Biomedical subjects

Hitoshi Kawamata

Publications and source records attributed to Hitoshi Kawamata.

35 records · Page 2Linked to original sources

PubMed2003

A case of primary esophageal tuberculosis diagnosed by identification of Mycobacteria in paraffin-embedded esophageal biopsy specimens by polymerase chain reaction.

We present a case of primary esophageal tuberculosis diagnosed by polymerase chain reaction (PCR) of paraffin-embedded esophageal biopsy specimens. A 42-year-old Japanese woman visited our clinic because of dysphagia. Radiologic and endoscopic examinations revealed a stenotic lesion with reddish mucosa and multiple ulcers in the middle esophagus. There was no associated lesion outside the esophagus. Histological and bacteriological studies of esophageal biopsy specimens and gastric aspirates did not give a definitive diagnosis. However, mycobacterial DNA was detected by PCR of paraffin-embedded esophageal biopsy specimens. She then was diagnosed as having primary esophageal tuberculosis. The esophageal mucosal lesion almost healed after 1 month of antituberculosis medication with residual annular stenosis which was resolved later by endoscopic balloon dilation.

Adult↗

PubMed2003

Evaluation of cell proliferation and apoptosis in Helicobacter pylori gastritis using an image analysis processor.

BACKGROUND: Infection of the gastric mucosa by helicobacter pylori is primarily responsible for gastritis, gastric ulcer, adenocarcinoma, and lymphoproliferative disorders. H. pylori appears to accelerate apoptosis and the proliferation of the gastric epithelium directly or indirectly. To precisely assess the proliferative and apoptotic profile of .H pylori-infected gastric mucosa, a quantitative imaging system is now required. METHODS: Fifty-two patients with H. pylori gastritis were the subjects of the study. Biopsy materials were taken from at least two sites (usually three to five sites) including the antrum and corpus. The grade of gastritis was evaluated by the updated Sydney System. The proliferative and apoptotic profile was examined by Ki-67 immunohistochemistry and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling method. In addition, Ki-67-positive cells were quantitated by an image processor for analytical pathology (IPAP) system. RESULTS: H. pylori density and polymorphonuclear neutrophil activity were significantly decreased after H. pylori eradication ( P< 0.0001). Chronic inflammation (P< 0.0001) and lymphoid follicle numbers ( P < 0.0005) were also significantly decreased after the eradication. Glandular atrophy and intestinal metaplasia were slightly decreased after eradication, but the decrease did not reach the significant level. the Ki-67 labeling index was significantly decreased after the eradication P< 0.0001). The apoptosis index was also decreased after the eradication, but this decrease did not reach the significant level ( P = 0.06). CONCLUSION: our data suggest that the activation of proliferative cells and induction of apoptosis in the gastric mucosa is a response to H. pylori-induced mucosal damage. Moreover, IPAP may be a useful technology for evaluating the results of immunohistochemistry, and it could provide quantitative and reliable data for studying H. pylori gastritis.

Adult↗

PubMed2003

Thiazolidinediones inhibit cell growth of human oral squamous cell carcinoma in vitro independent of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Recently, we have demonstrated that PPARgamma is expressed in human salivary gland tumors and its ligands inhibit the growth of cultured salivary gland cancer cells. However, expression and function of PPARgamma in normal and neoplastic human oral squamous epithelium remains unclear. In the present study, we examined PPARgamma expression in human oral squamous cell carcinoma (OSCC) and tested its ligands for any antitumor effect. PPARgamma mRNA was detected by RT-PCR in some OSCC tissues and cultured cells, but the PPARgamma protein showed neither expression nor ligand-induced transcriptional activity. Despite loss of PPARgamma function, synthetic PPARgamma ligands caused significant dose-dependent inhibition of cancer cell growth. These results suggest that PPARgamma function is inactivated in OSCC cells and the anti-proliferative effect of its synthetic ligands is independent of PPARgamma.

Carcinoma, Squamous Cell↗

PubMed2003

Vesnarinone: a differentiation-inducing anti-cancer drug.

Vesnarinone has been shown to be a unique anti-proliferating, differentiation-inducing and apoptosis inducing drug against several human malignancies, including leukemia and several solid tumors. Furthermore, vesnarinone potentiates the effect of conventional cytotoxic chemotherapy or radiation therapy. Combination of differentiation-inducing therapy by vesnarinone with conventional chemotherapy or radiation therapy might be second- or third-line therapy in patients with advanced cancer. Analysis of the molecular mechanisms of the tumor differentiation therapy by vesnarinone might provide selective and targeted molecules for novel tumor dormancy therapy.

