Cisplatinum and taxol induce different patterns of p53 phosphorylation

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.

Figure 1

Western blot analysis in HCT-116 cells treated with DDP or taxol. Extracts were obtained at different times after treatment. Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser15, Ser20, p53 (DO-1), p21, and mdm-2.

Figure 2

Western blot analysis in A2780 cells treated with DDP or taxol. Experimental conditions were as reported in Figure 1. Cells were also treated with 50 µM of LLnL for 3 hours and cellular extracts taken at the end of treatment.

Figure 3

Gel shift assay with extracts obtained from HCT-116 cells treated with DDP or taxol and performed with two different oligonucleotides (CON, panel A and p21, panel B), both containing a p53 binding site. Panel C reports the analysis with the CON oligonucleotide performed in the presence of a 50-fold molar excess of unlabeled specific (S) or unspecific (U) oligonucleotide.

Figure 4

Gel shift assay with extracts obtained from human ovarian cancer A2780 cells treated with DDP or taxol. Experiments were performed with two different oligonucleotides (CON, panel A, and p21, panel B).

Figure 5

HCT-116 cells were pretreated with wortmannin 35.5 µM for two hours before treatment with DDP or taxol. At the end of DDP or taxol treatment cells were washed in PBS and medium containing wortmannin (35.5 µM) was added. Panel A: Extracts for DNA-PK activity were obtained 8 hours after treatment with and without wortmannin and processed as described in Materials and Methods. Values are the means±S.E. of pmolATP incorporated per minute per microgram protein in the biotinylated substrate in the absence (black columns) or in the presence of double-strand DNA (white columns). Panel B: Total cellular extracts were taken at 6 and 24 hours after treatment with wortmannin (lanes 2 and 5), with DDP (lanes 3 and 6), and with wortmannin and DDP (lanes 4 and 7). Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser15, Ser20 and p53 (DO-1). Panel C: Total cellular extracts were taken at 6 and 24 hours after treatment with wortmannin (lanes 2 and 5), with taxol (lanes 3 and 6), and with wortmannin and taxol (lanes 4 and 7). Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser20 and p53 (DO-1).

Figure 6

Western blot analysis in IARC1663 and AT cell lines (AT11 and AT13) treated with DDP (Panel A) or taxol (Panel B). Extracts were taken at different times after treatment. Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser15, Ser20 and p53 (DO-1).

Figure 7

Panel A: Western blotting analysis in HCT-116 cells untreated (lane 1) and treated with DDP (lane 2) or taxol (lane 3). Extracts were obtained 24 hours after treatment. Panel B: Activation of ATR kinase activity by DDP (lane 2) but not by taxol (lane 3) 24 hours after treatment in HCT-116 cells (lane 1 reports untreated cells). ATP, was immunoprecipitated and kinase activity was assessed using PHAS substrate as described in the Materials and Methods section. The amounts of ATP, present in each reaction were determined by Western blot and are shown at the bottom of the figure.