A heterochromatic homogeneously staining region (HSR) in the karyotype of a human breast carcinoma cell line

A long heterochromatic region is described in the karyotype of a human breast carcinoma cell line. Cytochemical studies of this region suggest: 1) homogeneously staining regions (HSRs) in human tumor cells may show some heterogeneity in staining, 2) HSRs in human tumor cells may be euchromatic or heterochromatic by C-, G-, and Q-banding methods.     

Loss of retinoblastoma gene and amplification of N-myc gene in retinoblastoma.

We have analyzed paired samples of genomic DNA from peripheral leukocyte and primary tumor tissue from nine patients with retinoblastoma (RB) and from two RB cell lines, WERI-Rb-1 and Y79, to detect the molecular alterations of the retinoblastoma susceptibility gene (RB-1) and N-myc gene. In Southern analysis, RB-1 deletions in tumor tissues were detected in five patients (56%), one of these revealed a total loss of RB-1. N-myc amplification was found only in one (11.1%) out of nine patients. We also observed a total loss of RB-1 in WERI-Rb-1, and a more than 100-fold amplification of N-myc in Y79. The analysis of the relationship between molecular events and clinical characteristics such as age, sex, tumor laterality did not reveal any specific correlation. These results suggest that genetic backgrounds of RB in Korean patients are quite similar to those of reported cases elsewhere. The high sensitivity of our method in detecting the RB-1 loss indicates that this method can be a useful tool for initially screening a large number of tumors.     

Double minute chromosomes and a homogeneously staining chromosome region in C3H10T1/2 murine cells transformed "in vitro" by proton radiation

Four foci (type II or type III) of transformed cells, isolated from the murine line C3H10T1/2 after exposure to proton radiations, were expanded and cytogenetically examined. While the overall numerical chromosome distributions were similar, there were some differences between the various cell lines with regard to the presence and frequency of specific-marker chromosomes and to the colony-forming efficiency in soft-agarose medium. No association between any of these markers and the transformed phenotype could be established. However, in the line F4, derived from a type II focus, numerous double-minute chromosomes (DM) were observed after passage 22, and the phenomenon became more pronounced in the subclone C2. The finding of DMs in radiation-transformed cells is unusual. The DMs were observed in long-term subcultures, and in one of them they were partially replaced by a homogeneously staining chromosome region (HSR). DNAs from transformed cells of the line F4 and subclone C2 was digested with restriction enzymes and analyzed by Southern blotting with probes for seven oncogenes commonly amplified in cancer cells (c-myc, N-myc, N-ras, Ki-ras, Ha-ras, c-myb, c-abl) and with probes for the mouse MHC class I region. None of the regions tested was structurally altered or amplified in these transformed cells. The origin of the genetic material carried by DMs or homogeneously staining intrachromosomal regions (HSR) in cells of the line F4 and subclone C2, where it is believed to provide a selective advantage for in vitro growth, remains unknown.     

Formation of double minutes by breakdown of a homogeneously staining region in a refractory anemia with excess blasts

A patient suffering from refractory anemia with excess blasts in transformation had four different bone marrow karyotypes. These were 46,XY; 45,X,-Y; 45,X,-Y, 5q-,19q+; and 43,X,-Y,-9,-17,5q-,+dmin. The most plausible explanation for this is proposed to be formation of a homogeneously staining region on chromosome #19, followed by its breakdown into double minutes.     

Homogeneously staining regions (HSR) in a human malignant melanoma

A case of nodular malignant melanoma (level V of Clark's classification) with homogeneously staining regions (HSR) on the long arm of one chromosome #2 is described. Ultrastructural observation of melanosomic and promelanosomic granules near Golgi's vesicles confirmed the histologic diagnosis. Chromosome analysis was performed on nine metaphases from a bone marrow sample and 76 metaphases from culture of the malignant skin tumor. G-banding revealed the presence of a clone with trisomy #8 and another cell line with the HSR marker. This is the first report of HSR in human melanoma cells. As HSR has been found only in malignant cells, we believe that among the many factors that influence the patients' clinical evolution and poor response to treatment, the genic imbalance is of the utmost importance.     

