Imatinib-induced apoptosis: a possible link to topoisomerase enzyme inhibition.

WHAT IS KNOWN AND OBJECTIVE: Imatinib is a specific BCR/ABL inhibitor, commonly used for the treatment of chronic myeloid leukaemia (CML), a hematological malignancy resulting from a chromosomal translocation that generates the BCR/ABL fusion protein. Recent studies showed that the imatinib has cytotoxic and apoptotic effects on many BCR/ABL-negative cancers. Numerous compounds with cytotoxic potential exert their functions by interfering with the DNA topoisomerase. In this study, we examined the effects of imatinib on tumour cell-killing in relation to DNA topoisomerase enzyme inhibition. METHODS: We determined the cytotoxicity by cell proliferation assay (XTT; tetrazolium hydroxide), using the human K562 CML cells, and loss of mitochondrial membrane potential by monitoring the changes in caspase-3 enzyme activity. Type I and II topoisomerase activities were measured by supercoiled plasmid relaxation and minicircle DNA decatenation assays respectively. RESULTS AND DISCUSSION: Imatinib-induced apoptosis and inhibited cell proliferation in a dose-dependent manner. We also found that the imatinib was effective in both type I and type II topoisomerase reactions to a varying degree between 94% and 7% for the concentration range of 1 mm-0.02 mm in a dose-dependent manner. WHAT IS NEW AND CONCLUSION: Our results suggest that the inhibition of topoisomerases may be a significant factor in imatinib-induced apoptosis in CML.

Telomeric restriction analysis of vascular smooth muscle cells following balloon angioplasty in rabbits

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Intimal hyperplasia due to smooth muscle cell proliferation and migration has been reported to be responsible for the pathogenesis of atherosclerosis and restenosis, manifested following balloon angioplasty. In this study, we employed the balloon angioplasty model to study telomere length regulation in proliferating vascular smooth muscle cells. Our results showed that balloon angioplasty in iliac arteries resulted in intimal hyperplasia due to proliferation of the smooth muscle cells and small size telomeric restrictional fragments were evident in injured arteries (AU)

Telomeric restriction analysis of vascular smooth muscle cells following balloon angioplasty in rabbits.

Intimal hyperplasia due to smooth muscle cell proliferation and migration has been reported to be responsible for the pathogenesis of atherosclerosis and restenosis, manifested following balloon angioplasty. In this study, we employed the balloon angioplasty model to study telomere length regulation in proliferating vascular smooth muscle cells. Our results showed that balloon angioplasty in iliac arteries resulted in intimal hyperplasia due to proliferation of the smooth muscle cells and small size telomeric restrictional fragments were evident in injured arteries.

RT-PCR-based cytochrome P450 expression profile of oral tissue samples.

OBJECTIVE: To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. METHOD: Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. RESULTS: Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. CONCLUSIONS: These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.

DNA topoisomerases as targets for anticancer drugs.

DNA topoisomerases are essential enzymes that regulate the conformational changes in DNA topology by catalysing the concerted breakage and rejoining of DNA strands during normal cellular growth. Over the past few years there has been considerable pharmacological interest in these enzymes because inhibitors of DNA topoisomerases represent a major class of anticancer drugs. This review highlights topoisomerase-targeting drugs that have shown promising anticancer activities. The mechanisms by which those drugs interfere with the catalytic cycles of type I and type II DNA topoisomerases and the factors involved in the development of resistance to these drugs are discussed.

The yeast two-hybrid system and its pharmaceutical significance.

The detected phenotypes in many diseases are caused from dysfunction in protein-protein, protein-DNA and receptor-ligand interactions. Therefore, determination of these molecular interactions followed by designing or screening the compounds to target these interactions provides a significant challenge in drug development. This review aims to highlight the yeast two-hybrid system in determination of protein-protein interactions and its possible outcomes in pharmaceutical research. The variations of the basic methodology as one- and three-hybrid systems are also discussed in relation to their potential pharmaceutical applications.

Densitometric quantification of DNA topoisomers in ethidium bromide-stained agarose gels and chemiluminescence-detected X ray films.

This study investigates the contribution of deformational strain imposed by topological interconversions of DNA in ethidium bromide-binding on agarose gels. Closed-circular plasmid DNAs were nicked using UV exposure and the DNA bands were quantified by densitometry. The results show that the closed circular DNA binds the same amount of the dye as its nicked counterpart. The relationship between the band intensity on X-ray films of chemiluminescence-detected Southern blots and DNA concentration was shown to be linear.

An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors.

This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.

The promyelocytic leukemia protein PML interacts with the proline-rich homeodomain protein PRH: a RING may link hematopoiesis and growth control.

Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML is disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis.

Mammalian mitochondrial DNA topoisomerase I preferentially relaxes supercoils in plasmids containing specific mitochondrial DNA sequences.

Selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors and used as substrates in a kinetic analysis of the highly purified bovine mitochondrial type I topoisomerase. Recombinant plasmids containing the bovine mtDNA heavy and light strand origins of replication (pZT-Hori and pZT-Lori, respectively), a major transcription termination region (pZT-Term) and a portion of cytochrome b gene (pZT-Cytb) were prepared. Southern hybridization using probes specific for either control or mtDNA-containing plasmid indicated a relative preference by the mitochondrial topoisomerase I to relax supercoils in pZT-Hori and pZT-Term. Quantitative determination of kinetic parameters derived from double-reciprocal Lineweaver-Burk plots showed that recombinant plasmids containing the heavy and light strand origins and the transcription termination region were preferentially relaxed by the mitochondrial enzyme with Km values 2.3- to 3.3-fold lower than controls. The Km values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 +/- 0.9 microM, 25.2 +/- 1.0 microM and 17.0 +/- 0.8 microM, respectively, while those for control plasmids were 57.5 +/- 2.1 microM and 56.3 +/- 2.3 microM. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km = 53.4 +/- 2.0 microM vs. 56.3 +/- 2.3 microM, respectively) indicating that mitochondrial topoisomerase I preferentially interacts with certain mtDNA sequences but not others. Identical experiments with the purified nuclear enzyme did not differentiate between control or mtDNA containing plasmids.