Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^