ABSTRACT
Amifostine is used as a cytoprotective agent in cancer treatments. Amifostine protects from apoptosis in some models and has been used as hematopoiesis stimulator in myeloid malignancies. As the apoptosis induced by many antitumoral agents is mediated by p53, we studied the effect of amifostine on p53-mediated apoptosis. We used human myeloid leukemia K562 and NB4 cells expressing the temperature-conditional p53-Val(135) mutant. Both cell lines undergo apoptosis at 32 degrees C due to the presence of p53 in wild-type conformation. We found that amifostine dramatically reduced apoptosis by p53 in both cell lines, as assessed by cell morphology, annexin V binding, fraction of sub-G(1) cells, and DNA laddering. To explore the mechanism responsible for this apoptosis protection, we tested the effect of amifostine on p53 transcriptional activity. We found that amifostine reduced p53-mediated transactivation of target promoters in NB4 and K562. Macroarray analysis confirmed that several p53 target genes as p21(Waf1), mdm2, gadd45, pig8, and pig3 were down-regulated at the mRNA level by amifostine in NB4 and K562. Also, c-myc was up-regulated by amifostine in K562 in the presence of p53, consistently with the impairment of p53-mediated apoptosis exerted by c-Myc in these cells. We conclude that amifostine impairs p53-dependent apoptosis of myeloid leukemia cells by reducing the activation of apoptosis-related genes. Our results open the possibility that amifostine could reduce the effectiveness of antitumoral treatments when it is dependent on active p53.
Subject(s)
Amifostine/pharmacology , Apoptosis/drug effects , Gene Expression Regulation, Leukemic , Leukemia, Myeloid/metabolism , Radiation-Protective Agents/pharmacology , Tumor Suppressor Protein p53/metabolism , Annexin A5/metabolism , Blotting, Northern , Caspases/metabolism , Fluorescent Antibody Technique , Gene Expression Profiling , Humans , Immunoblotting , Leukemia, Myeloid/genetics , Luciferases/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Protein Transport , Proto-Oncogene Proteins c-bcl-2/metabolism , Transcriptional Activation , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Suppressor Protein p53/genetics , bcl-X ProteinABSTRACT
BACKGROUND AND OBJECTIVES: The apoptotic Bcl-2 family member Hrk is transcriptionally silenced via DREAM in hematopoietic progenitor cell lines, and is specifically induced after growth factor withdrawal. Given that expression of Hrk is sufficient to induce apoptosis, we studied the expression of this apoptotic protein and its regulatory mechanism in human leukemia cells. DESIGN AND METHODS: K562 chronic myeloid leukemia cells were treated with STI571, a Bcr-Abl kinase inhibitor, and the Jurkat T-cell leukemia cell line was incubated with agonistic anti-Fas antibodies. Following treatment, we correlated the expression of Hrk protein with the DNA binding capacity of DREAM, and the induction of apoptosis. RESULTS: We show that treatment of K562 with STI571 blocks the binding of DREAM to the Hrk gene and allows the expression of Hrk, which correlates with the induction of apoptosis. Similarly, treatment of Jurkat cells with agonistic anti-Fas antibodies triggers the expression of Hrk through DREAM inactivation. Interestingly, inhibition of caspases, by culturing Jurkat cells in the presence of z-VAD-fmk, abrogates Fas-mediated hrk expression and apoptosis. Furthermore, in vitro analysis shows that active recombinant caspase-3 releases a fragment from the DREAM protein, suggesting that caspase-3 may be upstream of DREAM. INTERPRETATION AND CONCLUSIONS: These data suggest that apoptosis inducers as diverse as oncoprotein inhibitors and cell death receptor activators trigger Hrk expression via blockade of DREAM in leukemia cells, and this apoptotic pathway may be regulated, at least in some systems, by the proteolytic activity of caspase-3.
