双微体的研究进展

基因扩增在细胞遗传学上常表现为双微体 (DMs)和均质染色区 (HSR)。DMs可见于多种类型的肿瘤细胞 ,是肿瘤病人主要的细胞遗传学标记。在多种不同类型的肿瘤中 ,癌基因的扩增和过表达与演进有很强的相关性 ,在特定的恶性肿瘤中是不良预后的分子标记。此外 ,基因扩增与肿瘤细胞的抗药性有关。本文综述了目前在 DMs的细胞及分子遗传学、DMs介导的抗药性、DMs与肿瘤的相关性等领域中的研究进展。     

双微体的研究进展

基因扩增在细胞遗传不寂常表现为双微体（DMs) 和均质染色区（HSR),DMs可见于多种类型的肿瘤细胞,是肿瘤病人主要的细胞遗传学标记,在多种不同类型的肿瘤中,癌基因的扩增和过表达与演进有很强的相关性,在特定的恶性肿瘤中是不良预后的分子标记,此外,基因扩增与肿瘤细胞的抗药性有关,本综述了目前在DMs的细胞及分子遗传学,DMs介导的抗药性,DMs与肿瘤的相关性等领域中的研究进展。     

基因扩增机制的研究进展

基因扩增在很多恶性肿瘤发生发展中起重要作用。它是肿瘤细胞获得无限制生长及耐药的主要机制。基因扩增在细胞遗传学上主要表现为双微体(doubleminutes,DMs)和均质染色区(homoge-neouslystainingregions,HSR),了解基因扩增的机制可以为肿瘤治疗提供新的有效的途径。本文综述了基因扩增始动的机制,影响扩增的因素及基因扩增在细胞遗传学上的变化。     

基因扩增机制的研究进展

基因扩增在很多恶性肿瘤发生发展中起重要作用。它是肿瘤细胞获得无限制生长及耐药的主要机制。基因扩增在细胞遗传学上主要表现为双微体（double minutes，DMs）和均质染色区（homogeneously staining regions，HSR），了解基因扩增的机制可以为肿瘤治疗提供新的有效的途径。本文综述了基因扩增始动的机制，影响扩增的因素及基因扩增在细胞遗传学上的变化。     

DNA双链断裂修复与基因扩增关系的研究进展

基因扩增在肿瘤的发生发展过程中,起着不可忽视的作用.基因扩增的主要形式包括双微体(double minutes chromosomes,DMs)和均质染色区(homogeneous staining regions,HSRs).DNA双链断裂(DNA double strands break,DSBs)是最为严重的DNA损伤之一,近年来有关其与基因扩增关系的研究越来越多.该文综述了基因扩增的始动,并重点关注DNA双链断裂修复机制与基因扩增关系的研究进展.     

基因扩增与人类恶性肿瘤

基因扩增(gene amplification)是细胞内染色体上特定基因拷贝数的大量增加,常见于生物发育、肿瘤发生及抗药性的形成过程。肿瘤发生的分子遗传学标志即为基因组不稳定,其中基因扩增作为基因组不稳定的主要形式在很多人类恶性肿瘤的发生发展中起着重要作用。肿瘤细胞中基因扩增是癌基因激活的主要机制,可调控肿瘤细胞逃避生长限制产生耐药。针对基因扩增的深入研究对肿瘤遗传学的发展及肿瘤的诊断、治疗有着重要的作用。本文就人类恶性肿瘤中基因扩增的组成成分、表现形式、扩增机制及研究手段进展做一简要综述。     

人卵巢癌细胞及小鼠MTX抗性细胞中DMs大小的测定

目的 测定肿瘤细胞及耐药性细胞中双微体（Double minutes，DMs）的大小。方法 应用PFGE （Pulsed field gradient gel electrophoresis）与Southern杂交技术，对人卵巢癌细胞UACC-1598及氨甲喋呤（Methotrexate，MTX）抗性的小鼠胚胎成纤维细胞中DMs的大小进行检测。结果 发现UACC-1598细胞中存在2.8 Mb、2.1 Mb及1.4 Mb的DMs群体，提示多拷贝的扩增基因及其邻近的染色体区域经染色体断裂、易位及重排形成较大的DMs。同时，用浓度逐渐增高的MTX对小鼠胚胎成纤维细胞进行体外诱导，分离出富含DMs的不同MTX抗性程度的细胞群体。在MTX抗性细胞中检测到2.5 Mb及1.4 Mb的DMs群体， MTX100细胞中以1.4 Mb的DMs群体为主，而MTX500细胞中以2.5 Mb的DMs群体为主。 结论 MTX细胞中产生的扩增子结构在演化过程中趋向于寡聚化或多聚化形成较大片段的DMs。     

染色体显微切割结合比较基因组杂交在检测肿瘤细胞基因扩增中的应用

基因扩增是肿瘤基因组不稳定的一个重要方面。随着染色体显微切割和比较基因组杂交等分子细胞遗传学技术的发展，大大推动了肿瘤细胞中基因扩增的研究。这两种技术的有机结合，能有效地检测出肿瘤细胞中基因扩增的染色体区域，并且为寻找肿瘤发展进程中新的扩增基因开辟了一条重要的途径。     

