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Gemcitabine eliminates double minute chromosomes from human ovarian cancer cells.

Yu L, Zhao Y, Quan C, Ji W, Zhu J, Huang Y, Guan R, Sun D, Jin Y, Meng X, Zhang C, Yu Y, Bai J, Sun W, Fu S - PLoS ONE (2013)

Bottom Line: Amplified genes present on the double minute chromosomes are decreased at the DNA level upon gemcitabine treatment.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.

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.

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Related in: MedlinePlus

The signal of EIF5A2, MYCN, and MCL1 genes is decreased in HU and GEM treated UACC-1598.A. Genes EIF5A2, MYCN, and MCL1 are present on DMs in UACC-1598 cells. Metaphase spread of UACC-1598 cells detected with DNA probe for EIF5A2, MYCN, and MCL1. For the overlapped pictures, EIF5A2 and MCL1 signals are shown in red, and MYCN signal is shown in green. Overlapped EIF5A2/MYCN and MCL1/MYCN are shown in yellow. B. Representative pictures of EIF5A2/MYCN and MCL1/MYCN FISH signals in interphase cells of UACC-1598 and how individual cells are separated into different groups. MYCN signal is shown in green, EIF5A2 and MCL1 shown in red, and the overlap is shown in yellow. The percentage of cells in Groups 1, 2, and 3 is based on EIF5A2/MYCN and MCL1/MYCN FISH signals. The statistical analysis showed the differences of cells in Group 1 and Group 2/3 compared to the control cells. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.
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pone-0071988-g002: The signal of EIF5A2, MYCN, and MCL1 genes is decreased in HU and GEM treated UACC-1598.A. Genes EIF5A2, MYCN, and MCL1 are present on DMs in UACC-1598 cells. Metaphase spread of UACC-1598 cells detected with DNA probe for EIF5A2, MYCN, and MCL1. For the overlapped pictures, EIF5A2 and MCL1 signals are shown in red, and MYCN signal is shown in green. Overlapped EIF5A2/MYCN and MCL1/MYCN are shown in yellow. B. Representative pictures of EIF5A2/MYCN and MCL1/MYCN FISH signals in interphase cells of UACC-1598 and how individual cells are separated into different groups. MYCN signal is shown in green, EIF5A2 and MCL1 shown in red, and the overlap is shown in yellow. The percentage of cells in Groups 1, 2, and 3 is based on EIF5A2/MYCN and MCL1/MYCN FISH signals. The statistical analysis showed the differences of cells in Group 1 and Group 2/3 compared to the control cells. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.

Mentions: Once we found that GEM can decrease the number of DMs in both UACC-1598 and UACC-1598-4, the next question we asked is whether genes amplified on the DMs are also decreased. It is known that oncogenes EIF5A2 and MYCN are two genes amplified in UACC-1598 [15], [16], and MCL1 was also found to be amplified in this cell line (unpublished data). We first verified that these three genes we analyzed are present on DMs with FISH experiments (Figure 2A). We then separated cells grown in HU or GEM into 3 groups according to the level of EIF5A2, MYCN, and MCL1 signals (Figure 2B). Group 1 includes cells with <30% signal, which corresponds to cells with less signals from EIF5A2, MYCN, and MCL1, and is an indicator of cells with less number of DMs; group 2 includes cells with 30–60% signal; and group 3 includes cells with >60% signal, which is an indicator of cells with more DMs. The percentage of cells in each group was plotted. The percentage of cells in Group 3, which contains cells with greater than 60% signals, decreased in the presence of either HU (from 11% to 6%) or GEM (from 16% to 7%). In addition, the percentage of cells in Group 2 decreased from 33% to 24% for HU treated cells and 37% to 25% in GEM treated cells. In contrast to the decrease in the percentages of cells in Group 2 and 3, the percentage of cells in Group 1 increased 14 to 21% in cells treated with HU (from 56% to 70%) or GEM (from 47% to 68%). The increase in the percentage of cells in group 1 in the HU or GEM treated cells indicates cells have started losing their DMs. In addition, we consolidated groups 2 and 3 into one large group, and statistical analysis showed that the differences observed between DMSO and HU or control and GEM treated cells is statistically significant (Figure 2B). These results indicate that the signal of the amplified genes MYCN, EIF5A2, and MCL1 on DMs in UACC-1598 cells decreased in the presence of both HU and GEM. A similar result was seen for GEM when analyzing MYCN and EIF5A2 in UACC-1598-4 cells (Figure S1B).


Gemcitabine eliminates double minute chromosomes from human ovarian cancer cells.

