Correlation of invasion and metastasis of cancer cells, and expression of the RAD21 gene in oral squamous cell carcinoma.

Although RAD21 is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth, its role in cancer has been unclear. In this study, the relevance of RAD21 gene expression to the invasion and metastasis of oral squamous cell carcinoma was clarified using laser microdissection and real-time polymerase chain reaction (PCR). Using two different metastatic potential oral squamous cells [high-metastatic-potential squamous cell carcinoma cells (SAS-Ly) and low-metastatic-potential squamous cell carcinoma cells (SAS)], the relation of RAD21 gene expression to apoptosis, invasion, and metastasis was examined. The results showed that RAD21 gene expression was significantly decreased in oral squamous cell carcinoma when it expressed the INFbeta and INFgamma invasion patterns in comparison with the INFalpha invasion pattern (p<0.01). In addition, in comparison with SAS cells, SAS-Ly cells indicated tolerance to cell death induced by an apoptosis induction reagent, while the expression level of the RAD21 gene in SAS cells was increased by the apoptosis induction reagent. However, in SAS-Ly cells, the reagent induced no significant difference. Our findings indicate that the RAD21 gene was closely related to the invasion and metastasis of cancer cells.

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.

Analysis by comparative genomic hybridization of gastric cancer with peritoneal dissemination and/or positive peritoneal cytology.

Peritoneal metastasis is an important prognostic factor in cases of gastric cancer. Although studies on comparative genomic hybridization (CGH) in gastric cancer have been reported, there are few reports on the peritoneal metastasis (P) and peritoneal cytology (CY) factors in this cancer. In this study, we analyzed the chromosomal changes in the primary tumor with a combination of laser microdissection analysis and CGH in an attempt to detect the unknown abnormal chromosomal regions. We analyzed 34 primary tumors, including 13 primary tumors with peritoneal metastasis (P1) and/or positive peritoneal cytology (CY1) using a combination of laser microdissection and CGH. The minimal overlapping regions in gains were assigned to 5p14 (46.2%), 7q21.3 (61.5%), 7q31 (46.2%), 7q36 (46.2%), 8q23 (53.8%), 15q26 (46.2%), 20q12 (61.5%), 20q13.1 (53.8%), and 20q13.2 (53.8%) in primary tumors with P1 and/or CY1. The minimal regions of losses that occurred most frequently were 4q34-q35 (23.1%) and 22q11.2 (23.1%). There were significant differences in the minimal regions of 5p14 (P=0.033), 7q21.3 (P < 0.0001), 7q31 (P=0.013), 7q36 (P=0.033), and 22q11.2 (P=0.048) between primary tumors with and without P1 and/or CY1. In this study, gain/amplification of 5p14, 7q21.3, 7q31, and 7q36, and loss of 22q11.2 were significant in gastric cancer cases with peritoneal dissemination and/or positive peritoneal cytology.

Expression of TIMP-1 and -2 in different growth patterns of adenoid cystic carcinoma.

Tissue inhibitors of metalloproteinases (TIMPs) are special inhibitors of matrix metalloproteinases. To evaluate their roles in adenoid cystic carcinoma (ACC), we compared TIMP-1 and -2 mRNA and protein expression in different histological pattern of ACC. We obtained carcinoma cells from each of cribriform and tubular pattern of ACCs using by laser microdissection (LM), to determine the mRNAs expression of TIMP-1 and -2 using by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and to confirm expression of them by immunohistochemical staining. Our results showed that mRNA expression of TIMP-1 tended to be decreased in cribriform pattern compared with tubular pattern in four cases, and TIMP-1 significantly decreased in three cases. TIMP-2 also significantly decreased in cribriform than in tubular pattern in three of four cases. Protein expression of TIMP-1 and -2 decreased in the cribriform pattern compared to tubular pattern. These results indicate that there is close relationship between TIMPs and growth patterns of ACC, and TIMP-1 and -2 may play important roles in morphogenesis and biological character of adenoid cystic carcinoma.

