Heterogeneous activation of p19Arf in pulmonary artery smooth muscle cells.
Heterogeneous activation of p19Arf in pulmonary artery smooth muscle cells.
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肺动脉平滑肌细胞中 p19Arf 的异质激活。
DOI：
10.1152/ajplung.00117.2010
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Fouty,Brian
中科院分区：
文献类型：
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作者：
Solodushko,Victor;Alvarez,DiegoF;Viator,Ryan;Messerall,Tiffany;Fouty,Brian
p19arfi是一种肿瘤抑制因子，通过稳定p53导致细胞周期阻滞或凋亡。在正常情况下，p19arfi对细胞周期调节并不重要，但在许多人类癌症中发现p19arfi的缺失，小鼠p19arf敲除模型导致恶性增殖和肿瘤形成；其在控制非恶性增殖中的作用尚不明确。为了研究这个问题，我们在培养中扩增了来自转基因小鼠的肺动脉平滑肌细胞（PASMC），其中p19Arfgene的编码序列被编码绿色荧光蛋白（GFP）的cDNA取代，使启动子保持完整。在培养的前10天，野生型、杂合型和敲除型PASMC的生长相似，但到第14天，p19arf缺陷型PASMC的增殖速度快于p19arf杂合型或野生型细胞；p19arf的再表达阻止了增殖的增加。这个时间过程与p19arf启动子的激活相关，p19arf缺陷PASMC中出现GFP阳性表明了这一点。第42天，约80%的p19arf缺陷细胞呈gfp阳性。当gfp阳性、p19arf缺陷的细胞被分选并分别传代培养时，它们仍保持gfp阳性，这表明一旦细胞激活了p19arf启动子，启动子在这些细胞和随后的所有子细胞中仍保持活性。相反，随着时间的推移，gfp阴性的p19arf缺陷细胞会产生gfp阳性和阴性子细胞的组合。这些结果表明，PASMC的一个亚群对激活p19Arfpromoter的信号具有抗性，而p19Arfpromoter通常是针对这些细胞进行阻滞或细胞死亡的事件。
p19ARFis a tumor suppressor that leads to cell cycle arrest or apoptosis by stabilizing p53. p19ARFis not critical for cell cycle regulation under normal conditions, but loss of p19ARFis seen in many human cancers, and a murine p19Arfknockout model leads to malignant proliferation and tumor formation; its role in controlling nonmalignant proliferation is less defined. To examine this question, pulmonary artery smooth muscle cells (PASMC) were expanded in culture from a transgenic mouse in which the coding sequence of the p19Arfgene was replaced with a cDNA encoding green fluorescent protein (GFP), leaving the promoter intact. During the first 10 days in culture, wild-type, heterozygous, and knockout PASMC grew similarly, but, byday 14, p19Arf-deficient PASMC proliferated faster than p19Arfheterozygous or wild-type cells; reexpression of p19Arfprevented the increased proliferation. This time course correlated with activation of the p19Arfpromoter, as indicated by the appearance of GFP positivity in p19Arf-deficient PASMC. Byday 42, ∼80% of p19Arf-deficient cells were GFP-positive. When GFP-positive, p19Arf-deficient cells were sorted and subcultured separately, they remained GFP-positive, indicating that once cells had activated the p19Arfpromoter, the promoter remained active in those and all subsequent daughter cells. In contrast, GFP-negative p19Arf-deficient cells gave rise to a combination of GFP-positive and -negative daughter cells over time. These results suggest that a subpopulation of PASMC are resistant to the signals that activate the p19Arfpromoter, an event that would normally target these cells for arrest or cell death.