从非转化人成纤维细胞增生四倍体细胞的建立
Özet
Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.
Abstract
Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.
Introduction
多倍体已经观察到，不仅在哺乳动物物种的专门的组织，而且在各种病理状况，如癌症和变性疾病。多倍体（主要是四倍体）细胞通常在人体组织中，诸如Barrett食管1,2或鳞状子宫颈3,4上皮内病变的癌前病变观察到的，并已经被认为是恶性非整倍体细胞的来源在那些组织5 6。虽然有人建议，四倍体转化为异倍体细胞可以是在肿瘤发生的早期阶段的关键事件，参与这一过程的机制尚未完全了解。这部分是因为没有体外模型已经可以在那里未转化多倍体人体细胞可以传播。
一些研究人员已经通过代双核细胞通过异烟肼诱导四倍体在非转化人类上皮细胞ibiting胞质7-9。在该方法中，然而，不必要的二倍体细胞，必须通过荧光激活细胞分选（FACS）消除7,8- 或通过有限稀释9克隆。因为这些程序是艰苦和不易执行，简单的方法来建立非转化四倍体细胞被期望在这一领域的研究。
在本报告中，我们描述了一个协议，通过相对简单的程序，以建立正常的人类成纤维细胞或端粒酶永生化人成纤维细胞增殖四倍体细胞。该过程使用的主轴毒药demecolcine（DC）逮捕由摆脱与DC进一步处理收集在有丝分裂二倍体细胞，并有丝分裂细胞。与DC治疗时间延长二倍体有丝分裂细胞转化为四倍体G1期细胞，而这些细胞增殖生长停滞了以下药物清除几天后的四倍体细胞。该协议提供用于创建一个有用的模型来研究染色体的不稳定性和多倍体的人类细胞的致瘤潜力之间的关系的有效方法。
Protocol
Representative Results
Discussion
从二倍体细胞通过化学试剂四倍体诱导的主要问题，无论是由胞质抑制剂或由主轴抑制剂，是细胞常成为二倍体和四倍体群体的混合物，和四倍体细胞必须从二倍体细胞中分离出来。免费二倍体细胞的四倍体种群隔离最常用的方法通过有限稀释使用FACS或克隆。然而，这些程序是费力和不容易执行。在这份报告中，我们提出了一个新的协议建立无正常人成纤维细胞二倍体人口增殖细胞四倍体。该协…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
We thank Mrs. Matsumoto for the technical assistance.
Materials
| MEM-α | Sigma-Aldrich | M8042-500ML | |
| Trypsin-EDTA | Sigma-Aldrich | T4174 | |
| FBS | Sigma-Aldrich | 172012-500ML | |
| Demecolcine solution (10 μg/mL in HBSS) | Sigma-Aldrich | D1925-10ML | |
| BD CycleTES Plus DNA Reagent Kits | BD Biosciences | #340242 | For examination of DNA ploidy by flow cytometry |
| Human chromosome multicolor FISH probe 24XCyte | MetaSystems | #D-0125-060-DI | Specialized filter set and software for mFISH analysis are necessary |
| Isis imaging system with mFISH software | MetaSystems | Specialized probe kit is necessary |
Referanslar
- Rabinovitch, P., et al. Predictors of progression in Barrett’s esophagus III: baseline flow cytometric variables. Am. J. Gastroenterol. 96 (11), 3071-3083 (2001).
- Galipeau, P., et al. NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma. PLoS Med. 4 (2), e67 (2007).
- Olaharski, A., et al. Tetraploidy and chromosomal instability are early events during cervical carcinogenesis. Carcinogenesis. 27, 337-343 (2006).
- Liu, Y., et al. p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy. Cancer Res. 67, 2603-2610 (2007).
- Davoli, T., de Lange, T. The causes and consequences of polyploidy in normal development and cancer. Annu Rev Cell Dev Biol. 27, 585-610 (2011).
- Fox, D., Duronio, R. Endoreplication and polyploidy: insights into development and disease. Development. 140, 3-12 (2013).
- Fujiwara, T., et al. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature. 437, 1043-1047 (2005).
- Ganem, N., et al. Cytokinesis failure triggers hippo tumor suppressor pathway activation. Cell. 158 (4), 833-848 (2014).
- Kuznetsova, A., et al. Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells. Cell cycle. 14 (17), 2810-2820 (2015).
- Vindeløv, L., Christensen, I. Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis. Methods Cell Biol. 41, 219-229 (1994).
- Darzynkiewicz, Z., Juan, G. DNA content measurement for DNA ploidy and cell cycle analysis. Curr Protoc Cytom. , Chapter 7: Unit 7.5 (2001).
- Knutsen, T., Bixenman, H., Lawce, H., Martin, P. Chromosome analysis guidelines preliminary report. Cancer Genet Cytogenet. 52 (1), 11-17 (1991).
- Liehr, T., et al. Multicolor FISH probe sets and their applications. Histol. Histopathol. 19 (1), 229-237 (2004).
- Ohshima, S., Seyama, A. Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts. Hum. Cell. 25 (3), 78-85 (2012).
- Ohshima, S., Seyama, A. Establishment of proliferative tetraploid cells from normal human fibroblasts. Front. Oncol. 3, 198 (2013).
- Ohshima, S., Seyama, A. Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts. Genes, Chromosome Cancer. 55 (6), 522-530 (2016).
- Di Leonardo, A., et al. DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function. Cancer Res. 57, 1013-1019 (1997).
- Andreassen, P., Lohez, O., Lacroix, F., Margolis, R. Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol. Biol. Cell. 12, 1315-1328 (2001).
- Vogel, C., et al. Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy. Oncogene. 23, 6845-6853 (2004).
- Aylon, Y., Oren, M. p53: Guardian of ploidy. Mol. Oncol. 5 (4), 315-323 (2011).
- Uetake, Y., Sluder, G. Cell cycle progression after cleavage failure : mammalian somatic cells do not possess a "tetraploidy checkpoint". J. Cell Biol. 165, 609-615 (2004).
- Ganem, N., Pellman, D. Limiting the proliferation of polyploid cells. Cell. 131, 437-440 (2007).
- Ho, C., Hau, P., Marxer, M., Poon, R. The requirement of p53 for maintaining chromosomal stability during tetraploidization. Oncotarget. 1 (7), 583-595 (2010).
