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Gene Amplification: Mechanisms and Involvement in Cancer

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Gene Amplification: Mechanisms and Involvement in Cancer DOI 10.1515/bmc-2013-0026 BioMol Concepts 2013; 4(6): 567–582 Review Atsuka Matsuia, Tatsuya Iharaa, Hiraku Sudaa, Hirofumi Mikamia and Kentaro Semba* Gene amplification: mechanisms and involvement in cancer Abstract: Gene amplification was recognized as a physi- DHFR, dihydrofolate reductase; DM, double minute chro- ological process during the development of Drosophila mosome; DRCR, double rolling-circle replication; DSB, melanogaster. Intriguingly, mammalian cells use this double-stranded break; EGFR, epidermal growth factor mechanism to overexpress particular genes for sur- receptor; EMSY, chromosome 11 open reading frame 30; vival under stress, such as during exposure to cytotoxic ERBB2, v-erb-b2 avian erythroblastic leukemia viral onco- drugs. One well-known example is the amplification of gene homolog 2; ERα, estrogen receptor alpha; FISH, the dihydrofolate reductase gene observed in methotrex- fluorescence in situ hybridization; FoSTeS, replication ate-resistant cells. Four models have been proposed for fork stalling and template switching; GRB7, growth factor the generation of amplifications: extrareplication and receptor-bound protein 7; HR, homologous recombina- recombination, the breakage-fusion-bridge cycle, dou- tion; HSR, homogeneously staining region; MAPK, mito- ble rolling-circle replication, and replication fork stalling gen-activated protein kinase; MAX, MYC associated factor and template switching. Gene amplification is a typical X; MDM2, mouse double minute 2 homolog; MEK, mito- genetic alteration in cancer, and historically many onco- gen-activated protein kinase kinase 1; MITF, microphthal- genes have been identified in the amplified regions. In mia-associated transcription factor; MTX, methotrexate; this regard, novel cancer-associated genes may remain to NGS, next-generation sequencing; PAK1, P21-activated be identified in the amplified regions. Recent comprehen- kinase 1; PI3K, phosphatidylinositol-4,5-bisphosphate sive approaches have further revealed that co-amplified 3-kinase; PLP1, proteolipid protein 1; RARα, retinoic acid genes also contribute to tumorigenesis in concert with receptor α; RAS, rat sarcoma viral oncogene homolog; known oncogenes in the same amplicons. Considering RB, retinoblastoma; StAR, steroidogenic acute regulatory that cancer develops through the alteration of multiple protein; STARD3, START domain containing 3; START, genes, gene amplification is an effective acceleration StAR-related lipid transfer; STAT3, signal transducer and machinery to promote tumorigenesis. Identification of activator of transcription 3; TKI, tyrosine kinase inhibitor; cancer-associated genes could provide novel and effective TOP2A, DNA topoisomerase 2-α. therapeutic targets. aThese authors contributed equally to this article. amplicon; cancer; development; drug resist- Keywords: *Corresponding author: Kentaro Semba, Department of Life ance; microarray. Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku, Tokyo 162-8480, Japan, e-mail: [email protected] List of abbreviations: Akt, v-akt murine thymoma viral Atsuka Matsui, Tatsuya Ihara, Hiraku Suda and Hirofumi Mikami: Department of Life Science and Medical Bioscience, School of oncogene homolog; AR, androgen receptor; BCL2, B-cell Advanced Science and Engineering, Waseda University, 2-2 CLL/lymphoma 2; BFB, breakage-fusion-bridge; bHLHZ, Wakamatsu-cho, Shinjuku, Tokyo 162-8480, Japan basic-helix-loop-helix zipper; BIR, break-induced rep- lication; BRAF, v-Raf murine sarcoma viral oncogene homolog B; BRCA2, breast cancer 2, early onset; BRD4, bromodomain-containing 4; CAD, carbamoyl-synthetase 2 aspartate transcarbamylase and dihydroorotase; CCND1, Introduction cyclin D1; CDK, cyclin-dependent kinase; CGH, compara- tive genomic hybridization; C-MYC, v-myc avian myelo- Gene amplification is defined as an increase in copy cytomatosis viral oncogene homolog; CNV, copy number number of a restricted region of a chromosome arm variation; CRPC, castration-resistant prostate cancer; (1). This amplified region is called an ‘amplicon’. Gene 568 A. Matsui et al.: Gene amplification and cancer amplification plays a crucial role in the normal develop- the use of cultured mammalian cells, a robust method for mental program, including in the amplification of chorion the production of large amounts of recombinant protein genes in Drosophila melanogaster ovaries (2) and ampli- in mammalian cells is critically important. In the general fication of rDNA in amphibian oocytes (3). Gene amplifi- method for mammalian cell-mediated production of cation in somatic cells was first observed by Breuer and recombinant proteins, the plasmid harboring