Böhnke A, Westphal F, Schmidt A, El-Awady R A, Dahm-Daphi J
University Hospital of Hamburg-Eppendorf, Department of Radiotherapy and Radiation Oncologyk Martinistr. 52k D-20246 Hamburgk Germany.
Int J Radiat Biol. 2004 Jan;80(1):53-63. doi: 10.1080/09553000310001642902.
The tumour suppressor protein p53 is considered to have an impact on the radiosensitivity of tumour cells. However, this concept does not easily translate to the tumour sensitivity in the clinics. The aim of the present study was to determine whether a functional or dysfunctional p53 is associated with a sensitive or resistant phenotype. It was further studied whether DNA damage might be an additive factor by which p53 has impact on cell survival.
Nine human tumour cell lines were studied for p53 mutation by direct sequencing of exons 4-9. Regulation of p53 and p21(cip1/waf1) protein was assessed by immunoblotting and cell cycle effects by combining 5-bromodeoxyuridine incorporation and flow cytometry.
Three strains (RT112, Du145, SCC4451) were found to have a missense-mutation in the core domain and one did not express p53 at all (HeLa), presumably due to HPV18 infection. Immunoblots of these cells showed neither a regulated p53 nor p21 expression. The cells did not arrest in G1 phase after X-irradiation but did arrest in G2/M. All cells expressing wild-type protein (LNCaP, T47D-B8, MCF-7 and sublines BB and Bus) showed an intact p53 and p21 regulation and a modest arrest in both G1 and G2/M. Thus, in contrast to other studies, all tumour cells investigated showed either a typical p53wt or mutant (mut) pattern. Protein function was compared with cell survival and DNA damage, as assessed previously. p53 wild-type cells were on average 1.3-times (n.s.) more radiosensitive than mutant cells, but there was a considerable overlap between both groups. Further, the 1.3-fold enhanced resistance of cells lacking wild-type p53 was paralleled by a 1.3-fold lower number of induced double-strand breaks. The results suggest that p53 could have impact on chromatin compaction and thus effect DNA damage induction and radiosensitivity of tumour cells.
肿瘤抑制蛋白p53被认为会影响肿瘤细胞的放射敏感性。然而，这一概念在临床中并不容易转化为肿瘤敏感性。本研究的目的是确定功能性或功能失调性p53是否与敏感或耐药表型相关。还进一步研究了DNA损伤是否可能是p53影响细胞存活的一个附加因素。
通过对4-9号外显子进行直接测序，研究了9种人肿瘤细胞系的p53突变情况。通过免疫印迹评估p53和p21（cip1/waf1）蛋白的调控，并结合5-溴脱氧尿苷掺入和流式细胞术评估细胞周期效应。
发现三株细胞系（RT112、Du145、SCC4451）在核心结构域存在错义突变，一株细胞系（HeLa）根本不表达p53，推测是由于HPV18感染所致。这些细胞的免疫印迹显示p53和p21表达均不受调控。X射线照射后，这些细胞未在G1期停滞，但在G2/M期停滞。所有表达野生型蛋白的细胞系（LNCaP、T47D-B8、MCF-7及其亚系BB和Bus）均显示p53和p21调控完整，且在G1和G2/M期均有适度停滞。因此，与其他研究不同，所有研究的肿瘤细胞均表现出典型的p53野生型或突变型模式。如先前评估的那样，将蛋白功能与细胞存活和DNA损伤进行了比较。p53野生型细胞的放射敏感性平均比突变型细胞高1.3倍（无显著性差异），但两组之间有相当大的重叠。此外，缺乏野生型p53的细胞系抗性增强1.3倍的同时，诱导的双链断裂数量减少1.3倍。结果表明，p53可能会影响染色质浓缩，从而影响肿瘤细胞的DNA损伤诱导和放射敏感性。
