br Structure Modeling br Homology modeling was
Homology modeling was used to build a structural model of the MATH domain of SPOP with the NANOG peptide (aa 66–70; PDSST). The structure of a SBC peptide from phosphatase Puc (PucSBC1) in a complex with the MATH domain of SPOP (PDB: 3IVV) (Zhuang et al., 2009) was used as a template. The homology modeling was performed in PyMOL (The PyMOL Molecular Graphics System; Version 188.8.131.52).
Statistical analyses were performed with a two-tailed unpaired Student’s t-test. The data are presented as the means ± SEM. The mean was calculated from truly independent experiments. P values < 0.05 were considered statistically significant.
Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization☆,☆☆
Akishi Ooi MD, PhD a,b, , Masafumi Inokuchi MD, PhD c, Shin-ichi Horike PhD d, Hiroko Kawashima MD, PhD e, Satoko Ishikawa MD, PhD c, Hiroko Ikeda MD, PhD b, Ritsuko Nakamura MD, PhD a, Takeru Oyama MD, PhD a, Yoh Dobashi MD, PhD f
aDepartment of Molecular and Cellular Pathology, Kanazawa University, Ishikawa 920-8641, Japan bPathology Section, University Hospital, Kanazawa University, Ishikawa 920-8641, Japan
cDepartment of Breast Oncology, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8641, Japan dAdvanced Science Research Center, Institute for Gene Research, Kanazawa University, Ishikawa 920-8641, Japan eSection of Breast Oncology, University Hospital, Kanazawa University, Ishikawa 920-8641, Japan fDepartment of Pathology, Saitama Medical Center, Jichi Medical University, Saitama, 330-8503, Japan
Summary Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded in-vasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as ‘gain’ or ‘amplified’ was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11–21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplifica-tion in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single 3Methyladenine was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12–21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH. © 2018 Elsevier Inc. All rights reserved.
☆ This laboratory study was approved by the Institutional Review Board at the Kanazawa University Hospital [Approval No. 265] and written informed consent was obtained from all patients. ☆☆ Competing interests: No conflicts of interests are declared. Corresponding author at: Department of Molecular and Cellular Pathology, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. E-mail address: [email protected] (A. Ooi).
Gene amplification is defined as an increase in the copy number of a restricted region of a chromosome arm, and the amplified DNA segment (amplicon) in cancer can attain a size of tens of megabases , covering multiple genes . The levels of amplification can range from a single additional copy to more than a hundred copies. Analysis of amplified DNA in mammalian cell lines and tumors has revealed that high-level gene amplification can occur either intrachromosomally (in the form of homogeneously staining regions, HSRs) or extra-chromosomally (as double-minute chromosomes, DMs). It is also known that fluorescence in situ hybridization (FISH) of interphase nuclei detects the amplified gene of interest in HSRs as one or more large clusters of signals, whereas those in DMs are detected as multiple scattered signals . Although little is known about the mechanism of amplification or, in par-ticular, about the processes that initiate gene amplification , at least two distinct initiating mechanisms have been proposed. Specifically, HSRs appear to be formed by breakage-fusion-bridge (BFB) cycles, whereas DMs are believed to result from “looping out” of extrachromosomal sequences. The BFB cycle consists of a series of recombination events and is physically initiated by a chromosome break. An initial break can occur on the telomeric side; such a break would occur following the fusion of broken chromatid ends, an event that leads to the formation of dicentromeric chromosomes. If the first fu-sions occur between chromatids of a single chromosome, the subsequent amplification would be entopical amplification (occurring or situated at the original chromosomal site, as op-posed to ectopic amplification) ; if the first fusions occur be-tween chromatids of different chromosomes, the subsequent amplification would produce chromosomal translocation and could lead to ectopic amplification . Dicentromeric chromo-somes then are separated by a second break; thus, intrachro-mosomal amplification has been speculated to depend on breakage of a given chromosome at a minimum of two sites. At least in the initial stages , the size and extent of the ampli-con is determined by the distance between the break sites. In contrast, in clinical tumors it has been speculated that ampli-con status changes in the process of cancer progression.