ABSTRACT
BACKGROUND: Interruption of the role of p53s as a tumour suppressor by MDM2 may be one of the mechanisms by which cancer cells evade current therapy. Blocking the inhibition of wild-type p53 by MDM2 in cancer cells should reactivate p53's tumour suppressor functions and enhance current cancer treatments. MI-63 is a novel non-peptide small molecule that has shown strong binding affinity (K(i)=3 nM) for MDM2; however, its effects on paediatric cancer cells and the specific mechanism of tumour suppressor reactivation have not been evaluated. METHODS: Rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, expresses either wild-type or mutant p53 protein. We examined the inhibitory effects of MI-63 in embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines expressing wild-type or mutated p53. RESULTS: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53. In contrast, RH30 and RD2 cells expressing p53 mutants are resistant to MI-63 treatment. An increased expression of p53, p21(WAF1), and Bax protein was observed after treatment with MI-63 in RMS cells with wild-type p53, and apoptosis was confirmed by cleaved PARP and caspase-3 expression. However, RD2 and RH30 RMS cells, as well as human normal skeletal muscle cells, showed a minimal increase in p53 signalling and no induction of cleaved PARP and caspase-3. MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability. Further, synergy was observed when MI-63 was used in combination with doxorubicin. CONCLUSION: These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Indoles/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma, Embryonal/pathology , Spiro Compounds/pharmacology , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/chemistry , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Screening Assays, Antitumor , Humans , Imidazoles/pharmacology , Indoles/chemistry , Molecular Weight , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/metabolism , Rhabdomyosarcoma, Alveolar/drug therapy , Rhabdomyosarcoma, Alveolar/metabolism , Rhabdomyosarcoma, Embryonal/drug therapy , Rhabdomyosarcoma, Embryonal/metabolism , Signal Transduction/drug effects , Spiro Compounds/chemistry , Structure-Activity Relationship , Tumor Suppressor Protein p53/drug effectsABSTRACT
Uracil-DNA glycosylase (UNG) is the primary enzyme responsible for removing uracil residues from DNA. Increasing evidence suggests that UNG may be a potential target for the development of novel antiviral and/or anticancer agents. To determine whether the uracil-DNA glycosylase inhibitor protein (UGI) could be used to specifically target UNGs intracellularly, we developed a construct that expresses UGI as a fusion protein with the TAT-protein transduction domain and described a novel method for the purification of recombinant TAT-UGI. Treatment of several cell types with TAT-UGI resulted in a dose- and time-dependent decrease in UNG activity. A somewhat surprising effect of TAT-UGI treatment was the decrease in cell proliferation, but not in cell viability. The results of this study support the premise that UNG can be used as a potential therapeutic target and also demonstrate that protein transduction can be used to modulate UNG activity.
Subject(s)
Apoptosis , DNA Glycosylases/deficiency , Viral Proteins/metabolism , Cell Proliferation , DNA Glycosylases/genetics , Enzyme Activation , Gene Transfer Techniques , HT29 Cells , HeLa Cells , Humans , Uracil-DNA Glycosidase , Viral Proteins/geneticsABSTRACT
Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is responsible for maintaining low intracellular levels of dUTP, thus preventing the incorporation of dUTP into DNA. A 21 bp double-stranded RNA molecule (siRNAdUT3) targeted against motif 3 of human dUTPase resulted in a time- and dose-dependent decrease in dUTPase activity in transfected cells. dUTPase activity was reduced approximately 95+/-5% in all cell lines tested 48 h after transfection with 2 microg siRNAdUT3 and it was maintained at this decreased level for at least 72 h. Down-regulation of dUTPase resulted in a significant increase in intracellular dUTP and a decreased proliferation of the transfected cells. Therefore, we conclude that dUTPase activity/expression can be down-regulated using siRNA specifically targeted to dUTPase mRNA and that this approach can be used to elucidate the role of dUTPase in DNA metabolism, as well as, to determine whether dUTPase is a valid target for drug development.
Subject(s)
Gene Expression Regulation , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , RNA, Small Interfering/metabolism , Cell Line, Tumor , Cell Proliferation , Humans , RNA, Small Interfering/genetics , TransfectionABSTRACT
A small interfering double stranded RNA molecule (siRNA, 21 bp) corresponding to a portion (nucleotides 337 to 357) of domain 3 of the human dUTPase was synthesized and used to determine whether it could down-regulate dUTPase activity in human cells. Transfection of the siRNA into HeLa and HT29 cells resulted in a 56 +/- 3.6% decrease in dUTPase activity, while transfection of SW620 cells resulted in a 27 +/- 6% decrease in dUTPase activity when compared to non-treated controls.
Subject(s)
Down-Regulation , Pyrophosphatases/biosynthesis , RNA, Small Interfering , Amino Acid Motifs , HT29 Cells , HeLa Cells , Humans , RNA, Messenger/metabolism , Time Factors , TransfectionABSTRACT
The human herpesviruses are a well characterized group of viruses that are responsible for a wide spectrum of human diseases. Included in this group of pathogens are the alphaherpesviruses (herpes simplex types 1 and 2 and varicella-zoster virus), the betaherpesviruses (cytomegalovirus, human herpesvirus types 6 and 7) and the gammaherpesviruses (Epstein-Barr virus and human herpesvirus 8). An important feature of these viruses is that they cause latent infections that can be reactivated to cause disease. The herpesviruses encode for a large number of structural and non-structural proteins, and several of the non-structural proteins, such as thymidine kinase, DNA polymerase, and ribonucleotide reductase, have been utilized as targets for the development of anti-herpesvirus agents. Another herpesvirus encoded enzyme that has received little attention as a potential target for the development of specific anti-herpesvirus agents is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Furthermore, little is known concerning the role of the herpesviruses' encoded dUTPases in virus replication and in modulating the chemotherapeutic efficiency of other anti-herpes agents. Because of recent advances in molecular virology and biochemistry, it is now possible to rationally develop "designer" drugs based upon the structural/functional interaction of the drug with a specific viral protein. The purpose of this review is to describe previous studies demonstrating the potential use of the herpesvirus encoded dUTPase as a drug target, to describe problems associated with using the dUTPase as a target and to discuss new approaches that can be used.
