ABSTRACT
Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21(WAF1/CIP1), a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21(WAF1/CIP1) in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21(WAF1/CIP1) expression in a dose-dependent manner (ED(50)=103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21(WAF1/CIP1) synergistically. Upregulation of p21(WAF1/CIP1) paralleled DAC-induced apoptosis (ED(50)=153 nM). Low doses of DAC induced gamma-H2AX expression (ED(50)=16.5 nM) and upregulated p21(WAF1/CIP1) in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-alpha or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21(WAF1/CIP1) upregulation, indicating that DAC upregulation of p21(WAF1/CIP1) was p53- and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21(WAF1/CIP1) in DNMT-independent manner via the DNA damage/ATM/p53 axis.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytosine/analogs & derivatives , DNA Damage , Gene Expression Regulation, Leukemic , Leukemia/drug therapy , Leukemia/metabolism , Caffeine/pharmacology , Cell Line, Tumor , CpG Islands , Cytosine/pharmacology , DNA Repair , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic , Humans , Models, Biological , Models, Genetic , Transcriptional ActivationABSTRACT
The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, alpha-crystallin-like protein in response to exposure to extreme temperatures, above 103 degrees C. The P. furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes. Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions. We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly. The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics. The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP. The amino terminal domain was not required for inter-species thermotolerance.
Subject(s)
Archaeal Proteins/chemistry , Heat-Shock Proteins/chemistry , Pyrococcus furiosus/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Base Sequence , DNA, Archaeal/genetics , Dimerization , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hot Temperature , Models, Molecular , Protein Structure, Quaternary , Protein Subunits , Pyrococcus furiosus/genetics , Pyrococcus furiosus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , alpha-Crystallins/chemistry , alpha-Crystallins/genetics , alpha-Crystallins/metabolismABSTRACT
To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif.
