The goal of our research is to define the initiation mechanism of gene amplification in cancer cells. Defining the initiation mechanism should provide important knowledge, as amplification very often drives tumor progression and therapy resistance. An important chromosomal structure at a very early step of gene amplification is an Inverted duplication of large chromosomal regions. This indicates a following process (breakage-fusion-bridge cycle, BFB cycle) as a potential initiation mechanism;(1) a chromosome break leads to the fusion of the broken ends after replication (sister chromatid fusion), resulting in a de novo palindromic (inverted duplication) chromosome with two centromeres;(2) subsequent mitosis causes a tension between the two centromeres (bridge), resulting in a partially duplicated chromosome with a broken end (break);and (3) the broken end enters into another cycle. Therefore, the initial step is a potential rate-limiting step, because, once a palindromic dicentric chromosome is generated, it would inevitably enters into a bad spiral (BFB cycle) and leads to the accumulation of specific genomic regions (palindromic gene amplification). In this proposal, we will define cis- (genomic) and trans- (genetic) acting factors that are important in the initiation of palindromic gene amplification in human tumors. We have previously shown in mammalian cell models that a DNA inverted repeat (DNA-IR) pre-existing in the genome, with an adjacent DSB, greatly promotes palindromic gene amplification. Thus, an initial step of gene amplification is DSB-initiated, illegitimate recombination between the repeats of a DNA-IR. This leads to our DNA-level model;fold-back (Intra-strand) annealing within a DNA-IR would generate a chromosome with a hairpin-capped end, and subsequent DNA replication would complete palindromic duplication. Based on our model, we will test potential determinants for palindromic gene amplification: pre-existing DNA-IRs in the human genome are cis- acting (genomic) determinant (Aim 1), and genes that process a hairpin-capped end and physiological conditions that induce DSBs are important trans-acting determinants (Aim 2). To accomplish our aims, we have established unique experimental systems that can overcome the difficulties in studying palindromic DNA. The genomic approach employs a novel technique for the enrichment of palindromic DNA and identifies important DNA-IRs for palindromic gene amplification in primary human tumors. Our cell culture system is designed to measure the occurrence of DSB-initiated illegitimate recombination at a DNA-IR in cells with a variety of genetic backgrounds. These experiments should collectively identify specific DNA-IRs as an At-Risk Motif for developing gene amplification. Given the impact of structural variations in the normal human genome, polymorphic DNA-IRs could be an important predictor of an individual's susceptibility to gene amplification.

Public Health Relevance

Project Narrative The major goal of our research is to identify the mechanisms generating gene amplification in human cancers. Gene amplification refers to the accumulation of extra copies of genes in cancer cells that often drives abnormal cell growth and aggressive behavior of tumors. Therefore, our identification of underlying mechanisms will provide better understanding of how cancers progress, and eventually will lead to the development of interventions for the early detection of gene amplification in patients.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Mietz, Judy
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Cleveland Clinic Lerner
Other Basic Sciences
Schools of Medicine
United States
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Watanabe, Takaaki; Marotta, Michael; Suzuki, Ryusuke et al. (2017) Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart. Cell Rep 21:2223-2235
Kondratova, Anna; Watanabe, Takaaki; Marotta, Michael et al. (2015) Replication fork integrity and intra-S phase checkpoint suppress gene amplification. Nucleic Acids Res 43:2678-90
Yang, Hui; Volfovsky, Natalia; Rattray, Alison et al. (2014) GAP-Seq: a method for identification of DNA palindromes. BMC Genomics 15:394
Marotta, Michael; Chen, Xiongfong; Watanabe, Takaaki et al. (2013) Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles. Nucleic Acids Res 41:9732-40
Marotta, Michael; Chen, Xiongfong; Inoshita, Ayako et al. (2012) A common copy-number breakpoint of ERBB2 amplification in breast cancer colocalizes with a complex block of segmental duplications. Breast Cancer Res 14:R150
Guenthoer, Jamie; Diede, Scott J; Tanaka, Hisashi et al. (2012) Assessment of palindromes as platforms for DNA amplification in breast cancer. Genome Res 22:232-45
Marotta, Michael; Piontkivska, Helen; Tanaka, Hisashi (2012) Molecular trajectories leading to the alternative fates of duplicate genes. PLoS One 7:e38958