Animals↗

PubMed2003

Redifferentiation and ZO-1 reexpression in liver-metastasized colorectal cancer: possible association with epidermal growth factor receptor-induced tyrosine phosphorylation of ZO-1.

Tubular gland structures of colorectal cancer (CRC) have been demonstrated to undergo dedifferentiation at the primary site, and then the gland structures are re-formed in the liver metastases. In this study, we examined the degree of differentiation of the gland structure of 48 cases of CRCs (24 cases with synchronous liver metastasis, 24 cases without metastasis) by the modified Gleason grading system. We also investigated the role of ZO-1, one of the tight junction proteins, in the morphological changes, i.e., dedifferentiation and redifferentiation, of CRCs at the primary site and liver metastases. Liver-metastasized CRCs (2.47+/-0.37) showed a lower score in the modified Gleason grading system than the corresponding primary tumors (3.28+/-0.36) did, i.e., the tumor cells had undergone redifferentiation at liver metastases. ZO-1 was expressed at the apical cell borders of normal colorectal epithelium, the luminal side of which has tubular gland structures. In comparison with this normal epithelium, the ZO-1 expression level was frequently reduced in primary CRC with liver metastasis (20.8%) and ZO-1 was reexpressed in liver metastasized cancers (79.2%). Furthermore, it was demonstrated by an immunoprecipitation-western blotting analysis on 5 cases of CRC with liver metastasis that ZO-1 bound to epidermal growth factor receptor (EGFR) irrespective of the phosphorylation status of EGFR, and that EGFR associated ZO-1 was highly tyrosine-phosphorylated only in the primary CRC, but was dephosphorylated in the liver-metastasized cancers. Our observations suggest that tyrosine phosphorylation of ZO-1 leads to down-regulation of the function of ZO-1 and dedifferentiation of the glands in CRCs, and these phenomena contribute to liver metastases, and redifferentiation of the glands occurs in the liver metastases.

Adenocarcinoma↗

PubMed2003

Identification of genes differentially expressed in a newly isolated human metastasizing esophageal cancer cell line, T.Tn-AT1, by cDNA microarray.

We isolated a metastasizing human esophageal squamous cell carcinoma (SCC) cell line, T.Tn-AT1, from a parental non-metastasizing cell line, T.Tn, by in vitro selection and by use of a nude mouse orthotopic inoculation model. Then, we compared the expression profiles of 9206 genes in T.Tn-AT1 and T.Tn by cDNA microarray analysis. The gene expression profiles of T.Tn and T.Tn-AT1 were very similar, and only 34 genes showed more than 3-fold differential expression. Among the 34 genes, 29 genes were down-regulated and only 5 genes were up-regulated in T.Tn-AT1 cells. Subsequently, we confirmed the expression levels of 14 of the 34 genes in T.Tn and T.Tn-AT1 cells by means of reverse transcription-polymerase chain reaction. The expression of 8 genes (KAL1, HPGD, NDN, REG1A, CXCR4, SPOCK, DIAPH2 and AIF1) was down-regulated and that of one gene (VNN2) was up-regulated in T.Tn-AT1 cells. These 9 genes encoded proteins associated with metastatic processes, such as adhesion, migration, inflammation, proliferation, and differentiation. Thus, these genes might regulate the metastasis of esophageal SCC, and could be predictive markers for lymph node metastasis of esophageal SCC.

Carcinoma, Squamous Cell↗

PubMed2003

Evaluation of the chemosensitivity of head and neck cancer cells based on the diverse function of mutated-p53.

