Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome.

BACKGROUND: Oral administration is the most common way to deliver drugs to the systemic circulation or target organs. Orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver. In the early stages of drug development, it is important to predict first-pass metabolism accurately to select candidate drugs with high bioavailability. The Caco-2 cell line derived from colorectal cancer is widely used as an intestinal model to assess drug membrane permeability. However, because the expression of major drug-metabolizing enzymes, such as cytochrome P450 (CYP), is extremely low in Caco-2 cells, it is difficult to predict intestinal metabolism, which is a significant factor in predicting oral drug bioavailability. Previously, we constructed a mouse artificial chromosome vector carrying the CYP (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P450 oxidoreductase (POR) (4CYPs-MAC) genes and increased CYP expression and metabolic activity in HepG2 cells via transfer of this vector. RESULTS: In the current study, to improve the Caco-2 cell assay model by taking metabolism into account, we attempted to increase CYP expression by transferring the 4CYPs-MAC into Caco-2 cells. The Caco-2 cells carrying the 4CYPs-MAC showed higher CYP mRNA expression and activity. In addition, high metabolic activity, availability for permeation test, and the potential to assess drug-drug interactions were confirmed. CONCLUSIONS: The established Caco-2 cells with the 4CYPs-MAC are expected to enable more accurate prediction of the absorption and metabolism in the human intestine than parental Caco-2 cells. The mammalian artificial chromosome vector system would provide useful models for drug development.

Transchromosomic technology for genomically humanized animals.

"Genomically" humanized animals are invaluable tools for generating human disease models and for biomedical research. Humanized animal models have generally been developed via conventional transgenic technologies; however, conventional gene delivery vectors such as viruses, plasmids, bacterial artificial chromosomes, P1 phase-derived artificial chromosomes, and yeast artificial chromosomes have limitations for transgenic animal creation as their loading gene capacity is restricted, and the expression of transgenes is unstable. Transchromosomic (Tc) techniques using mammalian artificial chromosomes, including human chromosome fragments, human artificial chromosomes, and mouse artificial chromosomes, have overcome these limitations. These tools can carry multiple genes or Mb-sized genomic loci and their associated regulatory elements, which has facilitated the creation of more useful and complex transgenic models for human disease, drug development, and humanized animal research. This review describes the history of Tc animal development, the applications of Tc animals, and future prospects.

Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.

Dendrimer mediated transfer of engineered chromosomes.

Gene therapy encounters important problems such as insertional mutagenesis caused by the integration of viral vectors. These problems could be circumvented by the use of mammalian artificial chromosomes (MACs) that are unique and high capacity gene delivery tools. MACs were delivered into various target cell lines including stem cells by microcell-mediated chromosome transfer (MMCT), microinjection, and cationic lipid and dendrimer mediated transfers. MACs were also cleansed to more than 95% purity before transfer with an expensive technology. We present here a method by which MACs can be delivered into murine embryonic stem (ES) cells with a nonexpensive, less tedious, but still efficient way.

CENP-B controls centromere formation depending on the chromatin context.

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.

Artificial chromosome transgenesis in pigmentary research.

Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ-line of mice using artificial-chromosome-type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position-independent and copy-number-dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild-type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial-chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard-type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.