Shoji Kitagawa

Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo.

To study cellular characteristics of human cementoblasts using a cellular model is important for understanding the mechanisms of homeostasis and regeneration of periodontal tissues. However, at present no immortalized human cementoblast cell line has been established due to limitation of the life span. In the present study, therefore, we attempted to establish human cementoblast-like cell lines by transfection with telomerase catalytic subunit hTERT gene. Two stable clones (HCEM-1 and -2) with high telomerase activity were obtained and they grew over 200 population doublings without significant growth retardation. The expression of mRNA for differentiation markers, type I collagen, alkaline phosphatase (ALP), runt-related transcription factor 2, osteocalcin, bone sialoprotein and cementum-derived protein was revealed in these clones by RT-PCR. Moreover, these cells showed high ALP activity and calcified nodule formation in vitro. Interestingly, HCEM-2 showed cementum like formation on the surface of hydroxyapatites granules by subcutaneous transplantation into immunodeficient mice with hydroxyapatite granules. Thus, we established human cementoblast-like cell lines. We suggest that HCEM cell lines can be useful cell models for investigating the characteristics of human cementoblasts.

Reduced expression of CD44 variant 9 is related to lymph node metastasis and poor survival in squamous cell carcinoma of tongue

Expression of CD44v9 was immunohistochemically studied in 120 biopsy specimens from primary squamous cell carcinoma (SCC) of the tongue and correlated with clinicopathological findings of the SCCs. The tumors were classified into three groups according to immunostaining pattern of CD44v9; 53 cases with distinct positivity in all cancer cells except for those in the central part of nests (Group 1, non-reduced group), 42 cases with reduced expression in peripheral cells of nests (Group 2, reduced group), and 25 cases with complete disappearance of the expression in one or more nests (Group 3, negative group). Nineteen of 25 (76%) tumors in Group 3 and 14 of 42 (33%) in Group 2 exhibited lymph node metastasis, compared with only 8 of 53 (15%) in Group 1. The average survival time in Groups 1, 2 and 3 was 4496+/-204, 3866+/-379 and 2719+/-359 days, respectively and became shorter with the reduction of CD44v9 expression. These results suggest that the down-regulation of CD44v9 in SCC of the tongue may relate to the detachment of tumor cells from primary lesions, establishment of lymph node metastasis and consequently the death of patients.

Synthetic ameloblastin peptide stimulates differentiation of human periodontal ligament cells.

OBJECTIVE This study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC). DESIGN We used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, we examined quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey-Lowry enzymological method, mineral nodule formation by Dahls method, and expression of mineralization-related genes by RT-PCR. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide. RESULTS At all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by an anti-ameloblastin antibody, which caused ALP levels to revert to their approximate levels in the untreated condition. At concentrations greater than 1ng/ml, the peptide promoted mineralized nodule formation of PDLC. And the peptide induced higher expressions of ALP and bone sialoprotein (BSP) than the control. CONCLUSION Our results show that the ameloblastin peptide upregulate ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide promotes the differentiation activity of PDLC.

Upregulated CD44v9 expression inhibits the invasion of oral squamous cell carcinoma cells.

Objectives: CD44 is one of the cell surface molecules that play an important role in cancer metastasis. In oral squamous cell carcinoma (OSCC), the downregulation of CD44v9 has been shown to be associated with tumor metastasis. We found that treatment with an anti-CD44v9 antibody enhanced the invasive potential of OSCC cell lines. Based on previous studies and our results, reduced expression of CD44v9 may be correlated with an increased invasive potential, i.e. the overexpression of CD44v9 may inhibit the invasive activity of OSCC cells. Methods: To study this correlation, we transfected the CD44v9 gene into HSC-4 cells with low CD44v9 expression and examined their invasive potential using the cell culture invasion assay and a three-dimensional culture invasion assay. Results: Overexpression of CD44v9 resulted in a downregulation of the invasive potential of HSC-4 cells. Moreover, CD44v9-transfected cells did not invade reorganized stroma, while parent HSC-4 cells exhibited diffuse invasion into reorganized stroma by the three-dimensional culture invasion assay. Conclusions: Overall, these findings suggest that the inhibition of the invasive potential by upregulation of CD44v9 expression may be due to enhanced cell-cell adhesion. In our opinion, the upregulation of CD44v9 may be a target for future cancer treatment.

Inhibition of CD44v9 upregulates the invasion ability of oral squamous cell carcinoma cells

The aim of the present study has been to determine the role of CD44v9 in the metastatic process of oral squamous cell carcinoma (OSCC). We have examined the expression intensity of CD44v9 in four OSCC cell lines, and using cell culture insert investigated the invasion ability of the cells expressing CD44v9 at higher levels (HSC-2, HSC-3), and the cells expressing this protein at lower levels (HSC-4, KB) with or without the treatment with an anti-CD44v9 antibody. In the highly expressing cells, the addition of anti-CD44v9 antibody enhanced their invasion ability, whereas it showed no effect on the invasion ability of the weakly expressing cells. These results suggest that the reduction of CD44v9 expression may weaken cell-to-cell adhesion in OSCC and make the tumor cells detach easily from their nests, resulting in the enhancement of their invasion ability. It may ultimately promote the establishment of a metastatic lesion.