In vivo stimulation by antitumor drugs of the topoisomerase II induced cleavage sites in c-myc protooncogene

Several antitumor drugs including DNA intercalative and non intercalative agents induce in vitro and in vivo double-stranded DNA breaks by stabilization of a topoisomerase II-DNA complex. In order to locate cleavage sites in an actively transcribed oncogene, N417 cells, originating from a human small cell lung carcinoma and containing 45-50 copies of c-myc oncogene, were treated with mAMSA, 9 hydroxyellipticine and VM 26. The presence of DNA lesions in c-myc was investigated by Southern blot hybridization with a human c-myc probe. In addition to normal bands, DNA patterns of drug treated-cells revealed the presence of new bands most likely corresponding to topoisomerase II-mediated cleavage as these bands were not found in untreated control DNA and in DNA treated with oAMSA, a biologically inactive stereoisomer of mAMSA. Major cleavage sites induced by drugs in the N417 cell c-myc locus were located in the 5' end of the c-myc exon 1 closely to some DNAse I hypersensitive sites which are assumed to reflect an activity of the gene. Therefore our data suggest that TopoII-mediated drug activity correlates with gene activity.     

Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II.

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.     

Effect of extracellular phosphatidylinositol on c-myc gene-expressed human renal cancer cell line.

Effect of exogenously added soybean phosphatidylinositol on c-myc gene expressed and unexpressed human cancer cell lines was investigated. When phosphatidylinositol liposomes were introduced into culture media, viability of c-myc unexpressed cells was reduced, while that of c-myc expressed cells was not. Death of c-myc unexpressed cells by phosphatidylinositol liposomes was found to be caused by abnormally accumulated intracellular Ca2+, and it seemed to be related to reduction of protein kinase C activity.     

DNA binding properties of 9-substituted harmine derivatives

The beta-carboline alkaloids have been characterized as a group of potential antitumor agents. The underlying mechanisms of harmine and its derivatives were investigated by DNA binding assay and Topoisomerase (Topo) inhibition assay. Meanwhile, the DNA photocleavage potential of these compounds and their cytotoxicity were also examined by DNA photocleavage assay and cytotoxicity assay in vitro. Harmine and its derivatives exhibited remarkable DNA intercalation capacity and significant Topo I inhibition activity but no effect with Topo II. Introducing an appropriate substituent into position-9 of beta-carboline nucleus enhanced the affinity of the drug to DNA resulting in remarkable Topo I inhibition effects. These results suggested that the ability of these compounds to act as intercalating agents and Topo I inhibitors was related to the antitumor activity. Moreover, these data showing a correlation between cytotoxicity and Topo I inhibition or DNA binding capacity are very important as they strongly suggested that the Topo I-mediated DNA cleavage assay and DNA binding assay could be used as a guide to design and develop superior analogues for antitumor activities.     

Differential expression of DNA topoisomerase I gene between CPT-11 acquired- and native-resistant human pancreatic tumor cell lines: detected by RNA/PCR-based quantitation assay.

RNA/PCR quantitation method was developed to determine DNA Topoisomerase I(Topo I)-specific mRNA in order to study its gene expression in CPT-11 sensitive, acquired- or native-resistant human pancreatic tumor cell lines. The results were supported by Northern blotting and Western blotting analyses. Acquired-resistant cells have shown decreased levels of Topo I mRNA, compared with their parental cells. On the contrary, in the wild type cells no correlation was shown between sensitivity and gene expression. On the other, specific Topo I activity of the native resistant cell lines was fairly lower than that of sensitive cell lines, suggesting that immunoreactive Topo I protein contains low levels of active form enzyme which could be targets of CPT-11 in these native-resistant ones. Finally, the different mechanisms might be operative between acquired- and native-resistant tumor cells.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.     

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.     

Degradation of DNA topoisomerase I by a novel trypsin-like serine protease in proliferating human T lymphocytes

DNA topoisomerase I (Topo I) contributes to various important biological functions, and its activity is therefore likely regulated in response to different physiological conditions. Increases in both the synthesis and degradation of Topo I were previously shown to accompany phytohemagglutinin stimulation of proliferation in human peripheral T lymphocytes. The mechanism of this degradation of Topo I has now been investigated with both in vivo and in vitro assays. The activity of a nuclear protease that specifically degrades Topo I was induced in proliferating T lymphocytes. The full-length Topo I protein (100 kDa) was sequentially degraded to 97- and 82-kDa fragments both in vivo and in vitro. The initial site of proteolytic cleavage was mapped to the NH(2)-terminal region of the enzyme. The degradation of Topo I in vitro was inhibited by aprotinin or soybean trypsin inhibitor, suggesting that the enzyme responsible is a trypsin-like serine protease. Furthermore, Topo I degradation by this protease was Mg(2+)-dependent. The Topo I-specific protease activity induced during T lymphocytes proliferation was not detected in Jurkat (human T cell leukemia) cells and various other tested human cancer cell lines, possibly explaining why the abundance of Topo I is increased in tumor cells.     

From GC-rich DNA binding to the repression of survivin gene for quercetin nickel (II) complex: implications for cancer therapy

The DNA binding and cleavage properties of quercetin nickel (II) complex have been studied, but little attention has been devoted to the relationship between antitumor activity of this complex and DNA-binding properties. In the present study, we report that quercetin nickel (II) complex showed significant cytotoxicity against three tumor cell lines (HepG2, SMMC7721 and A549). Hoechst33258 and AO/EB staining showed HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin nickel (II) complex. We also demonstrate that the levels of survivin and bcl-2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin nickel (II) complex by immunocytochemistry analysis. The relative activity of caspase-3 and caspase-9 increased significantly after treatment with the complex. Furthermore, fluorescence measurements and molecular modeling were performed to learn that the complex could be preferentially bound to DNA in GC region. These results imply that quercetin nickel (II) complex may intercalate into the GC-rich core promoter region of survivin, down-regulating survivin gene expression and promoting tumor cells apoptosis. So our results suggest that antitumor activity of quercetin nickel (II) complex might be related to its intercalation into DNA and DNA-binding selectivity, and that the complex may be a promising agent for cancer therapy.     

Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity

Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.