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Sci Rep. 2019;9(1):17889.

Non-SMC condensin I complex subunit H mediates mature chromosome condensation and DNA damage in pancreatic cancer cells.

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제1저자 김재형 · 교신저자 김재형, 황진혁

PMID 31784646 PMC6884527 DOI 10.1038/s41598-019-54478-3 IF 4.9 소화기내과
소속 (5)
  1. Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, 13620, Republic of Korea. kingfish12@naver.com.
  2. Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, 13620, Republic of Korea.
  3. Research Institute, National Cancer Center, Goyang-si, Gyeonggi-do, 10408, Republic of Korea.
  4. Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, 13620, Republic of Korea. woltoong@snu.ac.kr.
  5. Department of Internal Medicine, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea. woltoong@snu.ac.kr.

Abstract

Non-SMC condensin I complex subunit H (NCAPH) is a vital gene associated with chromosome stability and is required for proper chromosome condensation and segregation. However, the mechanisms through which NCAPH affects pancreatic cancer (PC) and its molecular function remain unclear. In this study, we examined the role of NCAPH in PC cells. Our results showed that NCAPH was overexpressed in clinical PC specimens (GEPIA) and cell lines. In addition, in NCAPH-knockdown cells, colony formation and proliferation were inhibited, and the cell cycle was arrested at the S and G2/M phases owing to failure of mature chromosome condensation (MCC) in poorly condensed chromosomes. Increased cell death in NCAPH-knockdown cells was found to help initiate apoptosis through the activation of caspase-3 and PARP cleavage. Furthermore, NCAPH-knockdown cells showed an increase in chromosomal aberrations and DNA damage via activation of the DNA damage response (Chk1/Chk2) signaling pathways. These data demonstrated that NCAPH played an important role in cell cycle progression and DNA damage by maintaining chromosomal stability through progression of MCC from poorly condensed chromosomes. Ultimately, NCAPH knockdown induced apoptotic cell death, which was partially mediated by caspase-dependent pathways. These findings highlight the potential role of NCAPH as a therapeutic target for PC.

Figures

Figure 4
Figure 4 NCAPH-knockdown cells fail to progress of mature chromosome condensation (MCC) from poorly condensed chromosomes. (A) General outline of the procedure followed for thymidine double block synchronization of MIA PaCa-2 cells and their cell cycle analysis. Cells were transfected with the indicated siRNAs, and after release from the second thymidine block, cells were assayed at different time points ranging from 0 to 8 h. (B) Cells were harvested for PI staining and analyzed by flow cytometry to determine the cell cycle fraction at 0, 4, and 8 h after release. Flow cytometry plots and data are representative of at least three separate experiments. (C) The percentages of cells in G1, S, and G2/M phases are indicated. (D,E) Frequencies of poorly condensed chromosomes and mature condensed chromosomes cells in control and NCAPH-knockdown cells. Cells were transfected with the indicated siRNA, treated with one thymidine block (36 h), released for 3 h and then harvested. For accurate quantification, more than 200 cells captured in at least three different fields were analyzed. Values represent means ± SEMs. *P <0.005, ***P <0.0001. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni’s multiple comparison tests.
Figure 5
Figure 5 Knockdown of NCAPH induces chromosomal aberrations and DNA damage. (A,B) To confirm the chromosome morphology, MIA PaCa-2 cells were transfected with control siRNA or NCAPH siRNA and arrested at metaphase by colcemid treatment for 4 h. The cells were spread onto slides, extracted, fixed, and stained with DAPI (blue). For accurate quantification, more than 50 cells captured in at three different fields were analyzed. Scale bar, 5 µm. (C) Western blot analysis of phospho-H2AX expression in control and NCAPH-knockdown cells. Cell lysates were immunoblotted with the indicated antibodies. (D) Phospho-H2AX fluorescence pattern (green) in control and NCAPH-knockdown cells was observed by confocal microscopy. DNA was stained using DAPI (blue). Scale bar, 20 µm. (E) Frequency of phospho-H2AX fluorescence intensity. For accurate quantification, more than 100 cells captured in at least two different fields were analyzed. Values represent means ± SEMs. ***P <0.0001, two-way analysis of variance. (F) Western blot analysis showing levels of phospho-Chk1 and phospho-Chk2 activation in response to DNA damage in MIA PaCa-2 cells after transfection with control siRNA and NCAPH siRNA for 6, 12, 24, and 30 h. (G) Cells were harvested for PI staining and analyzed by flow cytometry to determine the cell cycle fraction after release from the second thymidine block at 0, 4, and 8 h. ATMi (13 μM) was treated with a second thymidine block.

그림 출처: PubMed Central 공개 논문 (PMC6884527)

Conflict of interest statement

The authors declare no competing interests.

그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
Scientific reports · eISSN 2045-2322
전자 출판
2019-11-29
MeSH
Apoptosis · Caspase 3 / metabolism · Cell Cycle Checkpoints / drug effects · Cell Cycle Proteins / antagonists & inhibitors / genetics / metabolism · Cell Line, Tumor · Checkpoint Kinase 1 / metabolism · Checkpoint Kinase 2 / metabolism · Chromosome Aberrations* · DNA Damage* · Humans · Kaplan-Meier Estimate · Nuclear Proteins / antagonists & inhibitors / genetics / metabolism · Pancreatic Neoplasms / metabolism / mortality / pathology · RNA Interference · RNA, Small Interfering / metabolism
Substances
Cell Cycle Proteins · NCAPH protein, human · Nuclear Proteins · RNA, Small Interfering · Checkpoint Kinase 2 · Checkpoint Kinase 1 · Caspase 3
참고문헌
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