ABSTRACT
Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP) and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20), only DDP treatment induced p53 phosphorylation at serine 15 (Ser15). Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM) cell lines, the role of ATM in drug-induced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , DNA-Binding Proteins , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins , Paclitaxel/pharmacology , Tumor Cells, Cultured/metabolism , Tumor Suppressor Protein p53/metabolism , Androstadienes/pharmacology , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle Proteins/metabolism , Colonic Neoplasms/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA-Activated Protein Kinase , Female , Humans , Ovarian Neoplasms/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Transcription, Genetic , Tumor Cells, Cultured/drug effects , WortmanninABSTRACT
The cytotoxic activity of ecteinascidin 743 (ET-743), a natural product derived from the marine tunicate Ecteinascidia turbinata that exhibits potent anti-tumor activity in pre-clinical systems and promising activity in phase I and II clinical trials, was investigated in a number of cell systems with well-defined deficiencies in DNA-repair mechanisms. ET-743 binds to N2 of guanine in the minor groove, but its activity does not appear to be related to DNA-topoisomerase I poisoning as the drug is equally active in wild-type yeast and in yeast with a deletion in the DNA-topoisomerase I gene. Defects in the mismatch repair pathway, usually associated with increased resistance to methylating agents and cisplatin, did not affect the cytotoxic activity of ET-743. However, ET-743 did show decreased activity (from 2- to 8-fold) in nucleotide excision repair (NER)-deficient cell lines compared to NER-proficient cell lines, from either hamsters or humans. Restoration of NER function sensitized cells to ET-743 treatment. The DNA double-strand-break repair pathway was also investigated using human glioblastoma cell lines MO59K and MO59J, respectively, proficient and deficient in DNA-dependent protein kinase (DNA-PK). ET-743 was more effective in cells lacking DNA-PK; moreover, pre-treatment of HCT-116 colon carcinoma cells with wortmannin, a potent inhibitor of DNA-PK, sensitized cells to ET-743. An increase in ET-743 sensitivity was also observed in ataxia telangiectasia-mutated cells. Our data strongly suggest that ET-743 has a unique mechanism of interaction with DNA.
