P14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice
P14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice
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DOI：
10.1038/sj.onc.1206303
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发表时间：
2003-03-27
期刊：
影响因子：
8
通讯作者：
Gazzeri, S
中科院分区：
文献类型：
--
作者：
Eymin, B;Leduc, C;Gazzeri, S
直到最近，由INK 4A/ARF基因座编码的ARF（人p14（ARF），鼠p19（ARF））肿瘤抑制蛋白抑制响应于各种刺激的细胞生长的能力与其通过所谓的ARF/MDM 2/p53途径稳定p53的能力有关。然而，最近的数据表明，ARF是没有牵连在这个独特的p53依赖性途径。通过使用瞬时和稳定表达，我们在这里显示，人p14（ARF）抑制缺乏功能性p53的人肿瘤细胞的生长，诱导瞬时G（2）停滞和随后的凋亡。这种p14（ARF）诱导的G2期阻滞与CDC 2活性的抑制、CDC 25 C磷酸酶的失活和CDK抑制剂p21（WAF 1）的诱导相关。使用Hoechst 33352染色、半胱天冬酶-3的蛋白水解活化和PARP裂解来证明细胞凋亡。类似的结果，在实验中获得的细胞同步的羟基脲块。重要的是，我们能够通过显示p14（ARF）抑制裸鼠中p53缺失型人类肿瘤的生长并诱导p53 -/-建立的肿瘤的消退来再现这些“体内”效应。在这些实验中，肿瘤消退与细胞增殖的抑制以及细胞凋亡的诱导相关，证实了在细胞系中获得的数据。
Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G2 arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAF1). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.