Jaiswal Aruna S, Narayan Satya
Department of Anatomy and Cell Biology, UF Shands Cancer Center, College of Medicine, University of Florida, P.O. Box 100232, Gainesville, FL 32610, USA.
Mutat Res. 2002 Mar 20;500(1-2):17-30. doi: 10.1016/s0027-5107(01)00296-2.
p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.
p53是细胞对基因毒性应激反应中的一个重要因子，其功能受多个丝氨酸和苏氨酸残基磷酸化的调控。p53的磷酸化会影响其DNA结合和基因调控活性。本研究检测了用S(N)2 DNA烷化剂甲磺酸甲酯（MMS）处理的HCT-116（错配修复缺陷）和HCT-116+ch3（错配修复 proficient）人结肠癌细胞中的p53磷酸化情况。MMS以剂量和时间依赖的方式诱导p53在Ser15和Ser392位点的磷酸化。MMS诱导的p53磷酸化与DNA错配修复（MMR）活性无关。与未处理的细胞相比，MMS处理的HCT-116细胞的核提取物在体外具有更高的p21WAF1/Cip1（p21）启动子DNA结合活性。MMS处理后，在瞬时转染实验中克隆的p21启动子的激活以及HCT-116（p53+/+）与HCT-116（p53-/-）细胞中内源性p21 mRNA水平增加，这与磷酸化p53（Ser15）和磷酸化p53（Ser392）水平的增加相关。这些结果表明，SN2 DNA烷化剂诱导的p53在Ser15和Ser392位点的磷酸化增加了其DNA结合特性，从而导致p21表达增加，这可能在HCT-116细胞的细胞周期停滞和/或凋亡中发挥作用。
