세포융합에 따른 유전체 불안정성
Chromosomal instability induced by cell-to-cell fusion using HeLa cells
- 주제(키워드) cell fusion , apoptosis , survivin , asymmetric division , chromosomal instability
- 발행기관 아주대학교
- 지도교수 이재호
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025879
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Tetraploidy, a potential precursor of cancer-associated aneuploidy, is produced either by cell fusion or cytokinesis failure. These cells undergo cell-cycle arrest or apoptosis in a p53-dependent manner. Here, I used low p53-expressing HeLa cells as a model system to address the fate of cancer cells after fusion in the context of decreased influence of p53. I found that massive apoptotic cell death or growth arrest occurred a few days after fusion and was accompanied by an increase in p53. In addition, cells with larger nuclei preferentially died after fusion, suggesting that a larger deviation from normal DNA content is a strong inducer of apoptosis. Closer observation of the cells revealed that the division of fused cells immediately after cell fusion showed the formation of multipolar spindles due to increased number of centrosomes, resulting in various mitotic defects (DNA bridges, lagging chromosome, micronucleus, etc.) and asymmetric division with subsequent cytokinesis failure. After a series of unstable cell division processes, the surviving cells showed depolyploidization to have a little bit more chromosomes comparing to parental cells. The CIN (chromosomal instability) assay measuring the centromere of chromosomes 8 and 18 confirmed that chromosomal instability was increased in fused cells, and even in stable fused cell lines. Notably, a fraction of cells escaped from cell death and proliferated. These surviving fused cells were characterized by upregulation of survivin, reflecting increased survivin protein stability. Moreover, in fused cells, survivin became preferentially localized to the cytosol, where it is known to exert its anti-apoptotic function. Knockdown of survivin decreased survival to a greater extent in fused cells than in unfused cells, suggesting that fused cells became more dependent on survivin. Therefore, above findings indicate that, after cancer cell fusion, a subpopulation of fused cells with a higher level of cytosolic survivin are able to avoid apoptotic crisis and survive to proliferate continuously, a process that might contribute to human cancer progression. Regarding cancer progression, fused cells were superior to unfused cells in their overall cell migration ability, and their abilities varied according to each cell line. In addition, when the cells were grown under low concentration of cisplatin, fused cells got resistance more readily than unfused cells. Collectively, the fusion of cancer cells leads to chromosomal instability that induce p53-dependent cell death; however, a fraction of cells such as the cells having more cytosolic survivin survive and get genetic diversity, which probably confers the cancer cells more progressed characteristics such as acquisition of chemoresistance as well as migration ability.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
1. Cell cultures and plasmids 4
2. RNA extraction, reverse transcription-polymerase chain reaction 4
3. Antibodies 4
4. Cell fusion and FACS 4
5. Measurement of cellular DNA content 5
6. Clonogenic cell proliferation assays 5
7. Establishment of Cisplatin-resistance cells 5
8. Immunocytochemistry 6
9. Cytogenetics: karyotypes and FISH 6
10. Live cell imaging 6
III. RESULTS 8
1. Generation and isolation of fused cells. 8
2. Fused cells experience massive cell death. 10
3. Cells with enlarged nuclei are prone to apoptosis. 12
4. Depolyploidization after cell fusion. 17
5. Fused cells showed asymmetric division showing chromosome missegregations with spindle multipolarity. 19
6. Cell fusion induces ROS production and DNA damages. 32
7. Survivin is necessary for the survival of fused cells that escape apoptotic crisis. 35
8. Survivin protein in fused cells is localized in the cytosol and shows increased stability. 39
9. Fused cells increased cell migration and showed higher resistance than unfused cells in cisplatin treatment. 42
IV. DISCUSSION 45
V. CONCLUSION 48
REFFERENCE 49
국문요약 52
