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Intracellular amplification results in a selective increase in gene copy number with the consequence of elevated gene expression. Gene amplification has been seen in three different settings
- Scheduled amplification as part of a developmental gene expression program, e.g. chorion genes in ovaries of the fruitfly Drosophila melanogaster or actin genes during myogenesis in the chicken
- Unscheduled amplification during acquisition of cellular drug resistance. For example; amplification of the gene encoding dihydrofolate reductase (DHFR) can result in up to 1,000 gene copies per cell with the consequence of cellular resistance against the chemotherapy drug methotrexate.
- Unscheduled amplification of cellular genes involved in growth control (oncogenes) during tumor progression. Amplification of oncogenes can result in up to several hundred gene copies and enhanced gene expression. Usually large DNA stretches (from 100 Kb up to several Mb) are amplified, and therefore syntenic genes in addition to the particular oncogene can be co-amplified due to their close linkage to the oncogene. Alternatively, different non-syntenic oncogenes can amplify independently in the same cell. The prototypic human cancer with oncogene amplification is neuroblastoma. Here, the amplified gene, MYCN, is a Biomarker for patient management.
Amplification. Figure 1 Cytogenetics of MYCN amplification in neuroblastoma cells. Chromosomal fluorescence in situ hybridization (FISH). High-level MYCN amplification appears in human neuroblastoma cells as two alternative cytogenetic manifestations: (a). Double minutes (DMs) (left), this tumor cell has in addition to amplified MYCN (red) amplification of another oncogene MDM2 (green). The two oncogenes are non-syntenic (2p24, and 12q13–14, respectively), and the amplification is the result of two independent genetic events. (b). Homogeneously staining region (HSR) (right), multiple copies are amplified in an HSR on chromosome 12 (with strong signal), while single copy gene is retained on the two parental chromosomes (arrows). The retention of MYCN at 2p24 indicates that not the original MYCN gene but rather a copy, presumably the result of extra-replication, has been amplified. Note also the strong signal in interphase nuclei which allows detection of amplified MYCN in tumor biopsies when chromosomes cannot be prepared.
Amplification can follow different pathways, the “onion skin model” and “breakage fusion-bridge” (BFB) cycles (Fig. 2), both fit experimental observations. Little is known about genomic or environmental elements involved in amplification. Unscheduled amplification presumably is a sporadic event that can become stabilized under selective pressures, i.e. cytostatic drugs or if cells acquire a growth advantage within a certain tissue architecture.
Amplification. Figure 2 Breakage-fusion-bridge (BFB) cycles in early stages of amplification. (a). BFB cycles start from common fragile sites, where a DNA break can occur in both sister chromatids. DNA repair systems will be recruited to the break and may join the free DNA ends of the two sister chromatids to form a dicentric chromosome, one that has two centromers. At anaphase, where sister chromatids are moved to the daughter cells, the dicentric chromosome at some point will break. Of the two daughter cells, one will carry a deletion, the other an inverted duplication of DNA, which is equivalent to a low-level amplification. By subsequent BFB cycles, the level of amplification can increase. (b). Low level amplification as the result of BFB cycles. FISH image, where each color shows the position and copy-number of a particular DNA sequence.
Resistance against cytostatic drugs poses a big problem in cancer therapy. Amplified oncogenes contribute to tumor progression, many different oncogenes have been found amplified (e.g. RAS, MYC, MYCN, MYCL, HER-2 (HER-2/neu), ABL in some tumor types the oncogene status provides information about patient prognosis: Amplified MYCN indicates poor prognosis for stage 1–3 neuroblastoma; and amplified HER-2 indicates unfavorable outcome in a subgroup of breast cancer.
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