Differences in gene expression in the proliferative human endometrium.

OBJECTIVE: To use microdissection and DNA microarray technology to demonstrate differences in gene expression between epithelial and stromal areas in the proliferative human endometrium. DESIGN: Pilot study. SETTING: University hospital. PATIENT(S): Patients with normal menstrual cycles and at least one previous intrauterine pregnancy. INTERVENTION(S): Uterine endometrial biopsy. MAIN OUTCOME MEASURE(S): Gene expression. RESULT(S): From a total of 1,200 genes, 14 were strongly expressed in epithelial areas and 12 were strongly expressed in stromal areas. Among the genes strongly expressed in the stroma, expressions of decorin and discoidin domain receptor were confirmed by real-time polymerase chain reaction. Decorin was localized in the stromal areas by immunohistochemical staining. To confirm the effects of estrogen on gene expression, stromal cells were cultured. When E(2) was added to the culture media, expression of decorin mRNA was increased. CONCLUSION(S): The data demonstrated in this study help to understand the physiology of human endometrium. Decorin was strongly expressed in the stromal areas and was regulated by estrogen, and therefore it may be involved in restoration of the endometrium.

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, Crohn's 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'.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: In 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. CONCLUSIONS: Some 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 adenomatous polyposis coli (APC) in tumorigenesis of human oral squamous cell carcinoma.

The product of the adenomatous polyposis coli (APC) tumor suppressor gene has been observed to regulate the Wnt signaling pathway through beta-catenin. In the present study, we attempted to clarify the relation between APC and the canceration of oral squamous epithelium. Each target tissue of normal squamous epithelium, epithelial dysplasia, and squamous cell carcinoma (SCC) was recovered the oral SCC case by laser microdissection. In recovered cells, we examined the change in expression of APC and beta-catenin gene transcription products, as well as the existence of mutations in APC gene. We analyzed the localization of each protein of APC and beta-catenin by immunohistochemical study. We found a clear change in the expression level of the gene transcription product of APC in canceration of oral squamous epithelium and the differentiation of oral SCC. In addition, there was some change in the localization of the APC protein in canceration. It was not clear, however, whether the APC was mutated. A change was also observed in the expression level of the beta-catenin gene transcription product during the differentiation of oral SCC. Our results suggest that the changes in the expression level and the intracellular localization of APC are related to the canceration of oral squamous epithelium, and in malignant characterization of oral SCC. Mutations of the APC gene might not be indispensable, however, in canceration of oral squamous epithelium.

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.

Basic and clinical studies on quantitative analysis of lymph node micrometastasis in oral cancer.

Metastasis to cervical lymph nodes (LN) is significantly associated with the outcome of patients with oral cancer. To provide a useful method for the detection of micrometastases, we analyzed 115 LNs from 10 patients with oral cancer using real-time quantitative polymerase chain reaction (PCR) based on the expression of squamous cell carcinoma antigen (SCCA) and cytokeratin 13 (CK13). The sensitivity and quantification of this method were assessed by means of limited dilution of cultured oral cancer cells and a model of cervical LN-metastasis established by inoculating green fluorescent protein (GFP)-expressing cells into the tongue of nude mice. In both investigations, a few cancer cells were detected by real-time quantitative PCR, but not by conventional reverse transcription-PCR (RT-PCR). SCCA mRNA was detected at high levels in metastatic LNs. In contrast, 26 of the 30 control cervical LNs did not express the gene at all, and the rest showed fairly low levels. Of 108 histologically metastasis-negative LNs, 19 (17.6%) expressed SCCA mRNA levels higher than the cut-off value (1.0: mean expression of control LNs + 2SD). CK13 mRNA is not a suitable marker for the real-time PCR since it was detected frequently even in the control LNs. These findings suggest that genetic diagnosis by real-time quantitative PCR based on SCCA mRNA expression may be clinically useful for detecting occult tumor cells in cervical LNs.

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.