Amplification of the MLL gene on double minutes, a homogeneously staining region, and ring chromosomes in five patients with acute myeloid leukemia or myelodysplastic syndrome

Most entities of B-cell malignant non-Hodgkin's lymphomas (NHL) are characterized by typical primary chromosomal changes such as the t(14;18) in follicular lymphoma or the t(11;14) in mantle cell lymphoma. In contrast, marginal zone B-cell lymphomas (MZBL), arising at different nodal and extranodal sites, are poorly characterized on the genetic level. We performed cytogenetic investigations in 20 splenic and in 10 nodal MZBL and analyzed 52 MZBL (including 12 MALT-type lymphomas) for deletions of TP53, D13S25, and RB1 loci by fluorescence in situ hybridization. A new nonrandom chromosomal aberration, del(10)(q22q24), was found as a clonal anomaly in 3 out of 20 cases of splenic MZBL. Further recurring abnormalities such as del(7q) or trisomy 3 were found to be characteristic chromosomal changes in a subset of splenic MZBL. TP53 was deleted in 5/25 cases of splenic MZBL. Deletions involving band 13q14 were only rarely encountered, challenging a previous report that stated a dissociated D13S25-RB1 status as characteristic in splenic MZBL. There are fundamental differences between the different subtypes of marginal zone lymphomas as defined with current classification schemes. Splenic MZBL, in contrast to most other entities of B-cell NHL, seems to constitute a heterogeneous disease especially with regard to genetic alterations. del(10)(q22q24) could be of importance at least in a subset of this lymphoma entity.     

Chromosomal instability and double minute chromosomes in a breast cancer patient

Cytogenetic analysis was performed in peripheral blood lymphocytes (PBL) of a woman with ductal breast carcinoma, who as a hospital employee was exposed professionally for 15 years to low doses of ionizing radiation. The most important finding after the chemotherapy in combination with radiotherapy was the presence of double minutes (DM) chromosomes, in combination with other chromosomal abnormalities (on 200 scored metaphases were found 2 chromatid breaks, 10 dicentrics, 11 acentric fragments, 2 gaps, and 3 double min chromosomes). In a repeated analysis (after 6 months), DM chromosomes were still present. To rule out the possibility that the patient was overexposed to ionizing radiation at work, her blood test was compared with a group of coworkers as well as with a group of professionally unexposed people. The data rejected this possibility, but the retroactive analysis showed that the patient even at the time of employment had a moderately increased number of chromosomal aberrations (3.5%) consisting of 3 isochromatids and 4 gaps, suggesting that her initial genomic instability enhanced the later development. The finding of a continuous presence of rare DM chromosomes in her PBL (4 and 10 months after radiochemotherapy) was considered as an indicator of additional risk, which might have some prognostic significance.     

Relational mapping of MYCN and DDX1 in band 2p24 and analysis of amplicon arrays in double minute chromosomes and homogeneously staining regions by use of free chromatin FISH

MYCN amplification has been observed in diverse neuronal tumors including neuroblastoma, retinoblastoma, and small cell carcinoma of the lung, and has been correlated with a poor prognosis in advanced-stage neuroblastomas. Recent studies have shown a co-amplification of DDX1, a DEAD box gene, and MYCN in retinoblastoma and neuroblastoma. DDX1 has been mapped to within a megabase of the MYCN gene in band 2p24. In the present study, the relational map of DDX1 and MYCN by fluorescence in situ hybridization (FISH) mapping to metaphase cells and extended free chromatin fibers indicated that DDX1 is telomeric to MYCN. Dual-color FISH analysis of amplicons within arrays of extended chromatin fibers was performed to examine the physical relationship of MYCN and DDX1 within double minute chromosomes (dmins) and homogeneously staining regions (hsrs). No regular reiterated amplicon repeat unit was present in the hsrs, but detailed analysis of the configurations of DDX1 and MYCN within each array indicated that multiple rearrangements generated a complex hsr amplicon structure. Similarly, analysis of a cell line bearing dmins showed that a composite amplicon structure involving deletions and/or duplications of MYCN and DDX1 is a feature of dmin formation. These data are consistent with a molecular mechanism involving many rearrangements during the evolution of gene amplification, resulting in complex amplicon structures with distinct changes in relative gene copy number and considerable variation in intragenic distances between coamplified genes. Genes Chromosomes Cancer 20:243–252, 1997. © 1997 Wiley-Liss, Inc.     

Patterns of BrdU incorporation in homogeneously staining regions and double minutes

The replication chronology of two structural chromosome abnormalities linked to the amplification phenomenon of DNA sequences was investigated. Three cell lines containing homogeneously staining region (HSR) chromosomes (IMR-32, MK42, and COLO-320) and one line with double minutes (DM) (SW-613) were examined. Using a bromodeoxyuridine-Hoechst 33258-Giemsa method, the HSR in the three cell lines were shown to be composed of subunits that replicated their DNA throughout all portions of the S-phase of the cell cycle. The double minute chromosomes were observed to replicate randomly throughout the entire S-phase, with no pattern evident. These results are consistent with the suggestion that DNA from HSR and DM are structurally and functionally related. Moreover, this observation that these amplified regions replicate their DNA throughout the entire S-phase favors the idea that, during amplification processes, both early and late replicating sequences are included. The apparent discordance between staining characteristics and replication behavior exhibited by some HSR and DM are also discussed.     