Yu L, Zhao Y, Quan C, Ji W, Zhu J, Huang Y, Guan R, Sun D, Jin Y, Meng X, Zhang C, Yu Y, Bai J, Sun W, Fu S - PLoS ONE (2013)

The signal of EIF5A2, MYCN, and MCL1 genes is decreased in HU and GEM treated UACC-1598.A. Genes EIF5A2, MYCN, and MCL1 are present on DMs in UACC-1598 cells. Metaphase spread of UACC-1598 cells detected with DNA probe for EIF5A2, MYCN, and MCL1. For the overlapped pictures, EIF5A2 and MCL1 signals are shown in red, and MYCN signal is shown in green. Overlapped EIF5A2/MYCN and MCL1/MYCN are shown in yellow. B. Representative pictures of EIF5A2/MYCN and MCL1/MYCN FISH signals in interphase cells of UACC-1598 and how individual cells are separated into different groups. MYCN signal is shown in green, EIF5A2 and MCL1 shown in red, and the overlap is shown in yellow. The percentage of cells in Groups 1, 2, and 3 is based on EIF5A2/MYCN and MCL1/MYCN FISH signals. The statistical analysis showed the differences of cells in Group 1 and Group 2/3 compared to the control cells. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750019&req=5

pone-0071988-g002: The signal of EIF5A2, MYCN, and MCL1 genes is decreased in HU and GEM treated UACC-1598.A. Genes EIF5A2, MYCN, and MCL1 are present on DMs in UACC-1598 cells. Metaphase spread of UACC-1598 cells detected with DNA probe for EIF5A2, MYCN, and MCL1. For the overlapped pictures, EIF5A2 and MCL1 signals are shown in red, and MYCN signal is shown in green. Overlapped EIF5A2/MYCN and MCL1/MYCN are shown in yellow. B. Representative pictures of EIF5A2/MYCN and MCL1/MYCN FISH signals in interphase cells of UACC-1598 and how individual cells are separated into different groups. MYCN signal is shown in green, EIF5A2 and MCL1 shown in red, and the overlap is shown in yellow. The percentage of cells in Groups 1, 2, and 3 is based on EIF5A2/MYCN and MCL1/MYCN FISH signals. The statistical analysis showed the differences of cells in Group 1 and Group 2/3 compared to the control cells. *denotes a P value of 0.01 to 0.05 and **denotes a P value of 0.001 to 0.01.
Mentions: Once we found that GEM can decrease the number of DMs in both UACC-1598 and UACC-1598-4, the next question we asked is whether genes amplified on the DMs are also decreased. It is known that oncogenes EIF5A2 and MYCN are two genes amplified in UACC-1598 [15], [16], and MCL1 was also found to be amplified in this cell line (unpublished data). We first verified that these three genes we analyzed are present on DMs with FISH experiments (Figure 2A). We then separated cells grown in HU or GEM into 3 groups according to the level of EIF5A2, MYCN, and MCL1 signals (Figure 2B). Group 1 includes cells with <30% signal, which corresponds to cells with less signals from EIF5A2, MYCN, and MCL1, and is an indicator of cells with less number of DMs; group 2 includes cells with 30–60% signal; and group 3 includes cells with >60% signal, which is an indicator of cells with more DMs. The percentage of cells in each group was plotted. The percentage of cells in Group 3, which contains cells with greater than 60% signals, decreased in the presence of either HU (from 11% to 6%) or GEM (from 16% to 7%). In addition, the percentage of cells in Group 2 decreased from 33% to 24% for HU treated cells and 37% to 25% in GEM treated cells. In contrast to the decrease in the percentages of cells in Group 2 and 3, the percentage of cells in Group 1 increased 14 to 21% in cells treated with HU (from 56% to 70%) or GEM (from 47% to 68%). The increase in the percentage of cells in group 1 in the HU or GEM treated cells indicates cells have started losing their DMs. In addition, we consolidated groups 2 and 3 into one large group, and statistical analysis showed that the differences observed between DMSO and HU or control and GEM treated cells is statistically significant (Figure 2B). These results indicate that the signal of the amplified genes MYCN, EIF5A2, and MCL1 on DMs in UACC-1598 cells decreased in the presence of both HU and GEM. A similar result was seen for GEM when analyzing MYCN and EIF5A2 in UACC-1598-4 cells (Figure S1B).

Bottom Line: Amplified genes present on the double minute chromosomes are decreased at the DNA level upon gemcitabine treatment.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.

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.

Show MeSH
Related in: MedlinePlus