Pre-therapeutic evaluation of p53 gene is very important for treating patients with head and neck cancer. However, the analysis for p53 gene has generally been done by immunohistochemistry, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and direct sequencing. Functional analysis system for p53 transcriptional activity in mammalian cells is now required. We developed a functional analysis system for p53 transcriptional activity in cancer cells. We used two human head and neck cancer cell lines harboring mutated p53 gene, HSG (Asn30Ser) and TYS (Asp281His), and a human osteosarcoma cell line, Saos-2 as a control. We transfected these cells with luciferase reporter plasmids containing promoter sequence of p53 target genes (p21waf1, BAX, MDM2, p53AIP1 or PUMA). After treating the cells with chemotherapeutic drugs, alteration of the luciferase activity was measured. In HSG cells, none of the target gene promoters was activated by treatment with chemotherapeutic drugs. In TYS cells, p21waf1 promoter was markedly activated by treatment with chemotherapeutic drugs, but Bax and p53AIP1 promoter was not activated. This type of mutated-p53 in TYS cells prevents cell death from DNA damage, and probably accumulates genetic alterations and accelerates the malignant progression of the cells by DNA damaging therapy. Thus, analysis for the diverse function of mutated-p53 may help to determine the therapeutic strategy, especially for chemotherapy and radiation in the individual patients with head and neck cancer.

Amino Acid Substitution↗

PubMed2003

A MEK inhibitor (U0126) prolongs survival in nude mice bearing human gallbladder cancer cells with K-ras mutation: analysis in a novel orthotopic inoculation model.

Most cases of gallbladder cancer are found at an advanced stage accompanied by invasion to the liver, metastases to the lymph nodes and distant organs, and peritoneal dissemination. Then, the treatment for advanced gallbladder cancer is often ineffective, and the prognosis of this disease is poor. The specific aim of this study was to establish a model system for developing new therapeutic strategies, such as molecular target therapy, for human advanced gallbladder cancer. We used a human gallbladder cancer cell line, NOZ with K-ras mutation in this experiment. Then, we established a novel orthotopic inoculation model for gallbladder cancer by using NOZ cells in nude mice. Mitogen-activated protein kinase (MAPK) in NOZ cells was constitutively activated, and the activation of MAPK provided autonomous proliferation of NOZ cells. All of the mice orthotopically inoculated by NOZ cells developed tumors at the gallbladder. Direct invasion into the liver, and bloody ascites were observed. Metastases to the mediastinal lymph nodes were also recognized in all of the mice examined. Moreover, most of the mice showed lung metastases. Survival duration was 29-50 days after inoculation. Nude mice with NOZ tumor at gallbladder were treated by an intraperitoneal injection of a MAPK kinase inhibitor U0126 (25 micro mole/kg) twice a week. U0126 (p=0.0110, one-way ANOVA) significantly prolonged the survival duration of the mice with NOZ gallbladder tumor. Our orthotopic inoculation model is useful for developing new therapeutic strategies, such as molecular target therapy for advanced gallbladder cancer.

Animals↗

PubMed2002

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells.

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.

Active Transport, Cell Nucleus↗

PubMed2002

Pathological issues of gastric and lower esophageal cancer: helicobacter pylori infection and its eradication.

Gastric carcinoma is thought to develop via the actions of inducers and promoters of carcinogenesis. Tryptophan in charred fish or animal meat, ultraviolet rays, and irradiation, which damage genes of normal cells, have long been regarded as inducers of carcinoma, and agents such as alcohol, tobacco, aflatoxin, and nitrosoamine as promoters, with tobacco having both activities. The interaction between these environmental factors, principally diet, and Helicobacter pylori (Hp) is important in the genesis of gastric carcinoma. In this report, the histopathological feature of the Hp gastritis-carcinoma sequence is outlined, and the pathological characteristics of gastroesophageal reflux disease (GERD) and endoscopically negative reflux disease (ENRD) and the risk factors for lower esophageal carcinoma after Hp eradicated status in particular are discussed regarding aspects of cell cycle-associated factors. We conclude that (1) Infection with Hp increases the risk of gastric cancer in two histological phenotypes (i.e., diffuse undifferentiated type and intestinal differentiated type). Excessive cell replication and interrupting the mucus secretion mechanism may result in a large proportion of cells with genetic abnormalities. (2) Genetic alterations in gastric carcinogenesis may differ from those in colonic carcinogenesis. (3) The degree of GERD in Japanese patients is milder than that in patients from Western countries, although the incidence of GERD increases the status after successful eradication of Hp. It is also possible that accumulation of genetic abnormalities increases the number of cardiac and lower esophageal cancers. Investigation of cell cycle factors in GERD including ENRD can be expected to reveal the risk of carcinogenesis.

