Tadateru Aida

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.

Comparison in gene expression of secretory human endometrium using laser microdissection.

BackgroundThe endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique.MethodsThe epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR.ResultsIn this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR.ConclusionsSome of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium.

Expression of Protein Kinases C βI, βII, and VEGF during the Differentiation of Enamel Epithelium in Tooth Development

Protein kinase C (PKC) is an important molecule involved in various cell function, and mediates induced secretion of vascular endothelial growth factor (VEGF). It is hypothesized that PKC and VEGF may be associated with tooth development. Using the laser microdissection method and real-time reverse-transcription-polymerase chain-reaction (RT-PCR), we investigated the expression of PKC βI and βII, VEGF, and amelogenin (used as a marker of differentiation to ameloblasts) in the inner and outer enamel epithelia, stellate reticulum, and dental papilla in each stage of the dental germ. We found that the expression levels of PKC βI and βII were increased in the inner enamel epithelium during the early bell stage. In addition, the increased expression levels of PKC βI and βII were accompanied by increased VEGF expression. These results indicate that PKC βI, βII, and VEGF are closely associated with the differentiation of the inner enamel epithelium to ameloblasts.

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.

Gene expression profiling of oral squamous cell carcinoma using laser microdissection and cDNA microarray.

Cancer diagnosis and therapy are performed on the basis of clinical stage and clinicopathological findings; however, sensitivity to therapy and prognosis may not always be the same even when considering similar cancers because it is difficult to recognize adequate biological characteristics of a cancer when determining cancer therapy. To enable personalized medicine for cancer diagnosis and therapy, which may solve this problem, we used laser microdissection and cDNA microarrays to study the gene expression profile of oral squamous cell carcinoma. Moreover, to establish an objective evaluation with this system, we examined which type of gene expression profile corresponded to the biological characteristics of this cancer. We identified several genes that were up- or downregulated in the majority of cases and clarified genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases. It was suspected that the genes that were commonly up- or downregulated in the majority of cases were important for histogenesis and acquisition of invasion and proliferation capability and that the genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases played a large role in cancer metastasis. Using the expression profile of these genes, it may be possible to evaluate cellular state and metastatic potential and use them as tumor markers. Alternately, we showed averaged gene expression profiles in cases with or without metastasis; this may reveal a profile that could evaluate metastatic potential, which is an important element in the biological characteristics of cancer. In conclusion, our system using laser microdissection and cDNA microarray may contribute to cancer diagnosis and therapy and improvement in the quality of life of cancer patients.

cDNA microarray analysis after laser microdissection in proliferating islets of partially pancreatectomized mice

With islet transplantation having grown in popularity since the introduction of the Edmonton protocol, how to secure an unlimited source of islets has become an urgent problem. To resolve this problem, techniques to induce or proliferate islets are urgently required. To achieve this goal, gene expression analysis using a cDNA microarray in islets of partially pancreatectomized mice, in which the remaining islets regenerate and proliferate with insulin secretion and glucose responsiveness, provides us with valuable information. However, those experiments have two critical problems: first, how to selectively collect the regenerating or proliferating islets, and second, the shortage of total RNA extracted from one islet for a microarray analysis. A useful system was thus designed which combined laser microdissection, cDNA amplification by SMART PCR, which can maintain the relative expression profile of transcripts throughout reactions, and a cDNA microarray. Furthermore, this system is expected to contribute to future studies regarding not only islet regeneration but also the function of the islet itself, and this system may also be applicable to many other types of endocrine tissue. In this review, the details of this system are presented and discussed.

Multiple granulomatous inflammation in the minor salivary glands: A proposed new entity, allergic granulomatous sialadenitis

We report a patient who presented with multiple small submucosal nodules with granulomatous inflammation in the minor salivary glands of the oral cavity. A 43‐year‐old woman presented with a 1‐week history of multiple small submucosal nodules in her oral cavity after having taken medicine for abdominal pain. The patient did not have a history of fever, rectal bleeding, skin lesions or arthritis, but did have a history of drug allergy and bronchial asthma. Histopathological examination of the submucosal nodules showed sialadenitis with marked infiltration of lymphocytes, eosinophilic cells, macrophages and Langhans‐type or foreign‐body‐type multinucleate giant cells. The macrophages tended to be aggregated and appeared to have caused immature granuloma formation without caseous necrosis. Degranulated eosinophilic cells were numerous. Sarcoidosis, Crohns disease, tuberculosis and atypical mycobacterial infection were not identified by medical examination. Three weeks after discontinuing the medication the patient was seen again at a follow‐up visit. Multiple submucosal small nodules and other symptoms were not evident at that time. This case report may represent a new entity of salivary gland disease that we tentatively refer to as ‘allergic granulomatous sialadenitis’.