Differences in mutant p53 protein stability and functional activity in teniposide-sensitive and -resistant human leukemic CEM cells.
We examined p53 protein stability and DNA damage-induced p53-dependent responses in a human leukemic CEM cell line and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5 ( approximately 40 and 400-fold resistant to teniposide, respectively). Although all cell lines contain the same p53 mutations at codons 175 (Arg-->His) and 248 (Arg-->Gln), the constitutive levels of p53 were progressively increased with the resistance of the cells to teniposide. By pulse-chase experiments, we found that the half-lives of mutant p53 protein were approximately 12, 17, and >30 h in CEM, CEM/VM-1, and CEM/VM-1-5 cells, respectively. The prolonged half-lives of p53 in these cells is consistent with the fact that the protein harbors the indicated mutations. Of note, however, is the fact that the increased p53 protein half-lives in the two drug-resistant cell lines corresponds to a proportional decrease in MDM2 protein levels but an increase in p53-MDM2 binding interactions. This suggests that MDM2-mediated p53 degradation may be altered in our leukemic cell lines. The DNA damage-induced p53 response is fully functional in the drug-sensitive CEM cells containing a mutant p53, but this pathway is attenuated in the drug-resistant cells. Specifically, while the mutant p53 was phosphorylated at serine-15 in response to ionizing radiation in all these cell lines, mutant p53 induction in response to teniposide or ionizing radiation and induction of the p53-target genes, p21 and GADD45 only occurred in the drug-sensitive CEM cells. As assessed by MTT cytotoxicity assay, CEM cells were also significantly more sensitive to ionizing radiation, compared to the drug-resistant cell lines, and this correlated with p53 induction. Collectively, these results suggest that changes in constitutive mutant p53 protein levels, p53-MDM2 binding interactions, and altered regulation of the DNA damage-inducible p53-dependent pathway may play a role in drug- and radiation-responsiveness in these cells.
Morgan
S E
SE
Division of Molecular Pharmacology, Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, IL 60607, USA.
Kim
R
R
Wang
P C
PC
Bhat
U G
UG
Kusumoto
H
H
Lu
T
T
Beck
W T
WT
D000970:Antineoplastic Agents / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D004249:DNA Damage / Q000235:genetics*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D013713:Teniposide / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051028:bcl-2-Associated X Protein; D000097642:GADD45 Proteins
D000970:Antineoplastic Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051736:Proto-Oncogene Proteins c-mdm2; D013713:Teniposide; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human; C497206:MDM2 protein, human