Breast cancer -specific activation of topoisomerase IIalpha and its application in the tumor -targeting gene therapy

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we identified the breast cancer-specific activity of the topoisomerase IIα promoter. We further showed that cdk2 and cyclin A activate topoisomerase IIα promoter in a breast cancer-specific manner. An element containing an inverted CCAAT box (ICB) was shown to respond this signaling. When the ICB-harboring topoisomerase IIα minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent pro-apoptotic gene, was shown to selectively kill breast cancer cells in vitro and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs of CT90-BikDD-treated animals. Finally, we demonstrated that CT90-BikDD treatment potentially enhanced the sensitivity of breast cancer cells to chemotherapeutic agents, especially doxorubicin and taxol. The results indicate that liposomal CT90-BikDD is a novel and effective systemic breast cancer-targeting gene therapy, and its combination with chemotherapy may further improve the current adjuvant therapy for breast cancer. ^

The mechanism of action of a novel benzo[c]phenanthridine alkaloid, NK314 and the cellular responses

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that is currently in clinical trials as an antitumor compound, based on impressive activities in preclinical models. However, its mechanism of action is unknown. The present investigations were directed at determining the mechanism of action of this agent and cellular responses to NK314. My studies demonstrated that NK314 intercalated into DNA, trapped topoisomerase IIα in its cleavage complex intermediate, and inhibited the ability of topoisomerase IIα to relax super-coiled DNA. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIα, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. This indicates topoisomerase IIα is the target of NK314 in the cells. NK314 caused phosphorylation of the histone variant, H2AX, which is considered a marker of DNA double-strand breaks. DNA double-strand breaks were also evidenced by pulsed-field gel electrophoresis and visualized as chromosomal aberrations after cells were treated with NK314 and arrested in mitosis. Cell cycle checkpoints are activated following DNA damage. NK314 induced significant G2 cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G2 checkpoint pathway was activated in response to NK314, which can be abrogated by the Chk1 inhibitor UCN-01. Cell cycle checkpoint activation may be a defensive mechanism that provides time for DNA repair. DNA double-strand breaks are repaired either through ATM-mediated homologous recombination or DNA-PK-mediated non-homologous end-joining repair pathways. Clonogenic assays demonstrated a significant decrease of colony formation in both ATM deficient and DNA-PK deficient cells compared to ATM repleted and DNA-PK wild type cells respectively, indicating that both ATM and DNA-PK play important roles in the survival of the cells in response to NK314. The DNA-PK specific inhibitor NU7441 also significantly sensitized cells to NK314. In conclusion, the major mechanism of NK314 is to intercalate into DNA, trap and inhibit topoisomerase IIα, an action that leads to the generation of double-strand DNA breaks, which activate ATM and DNA-PK mediated DNA repair pathways and Chk1 mediated G2 checkpoint pathway. ^

Searching for the ideal DNA recognition code of covalent ligands

The combitiatorial approach restriction endonuclease protection selection and amplification REPSA was successfully used to determine ideal DNA interactions sites of covalent ligands. Unlike most other combinatorial methods, REPSA is based on inhibition of enzymatic cleavage by specific ligand-DNA complexes, which enables identification of binding sites of various ligands. However, the inherent nature of this technique posses a problem during selection of binding sites of covalent ligands. By modifying the technique according to the nature of the ligand, we demonstrate the flexibility of REPSA in identifying the preferred binding sites for monocovalent ligands, topoisomerase I and tallimustine, and the bicovalent ligand topoisomerase II. From among the preferred binding sites, we identified the consensus binding sequence of camptothecin induced topoisomerase I cleavage as ‘aGWT/Gc’, and tallimustine consensus sequences as ‘GTTCTA’ and ‘TTTTTTC’. We have shown for the first time that preferential binding of tallimustine occurs at sequences not previously reported. Furthermore, our data indicate that tallimustine is a novel DNA minor groove, guanine-specific alkylating agent. ^ Additionally, we have demonstrated in vivo that sequence-specific covalent DNA-binding small molecules have the ability to regulate transcription by inhibiting RNA polymerase II. Tallimustine, binding to its preferred sequences located in the 5′ untranslated region were an effective impediment for transcribing polymerase II. The ability of covalent binding small molecules to target predetermined DNA sequences located downstream of the promoter suggests a general approach for regulation of gene expression. ^