at an HSR

Amplification of sequences within mammalian chromosomes is often accompanied by the formation of homogeneously staining regions (HSRs). The arrangement of DNA sequences within such amplicons has been investigated, but little is known about the chromosome structure or behaviour of these unusual regions. We have analysed the metaphase chromosome structure of the dihydrofolate reductase (DHFR) amplicon of CHOC400 cells. The chromatin in this region contains hyperacetylated nucleosomes yet, at the same time, appears to be densely packed like heterochromatin. The region does not bind heterochromatin proteins. We show that the dense packing of the region is restricted to DNA located close to the chromosome core/scaffold. In contrast, levels of the chromosome scaffold protein topoisomerase II at HSRs are the same as those found at other euchromatic locations. Metaphase chromosome condensation of the HSR is shown to be sensitive to topoisomerase II inhibitors, and sister chromatids often appear to remain attached within the HSRs at metaphase. We suggest that these features underlie anaphase bridging and the aberrant interphase structure of the HSR. The DHFR amplicon is widely used as a model system to study mammalian DNA replication. We conclude that the higher-order chromosome structure of this amplicon is unusual and suggest that caution needs to be exercised in extrapolating data from HSRs to normal chromosomal loci. Received: 19 October 1999; in revised form: 13 December 1999 / Accepted: 27 December 1999     

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.     

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     

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.     

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.     

Double insertion of homogeneously staining regions in chromosome 1 of wild Mus musculus musculus: effects on chromosome pairing and recombination

An examination of the meiotic pattern of chromosome 1 isolated from a feral mouse population and containing a double insertion (Is) of homogeneously staining regions (HSRs) was carried out. The region delineated by the proximal breakpoint of Is(HSR;1C5) 1Icg and the distal breakpoint of Is(HSR;1E3)2Icg is desynapsed during the early pachytene stage and heterosynapsed at the midpachytene, as shown by electron microscopic analysis of synaptonemal complexes. The HSRs have no effect on the segregation of chromosome 1 in heterozygous mice. The lack of homosynapsis in the region under study causes chiasmata redistribution in heteromorphic bivalents. In normal males, single chiasmata are located in the medial part of the chromosome. In heterozygotes, this segment is heterosynapsed and unavailable for recombination. This leads to a significant decrease in the frequency of bivalents bearing single chiasmata. The total number of chiasmata per bivalent is much higher in heterozygous males than in normal ones. The recombination frequency between proximal markers fz and In also is higher in heterozygous animals. The increase in the total chiasma number in the heteromorphic bivalent is due to the addition of double chiasmata located mostly at precentromeric and pretelomeric regions of the chromosome.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

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.     

High-resolution in situ hybridization technique using biotinylated NMYC oncogene probe reveals periodic structure of HSRs in human neuroblastoma

Gene amplification in mammalian cells commonly manifests itself as homogeneously staining chromosomal regions (HSRs). These are frequently seen in neuroblastoma and have been shown to be the site of amplification of the NMYC oncogene. We have used a nonisotopic, high-resolution in situ hybridization technique to reveal a hitherto unrecognized periodic microstructure within HSRs of a human neuroblastoma cell line.     

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.     

When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation

Amplified genes are frequently localized on extrachromosomal double minutes (DMs) or in chromosomal homogeneously staining regions (HSRs). We previously showed that a plasmid bearing a mammalian replication initiation region could efficiently generate DMs and HSRs after transfection into human tumor cell lines. The Breakage-Fusion-Bridge (BFB) cycle model, a classical model that explains how HSRs form, could also be used to explain how the transfected plasmids generate HSRs. The BFB cycle model involves anaphase bridge formation due to the presence of dicentric chromosomes, followed by the breakage of the bridge. In this study, we used our plasmid-based model system to analyze how anaphase bridges break during mitosis. Dual-color fluorescence in situ hybridization analyses revealed that anaphase bridges were most frequently severed in their middle irrespective of their lengths, which suggests that a structurally fragile site exists in the middle of the anaphase bridge. Breakage of the chromosomal bridges occurred prior to nuclear membrane reformation and the completion of cytokinesis, which indicates that mechanical tension rather than cytokinesis is primarily responsible for severing anaphase bridges. Time-lapse observation of living cells revealed that the bridges rapidly shrink after being severed. If HSR length was extended too far, the bridge could no longer be resolved and became tangled depending on the tension. The unbroken bridge appeared to inhibit the completion of cytokinesis. These observations strongly suggest that anaphase bridges are highly elastic and that the length of the spindle axis determines the maximal HSR length.     

Nonrandom karyotype changes in chemically induced tumors of the Djungarian hamster

In the cells of tumors induced with methylcholanthrene in wild type and mutant (pink-eyed dilution) Djungarian hamsters non-random involvement in structural changes of certain chromosomes (Xp, 3p and 3q, 7q, 8q) was revealed. In addition, characteristic feature of the majority of tumors was varied number of double-minutes chromosomes (DMs). In some tumors, the markers with long homogeneously or differentially stained regions (HSRs and DSRs) were also present. The DMs, HSRs and DSRs are known as the structures containing amplified genes.     

Homogeneously staining regions (HSRs) of a rat hepatoma cell line are not early replicating

The rat hepatoma cell line H4-IIE-C3 (H4) has homogeneously staining regions (HSRs) which contain multiple, tandemly repeated copies of ribosomal RNA (rRNA) genes. We determined the time of replication of the DNA within these HSRs autoradiographically after incorporation of [3H]thymidine and by Hoechst 33258 and Giemsa staining after 5-bromodeoxyuridine (5-BrdU) incorporation. The DNA within the H4 HSRs is not early replicating, unlike that in other HSRs. It begins replicating later than much of the other nuclear DNA, continues replicating throughout most of the S phase, and is completed 1-2 h before mitosis.     

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.     

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.     

The causes of appearance of a double insertion of homogeneously staining regions in the chromosome 1 of house mouse (Mus musculus musculus)]

A high resolution analysis of G-band pattern of normal and aberrant chromosome 1 bearing two linked insertions of homogeneously staining regions (HSRs) in the house mouse (Mus musculus musculus) reveals an inverted pattern of the euchromatic region between the HSRs. On the basis of this analysis, a hypothesis on the causes for appearance of the aberrant chromosome was put forward: the double insertion is a result of inversion of the chromosome 1 of Mus musculus domesticus bearing a single long insertion. The proximal breakpoint is localized inside the HSR and the distal one--between subbands E3 and E4. From the point of view of these data, new symbols for the aberrations are proposed: Ls (HSR, 1C5) 1Icg--for the proximal insertion, Is(HSR, 1D)21cg--for the distal one, In (1) 1Icg--for the inverted region, including the bands D, E1-E3 and the insertion Is(HSR 1D)21cg.