Burger H, Nooter K, Boersma A W, Kortland C J, van den Berg A P, Stoter G
Department of Medical Oncology, University Hospital Rotterdam and Rotterdam Cancer Institute (Daniel den Hoed Kliniek), The Netherlands.
Int J Radiat Oncol Biol Phys. 1998 May 1;41(2):415-24. doi: 10.1016/s0360-3016(98)00065-0.
Testicular germ cell tumors (TGCTs) represent one of the few tumor types that are curable by antineoplastic therapy, probably due to the high sensitivity of this neoplasm to induction of apoptosis by chemotherapeutic agents and/or ionizing radiation. Here, we tested cell susceptibility to radiation-induced apoptosis in a panel of TGCT cell lines and attempted to correlate this with the known potentially relevant molecular determinants (p53 gene status and Bcl-2 family proteins) of apoptosis.
Induction of apoptosis by gamma-radiation was morphologically recognized in NT2, NCCIT, S2, and 2102 EP using Hoechst/PI staining and additionally confirmed by Western blot analysis of PARP cleavage. The p53 gene status was estimated by sequence analysis. Expression of p21/WAF/CIP was determined by Northern blot analysis and immunoblotting was used to monitor p53, Bax, Bcl-2, Bcl-x, and Bak protein levels. In vitro colony formation was studied to establish clonogenic survival curves.
NT2 and NCCIT appeared to be susceptible for radiation-induced apoptosis, contrasting 2102 EP and S2 which were highly resistant. Sequence analysis showed that NT2, S2, and 2102 EP are homozygous for wild-type p53 (wtp53), whereas NCCIT contains mutant p53 (mtp53). NT2 and 2102 EP cells showed radiation-induced p53 upregulation, while NCCIT (mtp53) and S2 (no p53 protein) cells did not. Consistently, gamma-radiation-induced DNA damage resulted in a p53-dependent transactivation of the p21/WAF/CIP gene in NT2 and 2102 EP, but not in mtp53-containing NCCIT cells and p53 nonexpressing S2 cells. Constitutive expression of Bax, Bcl-2, Bcl-x, and Bak was not affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis. A discrepancy was found between apoptosis and reproductive death.
The present study revealed that: i) the presence of wtp53 may not be absolutely required for the hypersensitivity for radiation-induced apoptosis in TGCT cell lines, ii) the molecular mechanism underlying the unique radiosensitivity was independent of the expression of Bcl-2 family proteins, and iii) cell susceptibility to apoptosis induction is not sufficiently informative to predict intrinsic radiosensitivity as determined by clonogenic survival.
睾丸生殖细胞肿瘤（TGCTs）是少数可用抗肿瘤治疗治愈的肿瘤类型之一，这可能是由于该肿瘤对化疗药物和/或电离辐射诱导的细胞凋亡高度敏感。在此，我们检测了一组TGCT细胞系对辐射诱导凋亡的细胞敏感性，并试图将其与已知的潜在相关分子决定因素（p53基因状态和Bcl-2家族蛋白）的细胞凋亡情况相关联。
使用Hoechst/PI染色在NT2、NCCIT、S2和2102 EP细胞中通过形态学识别γ辐射诱导的细胞凋亡，并通过PARP裂解的蛋白质印迹分析进一步证实。通过序列分析评估p53基因状态。通过Northern印迹分析确定p21/WAF/CIP的表达，并使用免疫印迹监测p53、Bax、Bcl-2、Bcl-x和Bak蛋白水平。研究体外集落形成以建立克隆存活曲线。
NT2和NCCIT似乎对辐射诱导的细胞凋亡敏感，而2102 EP和S2具有高度抗性。序列分析表明，NT2、S2和2102 EP为野生型p53（wtp53）纯合子，而NCCIT含有突变型p53（mtp53）。NT2和2102 EP细胞显示辐射诱导的p53上调，而NCCIT（mtp53）和S2（无p53蛋白）细胞则没有。一致地，γ辐射诱导的DNA损伤导致NT2和2102 EP中p21/WAF/CIP基因的p53依赖性反式激活，但在含有mtp53的NCCIT细胞和不表达p53的S2细胞中则没有。Bax、Bcl-2、Bcl-x和Bak的组成型表达不受辐射影响，并且与细胞对辐射诱导凋亡的敏感性无关。在细胞凋亡和生殖死亡之间发现了差异。
本研究表明：i）在TGCT细胞系中，对于辐射诱导凋亡的超敏感性，野生型p53的存在可能不是绝对必需的；ii）独特放射敏感性的分子机制独立于Bcl-2家族蛋白的表达；iii）细胞对凋亡诱导的敏感性不足以预测由克隆存活确定的内在放射敏感性。