the recom- Pavan (1955) during a morphological study of salivary binant gene and a second plasmid containing a selection gland chromosomes of Rhynchosciara americana larvae, gene are first co-introduced into cells. The DHFR gene is a species of fly (4). The authors found that certain loci on the most frequently used selection gene. By adding MTX salivary gland chromosomes swelled enormously, produc- to the cells, the target gene co-amplifies gradually (13). ing ‘DNA puffs’, and that the quantity of DNA in these loci Although almost all cells die by MTX treatment, the sur- increased compared with other loci, either simultaneously viving cells that overproduce DHFR frequently contain or after the swelling (5). In 1957, Rudkin and Corlette pro- several hundred to a few thousand copies of the recombi- vided the first experimental evidence of gene amplifica- nant gene (14). This method allows the production of cells tion in Rhynchosciara angelae by measuring the quantity that hyper-produce the target gene. Most DHFR-ampli- of absorbing material in a chromosome using microspec- fied cells produce up to 10- to 20-fold more recombinant trophotometry (6). Large quantities of these develop- protein than unamplified cells (12). mental genes encoding rRNAs and structural proteins for In 1976, Biedler and Spengler analyzed the ampli- eggshells and cocoons are necessary during development. fied DNA in the MTX-resistant mammalian cell line and In recent studies, two additional amplicons encoding a observed elongated chromosomal structures, which variety of proteins, including transporters and proteases, they named ‘homogeneously staining regions’ (HSRs) were identified in Drosophila follicle cells by a compara- (15). Trypsin-Giemsa staining was used to visualize the tive genomic hybridization (CGH) array approach (7). In differential banding pattern along the length of meta- addition to its functions in physiological processes, gene phase chromosomes, and staining showed that the amplification has attracted much attention for its involve- long chromosome segments in drug-resistant Chinese ment in cellular adaptation against cytotoxic drugs and, hamster cells did not show any discrete ‘bands’ as did interestingly, in tumorigenesis (1, 8). normal Chinese hamster cells (15). HSRs were thus easily The first demonstration of gene amplification in cul- recognizable. tured mammalian cells was the amplification of the dihy- Double minute chromosomes (DMs), which are small drofolate reductase (DHFR) gene in the AT3000 line of and often paired extrachromosomal elements, were also methotrexate (MTX)-resistant murine sarcoma 180 cells observed in MTX-resistant Chinese hamster cells (16). DMs in 1978 (9). DHFR catalyzes the reduction of dihydrofolate vary in size among different cell lines and even within to tetrahydrofolate, which is used in glycine, purine, and the same cells. They replicate in the cell cycle without thymidylate synthesis. Methotrexate, a 4-amino analog of centromeres (17). The DHFR gene tends to be amplified folic acid, inhibits DHFR activity and thus causes arrest either on DM or in a HSR configuration, depending on the of DNA replication and cytotoxicity (10). After exposure cell line, although the reason for such tendency remains of cells to methotrexate, the surviving cells exhibited an unclear (17). increased copy number of the DHFR gene. This phenome- In this review, we first discuss the mechanisms of non was observed in several cell types, including Chinese gene amplification and then focus on gene amplification hamster ovary cells as well as the AT3000 cell line. The in cancer. We will introduce typical oncogenes localized in occurrence of gene amplification in drug-resistant cells amplicons and discuss their therapeutic potential. was observed not only with DHFR but also with the car- bamoyl-synthetase 2 aspartate transcarbamylase and dihydroorotase (CAD) gene. Amplification of the CAD gene was observed in Syrian hamster cells resistant to N-phos- Mechanisms of generation phonacetyl-l-aspartate, an inhibitor of aspartate transcar- of amplicons bamylase (11). The amplification mechanism of the DHFR gene in As described above, gene amplification is detected in MTX-resistant cells has been frequently employed for two forms: HSRs and DMs. DMs are circular extrachro- recombinant protein production in mammalian cells (12, mosomal elements that replicate autonomously and lack 13). Because the production of protein pharmaceuticals, a centromere and telomeres (18). Although gene amplifi- including cytokines and humanized antibodies, requires cation is often observed in cancer and other degenerative A. Matsui et al.: Gene amplification and cancer 569 disorders, the mechanisms of amplicon generation Gene A Gene B remain largely unknown. Four hypotheses have been pro-
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