MYC amplification in two further cases of acute myeloid leukemia with trisomy 4 and double minute chromosomes

We report two cases of trisomy 4 with double minute chromosomes (dmin): one in a woman with acute myeloid leukemia (AML), French-American-British subtype M2, the other in a man with chronic myelomonocytic leukemia. In the former case, many cells without trisomy 4 but with dmin were present, a finding not observed in previously reported cases. In both cases, fluorescence in situ hybridization studies demonstrated the double minutes to be MYC amplicons. Ten cases of AML with trisomy 4 and dmin have now been described; in the five cases investigated, the dmin have been shown to be amplified MYC gene sequences.     

In situ hybridisation analysis of a homogeneously staining region at 11q23–24 in an acute myeloid leukaemia (M5) using yeast artificial chromosomes

An example of a homogeneously staining region (hsr), occurring in an acute myeloid leukaemia (M5) on chromosome 11 in the region of bands q23–q24, has been analysed. In situ hybridisation using yeast artificial chromosome (YAC) DNA demonstrated that the amplification did not include the CD3 gene cluster and did not affect the human trithorax gene known to be disrupted by translocations at 11q23. In contrast, the amplification was shown to include the sequence D11S543 which has been previously mapped to chromosome band 11q24. High resolution analysis using confocal microscopy allowed the individual amplicons to be visualised, and it was shown that the hsr consisted of an 8-fold amplification of the region surrounding the probe D11S543. From previous estimates of human chromosome size it was possible to calculate that the hsr was composed of amplicons approximately 10 megabases in length. It was concluded that the region amplified did not extend as far as the translocation breakpoints occurring at 11q23 in acute leukaemias. © 1993 Wiley-Liss, Inc.     

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.     

The proposed origin of double minutes from Homogeneously Staining Region (HSR)-marker chromosomes in human neuroblastoma hybrid cell lines

Double minute chromosomes have been reported in a number of human and animal tumors although their origin and function in the cell remain unclear. The fusion of two cell lines without double minutes—a human neuroblastoma line containing another chromosomal abnormality, the homogeneously staining region (HSR), and a mouse fibroblast line—resulted in hybrid clones containing varying numbers of double minutes. The appearance of double minutes occurred coincident with the disappearance of the HSR. We propose that double minutes can originate from the breakdown of an HSR. The possible functional significance of these two chromosome abnormalities is discussed.     

c-myc gene amplification and N-ras transforming gene in two cases of acute myelocytic leukemia with double minute chromosomes

We report two leukemia patients with double minutes (DMs) chromosomes. Both patients were diagnosed as having acute myelocytic leukemia (AML) FAB M2. Cytogenetic analysis showed normal chromosome karyotype with 1-53 DMs chromosomes in the first patient, and complex chromosome aberrations including deletion of chromosome 8 at 8q24 region and 1-84 DMs chromosomes in the second patient who had a history of extensive radiotherapy for laryngeal cancer 8 years prior to the development of leukemia. Analysis of DNA from the two patients revealed that oncogene of c-myc was amplified about 5 to 10 folds in the leukemic cells. The other fourteen oncogene of c-myc was c-myb, c-abl and N-myc, showed no increases of gene content. Furthermore, a transforming gene, N-ras was detected in the first patient by in vivo selection assay method. This is the second report on AML patients with c-myc gene amplification and DMs chromosomes.     

Identification of amplified DNA sequences on double minute chromosomes in a leukemic cell line KY821 by means of spectral karyotyping and comparative genomic hybridization

Double minute chromosomes (dmin) are cytogenetic hallmarks of amplified genes. Using spectral karyotyping (SKY) and comparative genomic hybridization (CGH), we identified the origin of amplified DNA in a leukemic cell line, KY821, that harbors numerous dmin. The SKY revealed that the DNA sequences of dmin are derived from materials of chromosome 8, and CGH showed a high degree of overrepresentation only at 8q22–24, indicating that in KY821 only chromosomal material of 8q22–24, containing MYC, is amplified in dmin. An approach combining SKY with CGH should facilitate efforts to identify novel chromosomal regions of gene amplification and contribute information about genetic lesions that underly neoplastic tumors. Received: March 11, 1998 / Accepted April 9, 1998     

Characterization of homogeneously staining regions in a small cell lung cancer cell line,using in situ hybridization with an MYCN probe

The cell line CIPL38 was derived from the pleural effusion of a patient with small cell lung cancer. The karyotype was hyperdiploid and complex with a variable number of marker chromosomes. Two of the markers had large homogeneously staining regions (hsr), which were shown to consist of amplified MYCN by in situ hybridization. One hsr bearing a marker chromosome could not be identified with G-banding, but the other was situated on a der(14). This was elucidated further with FISH analysis, which enabled the identification of sequences of chromosome I involved in a complex rearrangement with chromosome 14 and the hsr. Genes Chromosom Cancer 10:213–216 (1994). © 1994 Wiley-Liss, Inc.