Tatsushi Matsumura

Expression of cytokeratin 14 in ameloblast-lineage cells of the developing tooth of rat, both in vivo and in vitro

In the search for a cell marker useful for studying tooth development, immunohistochemical studies using antibodies against cytokeratin 14 (K14), c-Met/hepatocyte growth factor receptor and amelogenin were carried out in the developing tooth of the newborn rat and in primary cultured cells of the ameloblast lineage, including inner enamel epithelium cells, preameloblasts and ameloblasts, prepared from the mandibular incisors of postnatal 7-day-old rats. The appearance of K14 was cell- and differentiation-stage specific, i.e. there was a weak expression signal within inner enamel epithelial cells that were in the proliferating stage, and there were strong signals within preameloblasts and ameloblasts that were in the post-proliferating and amelogenesis stages, respectively. In the culture system, c-Met appeared in all cells, whereas K14 and amelogenin appeared mainly in clustered cells that were considered to be in the post-proliferating stage. K14 was detected earlier than amelogenin, and it was also confirmed by immunofluorostaining that c-Met, K14 and amelogenin were coexpressed in ameloblasts. These findings indicate that K14 is a good new marker for ameloblast-lineage cells during rat tooth development both in vivo and in vitro.

Fibronectin accelerates the growth and differentiation of ameloblast lineage cells in vitro.

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial–mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.

Comparative analysis of G2 arrest after irradiation with 75 keV carbon-ion beams and 137Cs γ-rays in a human lymphoblastoid cell line

Heavy-ion beams are more effective than gamma-rays in causing G2 arrest. In this study, we investigated the expression of Wee1 and Cdc2 protein levels in order to analyze the G2 arrest caused by carbon-ion beam irradiation. Human lymphoblastoid TK6 cells were exposed to a 75 keV carbon-ion beam or 137Cs gamma-rays. Although the levels of Wee1 and Cdc2 protein were increased after exposure to either beam, Wee1 protein levels were influenced more by carbon-ion beam irradiation than by gamma-rays. To the contrary, Cdc2 protein levels were increased more by gamma-rays than by carbon-ion beams. These findings suggest that the G2 arrest produced by heavy-ion beams, such as the carbon-ion irradiation used in this study, might be associated with the overexpression of the Wee1 protein and of Cdc2 phosphorylation regulated by Wee1. Together, these events may act to prolong the length of G2 arrest.