Recombinant toxins, hybrid proteins composed of a bacterial toxin and either a growth factor or a portion of a recombinant monoclonal antibody, have received significant attention in cancer therapeutics. This application proposes a unique study to develop and evaluate a recombinant toxin based on the similarities between a human digestive enzyme and a bacterial cytotoxin. Human DNase I has been used as a therapeutic agent for the treatment of wounds and ulcers, bronchitis, inflammatory conditions, herpes infection and most notably, cystic fibrosis. The cytolethal distending toxin (Cdt) is a genotoxin, produced by the periodontal pathogen Actinobacillus actinomycetemcomitans. The Cdt contains a subunit protein, CdtB, that has been shown to be a nuclease that is genetically and functionally related to mammalian type I DNases. Human DNase I has 100 to 1000 times the specific activity of CdtB. In contrast, CdtB has a built-in cell delivery system, a functional domain that targets the protein to the cell nucleus and a DNA-damaging activity that leads to the growth arrest or apoptosis of rapidly proliferating epithelial-like cells. Our hypothesis is that chimeras of human DNase I and bacterial CdtB can be genetically constructed to combine potent DNA- damaging activity, cell delivery and nuclear localization mechanisms. The objective of this study is to develop the efficacy of these chimeric proteins for use as the potential anti-cancer cell reagents.
The specific aims of the study are: (1) to construct human DNase l/CdtB chimeric genes and site-specific mutants of the chimeras and to isolate the mutant gene products. (2) To compare the in vitro biological activities of the chimeric and mutated chimeric gene products. (3) To evaluate the efficacy of the chimeric and mutant chimeric constructs in cell culture and transfection systems. Mutant chimeric proteins that demonstrate the ability to enter cells and induce cell cycle arrest will be evaluated using a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. If these studies are successful, future applications will be focused on the effects of these mutant chimeras on sqamous carcinoma cells from cancer patients and on tumors in appropriate mouse models. ? ? ?
| DiRienzo, Joseph M (2014) Uptake and processing of the cytolethal distending toxin by mammalian cells. Toxins (Basel) 6:3098-116 |
| Damek-Poprawa, M; Volgina, A; Korostoff, J et al. (2011) Targeted inhibition of CD133+ cells in oral cancer cell lines. J Dent Res 90:638-45 |
| DiRienzo, Joseph M; Cao, Linsen; Volgina, Alla et al. (2009) Functional and structural characterization of chimeras of a bacterial genotoxin and human type I DNAse. FEMS Microbiol Lett 291:222-31 |
| Xynogala, I; Volgina, A; DiRienzo, J M et al. (2009) Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis. Oral Microbiol Immunol 24:116-23 |
