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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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Classification of cells into different categories based on the presence of MN and γ-H2AX signal within MN.DAPI stained DNA is shown in blue, and γ-H2AX immunofluorescence signal is shown in red. The different groups: A, Cells without MN, B, Cells with MN, and C, Cells with MN (γ-H2AX+) are indicated.
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pone-0071988-g006: Classification of cells into different categories based on the presence of MN and γ-H2AX signal within MN.DAPI stained DNA is shown in blue, and γ-H2AX immunofluorescence signal is shown in red. The different groups: A, Cells without MN, B, Cells with MN, and C, Cells with MN (γ-H2AX+) are indicated.

Mentions: Studies have also indicated that DNA damage induced by HU can facilitate the formation of MN containing γ-H2AX signals [26], [54]. It is hypothesized that these MN selectively incorporates damaged DNA, and the incorporated DNA are then lost from cells. We incubated UACC-1598 cells in different compounds for 24 hours, washed away the compounds, released the cells into media without compounds for 24 and 48 hours, and detected MN frequency together with γ-H2AX signals (Figure 6). We found that 24 hours after release from GEM treatment, there was a statistically significant increase in MN frequency (from 4.87×10−2 to 10.36×10−2, a 2.13 fold increase) when compared to control cells (Table 2). This is in agreement with cells released from HU, in which the MN frequency showed a statistically significant increase from 4.65×10−2 to 8.44×10−2 (a 1.82 fold increase). We detected γ-H2AX by immunofluorescence and when we analyzed the frequency of MN in which there was bright uniform γ-H2AX signal, designated MN (γ-H2AX+) or MN (+) for short, we observed a statistically significant increase (from 3.06×10−2 to 7.10×10−2, a 2.32 fold increase for HU; from 4.10×10−2 to 7.32×10−2, a 1.79 fold increase for GEM) for cells released from HU or GEM when compared to control cells. A similar trend was seen in a repeat experiment in UACC-1598-4 (Table S2 and S3). For 48 hours after release, a similar trend is seen in MN and MN (+) frequency albeit some with lower levels of fold change when compared with their respective controls (Table 3). Our results show that after cells are released from GEM treatment, a significant proportion of cells accumulate MN with bright γ-H2AX staining.


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)

Classification of cells into different categories based on the presence of MN and γ-H2AX signal within MN.DAPI stained DNA is shown in blue, and γ-H2AX immunofluorescence signal is shown in red. The different groups: A, Cells without MN, B, Cells with MN, and C, Cells with MN (γ-H2AX+) are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071988-g006: Classification of cells into different categories based on the presence of MN and γ-H2AX signal within MN.DAPI stained DNA is shown in blue, and γ-H2AX immunofluorescence signal is shown in red. The different groups: A, Cells without MN, B, Cells with MN, and C, Cells with MN (γ-H2AX+) are indicated.
Mentions: Studies have also indicated that DNA damage induced by HU can facilitate the formation of MN containing γ-H2AX signals [26], [54]. It is hypothesized that these MN selectively incorporates damaged DNA, and the incorporated DNA are then lost from cells. We incubated UACC-1598 cells in different compounds for 24 hours, washed away the compounds, released the cells into media without compounds for 24 and 48 hours, and detected MN frequency together with γ-H2AX signals (Figure 6). We found that 24 hours after release from GEM treatment, there was a statistically significant increase in MN frequency (from 4.87×10−2 to 10.36×10−2, a 2.13 fold increase) when compared to control cells (Table 2). This is in agreement with cells released from HU, in which the MN frequency showed a statistically significant increase from 4.65×10−2 to 8.44×10−2 (a 1.82 fold increase). We detected γ-H2AX by immunofluorescence and when we analyzed the frequency of MN in which there was bright uniform γ-H2AX signal, designated MN (γ-H2AX+) or MN (+) for short, we observed a statistically significant increase (from 3.06×10−2 to 7.10×10−2, a 2.32 fold increase for HU; from 4.10×10−2 to 7.32×10−2, a 1.79 fold increase for GEM) for cells released from HU or GEM when compared to control cells. A similar trend was seen in a repeat experiment in UACC-1598-4 (Table S2 and S3). For 48 hours after release, a similar trend is seen in MN and MN (+) frequency albeit some with lower levels of fold change when compared with their respective controls (Table 3). Our results show that after cells are released from GEM treatment, a significant proportion of cells accumulate MN with bright γ-H2AX staining.

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