染色体显微切割结合比较基因组杂交在检测肿瘤细胞基因扩？…

基因扩增是肿瘤基因组不稳定的一个重要方面。随着染色体显微切割和比较基因组杂交等分子细胞遗传学技术的发展。大大推动了肿瘤基因扩增的研究。这两个技术的有机结合，能有效地检测出肿瘤细胞中基因扩增的染色体区域，并且来寻找肿瘤发展进程中新的扩增基因开辟了一条重要的途径     

双微体的研究进展

双微体(double minute chromosomes,DMs)是基因扩增的主要细胞遗传学标志,是染色体外遗传单位的一种主要存在形式.大量研究表明,双微体与基因组不稳定、恶性肿瘤、细胞耐药及衰老密切相关.针对双微体的深入研究对于肿瘤遗传学的发展及其临床应用有着重要的意义.人类基因组计划的完成与功能基因组计划的深入,大大地加快了在序列水平对双微体的了解.对双微体的全面认识,将有助于理解基因扩增机制及基因组可塑性,并为后两者的研究提供全新的切入点.     

Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization.

An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This study was conducted to evaluate the potential of fluorescence in situ hybridization (FISH) to identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding to the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO III and SNU-16), a neuroblastoma (NUB-7), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO III, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachromosomal location: in KATO III and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU-16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosomal double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified DNA sequences in nondividing cells, enabling us to distinguish, at interphase, whether the amplification gave rise to intrachromosomal amplified regions (IARs) or to extrachromosomal DMs. The FISH technique also allowed us to determine at metaphase as well as at interphase the extent of amplification and the size of the IARs.     

Cytogenetics and DNA amplification in colorectal cancers

In vitro cultivated cells of 28 colorectal cancers were analyzed for chromosomal abnormalities that might signal amplification of DNA, either as double minutes (DMs) or homogeneously staining chromosomal regions (HSRs). Cells derived from 18 tumors showed DMs in 10 to 100% of all metaphases examined. Surveys that employed a panel of available oncogene probes failed to detect amplification of a known cellular oncogene with the exception of three cases where the ERBB2 gene was amplified. In one of these three cases neither DMs nor HSRs were detectable. Our studies show that from 28 lines established in culture, 19 (68%) show amplification of DNA, and indicate that DNA amplification is a frequent genetic alteration in colorectal cancers in addition to other genetic changes. Amplification is correlated with high Dukes stage, but not with histological grade. The identity of the amplified DNA remains to be established for most cases.     

Double‐strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus,micronucleation, and morphological transformation

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low‐dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low‐dose hydroxyurea also induces random double‐strand breakage throughout the nucleus. In the present study, we found that double‐strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double‐strand breakage in cancer cells. Because double‐strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.     

Double minutes in prematurely condensed chromatin of human tumor cells

Comparative studies on the occurrence of double minutes (DMs) were performed on metaphases and prematurely condensed interphases (G1, G2) of cells from 28 cancerous effusions of 24 carcinoma patients. In 21 of these effusions, agreement between metaphase and prematurely condensed chromatin (PCC) data was obtained concerning occurrence or nonoccurrence of DMs (10 DM positive, 11 DM negative). In two cases, DMs were observed in metaphases only, whereas in five cases, they were found in interphases only. In normal cells, no DMs could be found, neither in metaphase nor in PCC. No causal correlation of the occurrence of DMs and cytostatic therapy was found. The data suggest that cytogenetic screening of prematurely condensed interphase cells from human tumors or cancerous effusions provides a valuable method for estimating the incidence of gene amplification in malignant cells, particularly in those with poor mitotic yield.     

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.     

Amplification of the MYCN oncogene and deletion of putative tumour suppressor gene in human neuroblastomas

Human neuroblastoma cells often carry non-random chromosomal abnormalities signalling genetic alterations. Quite frequent are 'double minutes' (DMs) and homogeneously staining regions (HSRs), both cytogenetic manifestations of amplified DNA, and chromosome 1p-deletions indicating loss of genetic information. With the identification of amplified MYCN and the demonstration of a consensus deletion spanning the chromosome 1p36.1-2 region it appears now likely that both amplification of a cellular oncogene and loss of a tumour-suppressor gene play an important role in neuroblastoma. Amplification of MYCN is an indicator for poor prognosis, even when classical morphological criteria would suggest a better outcome. Consequently, patients with amplification are subjected to more intensive therapeutic regimens. Amplification of MYCN is a paradigm for the clinical use of an oncogene alteration.     

Oncogene mobility in a human leukemia line HL-60

HL-60, a cell line derived from a human promyelocytic leukemia, shows amplification of the oncogene c-myc. Chromosome aberrations reported in HL-60 include double minutes (DMs) and an abnormally banded region (ABR) on chromosome #8. A relationship between these chromosomal aberrations and amplification of c-myc DNA has been suggested. We report the localization by cytologic hybridization of amplified c-myc DNA to a marker chromosome, M3q+, in an early passage of HL-60. The localization of c-myc to an ABR on an 8q+ chromosome was confirmed in later passage clones. The most probable derivation of the M3q+ chromosome is t(5p;17q) with additional material associated with c-myc amplification inserted into 17q. This localization is of interest in light of the association between t(15:17) and promyelocytic leukemia. The results indicate that amplification and chromosome integration can occur at a site other than the native gene locus and at different integration sites in different lineages of the same tumor.     

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.