Counteraction of pRb-dependent protection after extreme hypoxia by elevated ribonucleotide reductase
Counteraction of pRb-dependent protection after extreme hypoxia by elevated ribonucleotide reductase
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DOI：
10.1111/j.1365-2184.2004.00319.x
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发表时间：
2004-10-01
影响因子：
8.5
通讯作者：
Pettersen, EO
中科院分区：
文献类型：
--
作者：
Graff, P;Seim, J;Pettersen, EO
我们已经研究了缺氧诱导的细胞周期停滞在人类细胞中，视网膜母细胞瘤肿瘤抑制蛋白（pRb）是功能性的（T-47 D和T-47 DHU-res细胞）或废除的HPV 18 E7癌蛋白的表达（NHIK 3025细胞）。我们以前已经发现，pRb是去磷酸化和反弹在T-47 D细胞的细胞核在S期缺氧期间被捕，这种结合是延长，甚至在复氧。然而，在本研究中，我们表明，持久的逮捕后再氧合诱导的pRb结合在细胞核中可能被推翻的核糖核苷酸还原酶（RNR）的水平升高。这似乎创造了一个强制的DNA合成，与细胞分裂不协调，诱导DNA的核内复制。这些数据表明，启动核内复制的细胞继续DNA合成，直到所有DNA复制一次，然后可以开始循环和细胞分裂，DNA含量加倍。在pRb缺陷型NHIK 3025-细胞上的相应数据显示在这些细胞中类似的核内复制。因此，数据表明，再氧合后DNA的核内复制可能是由于细胞中不存在pRb功能时DNA合成的缺氧停滞，或者如果它被增加的RNR所支配。本研究进一步表明，pRb不仅通过阻止大多数暴露于极端缺氧的细胞处于S期来保护培养物，而且通过增加这些细胞的克隆形成能力来增加细胞存活。然而，有趣的是，具有升高水平的RNR的细胞在20小时极端缺氧后具有与野生型细胞同样高的存活率。如果RNR-推翻pRb介导的逮捕后，再氧合的结果在一个不稳定的基因组，这可能因此代表致癌选择的危险，作为保护作用的pRb细胞存活似乎是维持。
We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRb) is either functional (T-47D and T-47DHU-res cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). We have previously found that pRb is dephosphorylated and rebound in the nucleus in T-47D cells arrested in S-phase during hypoxia and that this binding is protracted even following re-oxygenation. In the present study, however, we show that the long-lasting arrest following re-oxygenation induced by pRb-binding in the cell nuclei may be overruled by an elevated level of ribonucleotide reductase (RNR). This seems to create a forced DNA-synthesis, uncoordinated with cell division, which induces endoreduplication of the DNA. The data indicate that the cells initiating endoreduplication continue DNA-synthesis until all DNA is replicated once and then may start cycling and cell division with a doubled DNA-content. Corresponding data on the pRb-incompetent NHIK 3025-cells show similar endoreduplication in these. Thus, the data indicate that endoreduplication of DNA following re-oxygenation may come, either as a result of hypoxic arrest of DNA-synthesis when pRb-function is absent in the cells, or if it is overruled by increased RNR. The present study further shows that pRb not only protects the culture by arresting most of the cells that are exposed to extreme hypoxia in S-phase, but also increases cell survival by means of increased clonogenic ability of these cells. Interestingly, however, cells having an elevated level of RNR have equally high survival as wild-type cells following 20 h extreme hypoxia. If RNR-overruling of pRb-mediated arrest following re-oxygenation results in an unstable genome, this may therefore represent a danger of oncogenic selection as the protective effect of pRb on cell survival seems to be maintained.