Gemcitabine Eliminates Double Minute Chromosomes from Human Ovarian Cancer Cells

Abstract

Double minute chromosomes are cytogenetic manifestations of gene amplification frequently seen in cancer cells. Genes amplified on double minute chromosomes include oncogenes and multi-drug resistant genes. These genes encode proteins which contribute to cancer formation, cancer progression, and development of resistance to drugs used in cancer treatment. Elimination of double minute chromosomes, and therefore genes amplified on them, is an effective way to decrease the malignancy of cancer cells. We investigated the effectiveness of a cancer drug, gemcitabine, on the loss of double minute chromosomes from the ovarian cancer cell line UACC-1598. Gemcitabine is able to decrease the number of double minute chromosomes in cells at a 7500X lower concentration than the commonly used cancer drug hydroxyurea. Amplified genes present on the double minute chromosomes are decreased at the DNA level upon gemcitabine treatment. Gemcitabine, even at a low nanomolar concentration, is able to cause DNA damage. The selective incorporation of double minutes chromatin and γ-H2AX signals into micronuclei provides a strong link between DNA damage and the loss of double minute chromosomes from gemcitabine treated cells. Cells treated with gemcitabine also showed decreased cell growth, colony formation, and invasion. Together, our results suggest that gemcitabine is effective in decreasing double minute chromosomes and this affects the biology of ovarian cancer cells.

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate‐resistant colon cancer cells

“…The accumulation of DSBs induced by external pressures, such as hydroxyurea or low‐dose radiation, often promotes the formation of MN/NBUDs that entrap DNA fragments . Notably, a dysfunction in DNA repair pathways usually increases DNA breaks, thereby promoting the capture of DMs by MN/NBUDs formation .…”

“…Micronuclei (MNs) and nuclear buds (NBUDs) are the main pathways for export of nuclear material, including amplified DNA, DNA repair complexes and excess chromosomes . In previous studies, the application of DNA synthesis or repair inhibitors, such as hydroxyurea, gemcitabine and NU7026, has been found to eliminate gene amplification by forming MN/NBUDs . The formation of MN/NBUDs tightly depends on the cell cycle, and the HR core protein, BRCA1, is a regulator of cell cycle checkpoints .…”

“…27,28 In previous studies, the application of DNA synthesis or repair inhibitors, such as hydroxyurea, gemcitabine and NU7026, has been found to eliminate gene amplification by forming MN/NBUDs. 13,29,30 The formation of MN/NBUDs tightly depends on the cell cycle, 27 and the HR core protein, BRCA1, is a regulator of cell cycle checkpoints. 31 However, the influence of HR on the formation of MNs/NBUDs via cell cycle progression has not yet been elucidated.…”

Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

“…Based on the result that inhibiting constitutive phosphorylation of ERK1/2 is able to decrease the number of DMs, we next addressed whether inhibiting ERK1/2 phosphorylation would affect the amplification of DM‐carried genes. The oncogenes MCL1 , MYCN and EIF5A2 are highly amplified via DMs in UACC‐1598 cells, and FAM84B and MYC are highly amplified via DMs in Colo320DM . After 2 weeks of U0126 and PD98059 treatment, qPCR showed that amplification levels of these genes were significantly reduced in the respective cells compared to controls (Figure A–E) and that their expression levels were also decreased (Figure F–H).…”

“…We selected the BAC clones RP11‐54A4 (GRCh37/hg19, Chr1:150445215–150628209) and RP11‐115 J24 (Chr3:170607523–170769158), specific for the UACC‐1598 DM‐carried genes MCL1 (Chr1:150547032–150552066) and EIF5A2 (Chr3:170606204–170626426), labelled green or with Cy3, respectively; and BACs RP11‐89 K10 (Chr8:127567520–127730253) and RP11‐440 N18 (Chr8:128596756–128777986), specific for the Colo320DM DM‐carried genes FAM84B (Chr8:127564683–127570711) and MYC (Chr8:128748315–128753680), as probes to perform FISH analysis of inhibitor‐treated and control interphase and metaphase cells. To quantify the coverage area and distribution of fluorescence, cells were divided into five groups: dispersed 0–30% coverage; clustered 0–30% coverage; dispersed 30–60% coverage; clustered 30–60% coverage; and 60–100% coverage, indicating a dispersed or clustered distribution of the signal in the cells (Figure I) ; > 100 cells for each treatment and control group were counted and the data are illustrated in Figure I. In control UACC‐1598 cells, more cells hybridized with dispersed signals in the 0–30% and 30–60% groups (21.0% and 18.1%) than with clustered signals (12.3% and 13.8%), indicating that the amplified genes are located on DMs in a dispersed distribution.…”