Tetsuhiko Tachikawa

Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches.

Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.

Frequent silencing of a putative tumor suppressor gene melatonin receptor 1 A (MTNR1A) in oral squamous‐cell carcinoma

Array‐based comparative genomic hybridization (array‐CGH) has good potential for the high‐throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous‐cell carcinoma (OSCC) cell lines for genome‐wide copy‐number aberrations by array‐CGH using our in‐house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A (MTNR1A) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene‐silenced OSCC cells without its homozygous loss after treatment with 5‐aza‐2′‐deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor‐suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis. (Cancer Sci 2008; 99: 1390–1400)

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27KIP1 and p15INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27KIP1 and p15INK4B. These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27KIP1 and p15INK4B.

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air‐liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three‐dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV‐transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

K-ras mutation may promote carcinogenesis of endometriosis leading to ovarian clear cell carcinoma

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.

Malignant transformation of endometriosis and genetic alterations of K-ras and microsatellite instability

Objectives: To clarify the role of specific genetic alterations in the multi‐step process of malignant transformation of endometriosis. Methods: In cases of ovarian endometrioid carcinoma, we separated regions of normal endometriosis, atypical endometriosis and ovarian endometrioid carcinoma by laser microdissection, and examined K‐ras mutation and microsatellite instability in each separated tissue sample. Results: We detected K‐ras mutation and microsatellite instability in endometrioid carcinoma tissue, but not in normal or atypical endometriosis bordering the cancerous region. Conclusions: The present findings suggest that K‐ras mutation and microsatellite instability are associated with malignant transformation from atypical endometriosis to ovarian endometrioid carcinoma.

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development.

Effects of mastication on mandibular growth evaluated by microcomputed tomography.

It is well known that mastication has a significant influence on mandibular growth and development, but the mechanism behind this effect has not yet been clarified. Furthermore, no studies have examined the effects of changes in mastication on the three-dimensional (3D) morphometry of the mandible. The aim of the present study was to investigate the influences of changes in mastication on mandibular growth and morphology. Twenty-five 3-week-old (at the time of weaning) imprinting control region mice were randomly divided into three groups: mice fed a hard diet (HD), mice fed a soft diet (SD), and mice alternately fed hard and soft diets (HSDs) every week for 4 weeks. The morphometry of the mandible was analysed using 3D microcomputed tomography (muCT). Statistical analysis was undertaken using a t-test. muCT analysis showed that the condylar width was significantly greater in the HD group than in the SD group after 1 week. After 4 weeks, mandibular length was significantly longer and ramus height was greater in the HSD group than in the other two groups. Bone volume was significantly less in the SD group than in the other two groups after 4 weeks. These findings suggest that changes in mastication markedly affect mandibular condylar cartilage growth and mandibular morphology. It is considered that dietary education at an early age is important in order to prevent disruption of the development of the mandible.

Histomorphometric examination of healing around hydroxylapatite implants in 60Co-irradiated bone

The purpose of this study was to histomorphometrically evaluate the use of hydroxylapatite (HA) implants in 60Co-irradiated bone. HA implants were installed in rabbit mandibles 3 months, 6 months, and 12 months after 15-Gy irradiation. Nonirradiated rabbits served as controls. The rabbits were killed 7, 14, 30, 60, and 90 days after the HA implantation. The histologic features of the healing process were examined and histomorphometric measurements were made to quantify the percentage of HA-bone contact and trabecular bone in the medullary cavity. In the irradiated groups, although HA-bone contact was observed later than that in the controls, recovery increased with time after irradiation and the rate of HA-bone contact bone-contacting implant surface ratio; BCSR exceeded 90% in all groups examined before 90 days. In the radiated groups, the average trabecular bone-specific volume was lower than that in the controls and began to decrease before BCSR exceeded 90%. Based on the present data, as well as data from the literature, it is suggested that the success rate of HA implants in irradiated bone increases with the interval after radiotherapy. It is also recommended that HA implants in irradiated bone be installed so that bearing by the cortical bone is increased.

Association of genetic, psychological and behavioral factors with sleep bruxism in a Japanese population.

Sleep bruxism is a sleep‐related movement disorder that can be responsible for various pains and dysfunctions in the orofacial region. The aim of the current case–control association study was to investigate the association of genetic, psychological and behavioral factors with sleep bruxism in a Japanese population. Non‐related participants were recruited and divided into either a sleep bruxism group (n = 66) or control group (n = 48) by clinical diagnoses and 3‐night masseter electromyographic recordings by means of a portable miniature device. The Epworth Sleepiness Scale, Temperament and Character Inventory, NEO‐Five Factor Inventory and custom‐made questionnaires that asked about familial aggregation, alcohol intake, caffeine intake, cigarette smoking, past stressful life events, daytime tooth‐contacting habit, temporomandibular disorder, daily headache, snoring, apnea/hypopnea symptoms, leg‐restlessness symptoms and nocturnal‐myoclonus symptoms were administered. In addition, 13 polymorphisms in four genes related to serotonergic neurotransmission (SLC6A4, HTR1A, HTR2A and HTR2C) were genotyped. These factors were compared between case (sleep bruxism) and control groups in order to select potential predictors of sleep‐bruxism status. The statistical procedure selected five predictors: Epworth Sleepiness Scale, leg‐restlessness symptoms, rs6313 genotypes, rs2770304 genotypes and rs4941573 genotypes. A multivariate stepwise logistic regression analysis between the selected predictors and sleep‐bruxism status was then conducted. This analysis revealed that only the C allele carrier of HTR2A single nucleotide polymorphism rs6313 (102C>T) was associated significantly with an increased risk of sleep bruxism (odds ratio = 4.250, 95% confidence interval: 1.599–11.297, P = 0.004).This finding suggests a possible genetic contribution to the etiology of sleep bruxism.