Esophageal Neoplasms↗

PubMed2002

Expression of peroxisome proliferator-activated receptor gamma and the growth inhibitory effect of its synthetic ligands in human salivary gland cancer cell lines.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. It is expressed in several tissue types, including adipose tissue in which it stimulates adipogenesis. Recent studies have demonstrated that PPARgamma ligands induce cellular differentiation and inhibit cell growth in carcinomas of various organs including the breast, prostate, lung, colon, stomach, bladder, and pancreas. However, whether PPARgamma is expressed in human salivary gland tumors and its function in this tissue is unknown. In the present study, we examined PPARgamma gene expression in human salivary gland cancer cells and tested its ligands for any antitumor effect. PPARgamma mRNA was detected by RT-PCR in both benign and malignant salivary gland tumor tissues. The effect of PPARgamma on cell growth was investigated using four human salivary gland cancer cell lines; HSG, AZA3, HSY and TYS, which were confirmed to express PPARgamma1 mRNA and protein. Retinoid X receptor alpha protein, which forms heterodimers with PPARgamma, was also detected in all the cells tested. Data obtained by luciferase assay indicated that the intrinsic PPARgamma protein was activated by the synthetic ligands, troglitazone and pioglitazone, but not by the natural ligand, 15-deoxy-delta12, 14-prostaglandin J2. The synthetic PPARgamma ligands, particularly troglitazone, caused significant dose-dependent inhibition of cancer cell growth. Furthermore, the overexpression of PPARgamma1 or PPARgamma2 in cancer cells also reduced significantly their growth rate. These results suggest that PPARgamma and its synthetic ligands suppress the growth of human salivary gland cancer cells and it may be a useful molecular target for cancer treatment.

Blotting, Western↗

PubMed2002

Evaluation of the malignant potential of aberrant crypt foci by immunohistochemical staining for beta-catenin in inflammation-induced rat colon carcinogenesis.

We previously showed that colitis enhanced the development of cancer and aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in Fischer 344 rats. In this study, we examined the effect of two different anti-inflammatory drugs [non-steroidal anti-inflammatory drugs (NSAIDs: Fenbufen) and a platelet activating factor-receptor antagonist (PAF-RA)] on the inflammation-induced rat colon carcinogenesis. Furthermore, we examined the expression and the localization of beta-catenin protein, and the proliferating cell nuclear antigen (PCNA)-labeling index (LI) in ACF and cancer. PAF-RA significantly decreased the incidence of ACF in the rats (p<0.05), but Fenbufen did not affect the incidence of ACF and cancer. In most of the ACF (91%), beta-catenin was localized at the cell membrane like in normal colon epithelium. In about 9% of the ACF, beta-catenin was overexpressed not only on the cell membrane but also in the cytoplasm. In all of the cancer cells, beta-catenin was overexpressed in the nucleus. When we compared the PCNA-LI in the ACF showing normal beta-catenin expression pattern with that in the ACF showing abnormal beta-catenin expression pattern (overexpression in cytoplasm), there was no significant difference of the PCNA-LI in these two different types of ACF. These findings suggest that immunohistochemical staining of ACF for beta-catenin can evaluate the malignant potential of ACF, and that PAF-RA can be used for preventing the development of ACF in inflammation-induced carcinogenesis.

Animals↗

PubMed2002

Frequent polymorphism of peroxisome proliferator activated receptor gamma gene in colorectal cancer containing wild-type K-ras gene.

We analyzed the K-ras gene mutation and the polymorphism of peroxisome proliferator activated receptor gamma (PPARgamma) gene, which is known as a master gene for adipocyte differentiation and a tumor suppressor gene, in patients with colorectal cancer by polymerase chain reaction-restriction fragment length polymorphism. We detected a mutation of K-ras gene at codon 12 in 8 of 39 colorectal cancers. We also detected a polymorphism of PPARgamma gene at codon 12 in 10 of 39 patients with colorectal cancer. Homozygous polymorphism (Ala12Ala) in PPARgamma gene was found only in one patient, but heterozygous polymorphism (Pro12Ala) was found in 9 of 29 patients. Surprisingly, all 10 colorectal cancers developed in the patients with polymorphism of PPARgamma gene carrying the wild-type K-ras gene. These findings suggest that PPARgamma gene polymorphism may be implicated with the development of colorectal cancers, in which K-ras gene is not mutated.

