Bae I, Smith M L, Sheikh M S, Zhan Q, Scudiero D A, Friend S H, O'Connor P M, Fornace A J
Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Maryland 20892, USA.
Cancer Res. 1996 Feb 15;56(4):840-7.
DNA-damaging agents such as ionizing radiation (IR) activate the tumor suppressor p53, and, in turn, p53 transactivates a number of downstream effector genes such as GADD45, CIP1/WAF1, and MDM2. The induction of these downstream genes following IR appears to be strictly dependent upon the presence of wild-type functional p53 known to evoke G1 arrest. In this study, we characterized 56 cell lines from 9 different tumor types with predetermined p53 genotype by measuring the induction of GADD45, CIP1/WAF1, and MDM2 relative mRNA levels after IR. A higher fraction of melanoma lines had wild-type (wt) p53 (5/8, or 63%) compared to the nonmelanoma lines (11/48, or 23%). Most wt p53 (nonmelanoma) cell lines (11/12, or 92%) showed clear induction of both GADD45 and CIP1/WAF1. On the other hand, many wt p53 melanoma lines (4/5, or 80%) showed normal induction of CIP1/WAF1, but little or no induction of GADD45. Despite this defect in GADD45 induction, we found that all wt p53 melanoma lines exhibited strong G1 arrest and increased levels of p53 protein after IR. The results demonstrated that radiation-induced G1 arrest could occur by the p53-CIP1/WAF1 pathway without appreciable induction of GADD45 in melanoma lines. Time course experiments demonstrated prolonged induced expression of CIP1/WAF1 mRNA transcripts in melanoma lines in which GADD45 induction was lacking, suggesting some sort of compensatory mechanism involving CIP1/WAF1, in cell lines with defective GADD45 induction. We could reproduce this compensatory effect in RKO colon carcinoma cells in which GADD45 expression was blocked by constitutive antisense vectors. These findings reveal that defective induction of GADD45 following IR is common in human melanoma cell lines.
诸如电离辐射（IR）等DNA损伤剂可激活肿瘤抑制因子p53，进而p53反式激活许多下游效应基因，如GADD45、CIP1/WAF1和MDM2。IR照射后这些下游基因的诱导似乎严格依赖于已知能引起G1期阻滞的野生型功能性p53的存在。在本研究中，我们通过测量IR照射后GADD45、CIP1/WAF1和MDM2相对mRNA水平，对来自9种不同肿瘤类型的56个具有预先确定p53基因型的细胞系进行了特征分析。与非黑色素瘤细胞系（11/48，即23%）相比，黑色素瘤细胞系中具有野生型（wt）p53的比例更高（5/8，即63%）。大多数wt p53（非黑色素瘤）细胞系（11/12，即92%）显示出GADD45和CIP1/WAF1均有明显诱导。另一方面，许多wt p53黑色素瘤细胞系（4/5，即80%）显示出CIP1/WAF1正常诱导，但GADD45诱导很少或没有。尽管GADD45诱导存在这种缺陷，但我们发现所有wt p53黑色素瘤细胞系在IR照射后均表现出强烈的G1期阻滞和p53蛋白水平升高。结果表明在黑色素瘤细胞系中，辐射诱导的G1期阻滞可通过p53 - CIP1/WAF1途径发生，而无需明显诱导GADD45。时间进程实验表明，在缺乏GADD45诱导的黑色素瘤细胞系中，CIP/WAF1 mRNA转录本的诱导表达持续延长，这表明在GADD45诱导缺陷的细胞系中存在某种涉及CIP1/WAF1的补偿机制。我们可以在RKO结肠癌细胞中重现这种补偿效应，在该细胞系中GADD45表达被组成型反义载体阻断。这些发现揭示了IR照射后GADD45诱导缺陷在人黑色素瘤细胞系中很常见。