Adult↗

PubMed2002

Dysfunction of the p53 tumor suppressor pathway in head and neck cancer.

It is important to examine the abnormality of the entire p53 tumor suppressor pathway in head and neck cancer. We examined the mRNA expressions of p53 regulatory factors, p33ING1 and p14ARF, and a p53-target gene, p21WAF1 in head and neck cancer. Nine of 14 benign pleomorphic adenomas (PAs) and 7 of 8 malignant salivary gland tumors (MSGTs) expressed p33ING1 mRNA. Thirteen of 14 PAs expressed p14ARF mRNA, however, only 1 of 8 MSGTs expressed p14ARF mRNA. Eight of 14 PAs and 7 of 8 MSGTs expressed p21WAF1 mRNA. In salivary gland tumors, there was clear correlation between the expression of p33ING1 and p21WAF1 (p<0.0001, r2=0.53). However, there was no correlation between the expression of p14ARF and p21WAF1 (p=0.6543, r2=0.009). Twenty-six of 28 oral squamous cell carcinomas (SCCs) expressed p33ING1 mRNA. Nineteen of 28 oral SCCs expressed p14ARF mRNA. All of the oral SCCs expressed p21WAF1 mRNA. In oral SCCs, the expressions of both p33ING1 (p=0.009, r2=0.181) and p14ARF (p=0.0009, r2=0.271) correlated with the expression of p21WAF1. Interestingly, 24 of 26 oral SCCs (92%) showed either abnormality of p53 itself or loss of expression of p53 regulatory factors, p33ING or p14ARF. These results suggest that head and neck cancer often involve the dysfunction of p53 tumor suppressor pathway.

Blotting, Southern↗

PubMed2002

Molecular and genetic characterization of a non-metastatic human esophageal cancer cell line, T.Tn expressing non-functional mutated p53.

Lymph node metastasis is commonly found in esophageal squamous cell carcinoma (SCC). In this study, we examined the molecular and genetic characteristics of a human esophageal SCC cell line, T.Tn. T.Tn cells formed tumors at s.c. tissue in nude mice when inoculated with Matrigel, but did not metastasize to any organs. T.Tn cells expressed low level of proMMP2 and a trace level of proMMP9. However, T.Tn cells expressed high level of TIMP1 and TIMP2, and beta-catenin and E-cadherin. We found a point mutation of p53 gene at codon 213 (CAT-->CGT) in T.Tn cells. The mutated-p53 protein did not show transcriptional activity on p21(waf1), MDM2 and Bax promoters. Thus, T.Tn cells are low tumorigenic and weakly invasive but not metastasizing in nude mice, and T.Tn cells are suitable parental cells for establishing a model system to study invasion and metastasis of esophageal SCC.

Animals↗

PubMedDate not supplied

Leucine zipper structure of TSC-22 (TGF-beta stimulated clone-22) markedly inhibits the anchorage-independent growth of salivary gland cancer cells.

Several investigators have demonstrated that TGF-beta stimulated clone-22 (TSC-22) regulates cell growth and differentiation, and cell death. TSC-22 is a putative transcriptional regulator containing a leucine zipper-like structure and a nuclear export signal. We previously showed the cytoplasmic localization of TSC-22 and the nuclear translocation of TSC-22 concomitant with induction of apoptosis in salivary gland cancer cells. In the present study, we attempted to identify the active domain of TSC-22 protein that regulated the biological functions of TSC-22. We constructed three mammalian expression vectors, which could produce full length TSC-22 only in cytoplasm, the leucine zipper structure of TSC-22 in both cytoplasm and nucleus, and the leucine zipper structure only in nucleus. Then, we transfected a salivary gland cancer cell line, HSG with these expression vectors, and investigated the growth profile of the transfectants. None of the TSC-22 constructs inhibited the monolayer growth and the anchorage-dependent colony formation of HSG cells. However, the leucine zipper structure of TSC-22 markedly inhibited the anchorage-independent colony formation of HSG cells (p<0.001; one way ANOVA). Full length TSC-22 also suppressed the anchorage-independent colony formation of HSG cells, although the effect of full length TSC-22 was much lower than those of the leucine zipper constructs. These observations suggest that the leucine zipper structure in TSC-22 protein is an active domain that negatively regulates the growth of salivary gland cancer cells.

Amino Acid Motifs↗

PubMedDate not supplied

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Animals↗