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Profile of TCR Vb gene usage of cytotoxic T cells induced by intrapleural administration of streptococcal a preparation, OK432, in malignant effusions
Biology
[6-•
Expression of a putative human mismatch repair gene ME01 in small cell lung cancer cell lines
•8]
L.R. Hansen, M. Spang-Thomsen, L.N. Petersen. Institute of
Molecular Pathology, Copenhagen, Denmark Recently a putative human mismatch repair (MMR) gene MED1 (methyl CpG-binding endonuclease) has been cloned and shown to interact with MLH1 as well as being involved in microsatellite stability. Microsatellite instability (MSI) is thought to reflect cellular mismatch repair (MMR) deficiency, tn small call lung cancer (SCLC) the reports on MSl are conflicting with frequencies ranging from 0-76%. Thus, the role of MMR in the development of SCLC is still controversial. To investigate the role of MMR and particular MED1 in SCLC we designed a MED1 probe and analyzed the gene expression in a panel of 21 SCLC cell lines. Cells were harvested when subconfluent, and DNA, mRNA, and total cellular RNA was extracted and analyzed by Southern and northern blotting. The RNA expression of the MED1 gene was normalized to the level of the GAPDH or 28S ribosomal expression. In preliminary studies no aberrant MED1 bands on the Southern blots were detected and we therefore conclude that MED1 contains no gross genetic alterations in these SCLC cell lines. Furthermore a MED1 expression of the expected size was detected in all SCLC cell lines and only with a minor variation among them. This is in contrast to the highly heterogeneously expression pattern we previously have shown for other MMR genes in these cell lines. Ongoing studies include microsatellite analysis and determination of the in vitro MMR capacity in the SCLC cell lines.
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Down-regulation of KAI1 messenger RNA expression is not associated with loss of heterozygosity of the KAI1 gene region in lung adenocarcinoma
199
Profile of TCR Vb gene usage of cytotoxic T cells induced by intrapleural administration of streptococcal a preparation, O1(432, in malignant effusions
E. Miyahara, Y. Yamaguchi, J. Hihara, T. Toge. Research Institute for Radiation Biology and Medicine, Hiroshima University, Department of Surgical Oncology, Hiroshima, Japan T cell receptor (TCR) gene rearrangements were analyzed in tumor infiltrating lymphocytes (TIL) using the reverse transcription-polymerase chain reaction (RT-PCR) method to understand whether oligoclonal usage of TCRVb is present in TIL and whether the usage is involved in clinical response and action mechanism of the Iocoregional immunotherapy with a streptococcal preparation, OK432. Patients with malignant effusion were treated with the intrapleural administration of OK432 and clinical responses were assessed by cytological and chest X-ray examinations. Pleural exudate cells (PEC) were obtained before and after the administration of OK432, designated as pre- and OK432PEC, respectively, and subjected to TCR analysis. Both pre- and OK432-PEC showed highly diverse expressions of TCRVb gene usage and there was no relationship between clinical responses and TCRVb gene usage in pre- and OK432-PEC. The frequency of TCRVb20 gene usage in OK432-PEC was significantly higher than that in pre-PEC. Moreover, the overexpression of TCRVb20 gene usage was also induced in peripheral blood lymphocytes (PBL) and pre-PEC of patients by the in vitro stimulation with OK432, but not in healthy PBL. Single strand conformational polymorphism (SSCP) analysis revealed the clonotypes in these TCRVb20 genes. Autologous tumor-specific killing activity could be detected in OK432-PEC and was significantly reduced by the treatment with a TCRVb20-specific monoclonal antibody. It is suggested that the rearrangement of TCRVb20 gene usage may be highly involved in the autologous tumor-specific action of malignant effusions in the treatment with OK432.
K. Tagawa, Y. Takeshima, E. Hiyama, M. Yamasaki, K. Inai. Second
Department of Pathology, Hiroshima University School of Medicine, Hiroshima; Department of General Medicine, Hiroshima University School of Medicine, Hiroshima, Japan KAI1, a metastasis suppressor gene of prostate cancer, is located on human chromosome 1 l p l 1.2. Down-regulation of KAI1 mRNA during tumor progression and metastasis has been reported for several kinds of cancer, however the mechanism of this down-regulation still remains unknown. In our study, the aim is to ascertain the relationship between down-regulation of KAI1 mRNA expression and KAI1 gene alterations in lung cancer. Forty-nine cases of adenocarcinoma of the lung were studied by reverse-transcriptase polymerase chain reaction (RT-PCR) assay of KAI1 mRNA and by immunohistochemical detection of KAI1 protein. In addition, markers of the microsatellite loci, DllS1344 and D1 lS1326, were used to investigate loss of heterozygosity (LOH) and replication errors (RERs) of the KAI1 gene region. The RT-PCR assay showed that there was no correlation between KAI1 mRNA expression and either the age of the patients or tumor size. By contrast, KAI1 mRNA expression was significantly correlated with patient gender (P = 0.047), metastasis to the lymph nodes or other organs (P = 0.004), the histological grade of the tumor (P = 0.036) and the pathological stage (P = 0.049), Immunohistochemical staining showed that loss of KAI1 mRNA was associated with invasion of the stroma by KAI1 proteinnegative cancer cells in one case without metastasis. The numbers of informative cases by microsatellite analysis were 14 (28.6%) of 49 at D11S1344 and 27 (55.1%) of 49 at D11S1326; none of 49 adenocarcinomas showed LOH or RERs at these loci. These results suggest that down-regulation of KAI1 mRNA expression rarely related to the LOH or RERs of the KAI1 gene region in primary lung adenocarcinoma.
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Role of extracellular matrix in the brain metastasis of non-small cell lung cancer; analysis in the newly developed brain metastatic model
T. Sakurai, T. Yoshimasu, N. Matsuura, I. Ota. Wakayama Medical
College, Wakayama; Osaka University, Osaka, Japan Extracellular matrices (ECMs) play important roles in the metastatic process. Lung cancer is highly metastatic to the brain. The mechanism of brain metastasis of lung cancer, however, has not been fully explained. One of the reasons is that the experimental model for brain metastasis of lung cancer has not been developed up to now. We have established an in vivo experimental model for brain metastasis of human non-small call lung cancer (NSCLC). The objective of this study was to investigate the roles of ECMs in brain metastasis of NSCLC using our brain metastatic model. A NSCLC cell line EBC-1 was injected into the nude mouse left ventricle. Two or three months after transplantation, brain metastases were developed. Using serial intraventricular passages of the brain metastasis, a highly brain metastatic subclone EBC-1/brain was selected. EBC-1 can also develop the bone metastasis after intraventricular injection. Highly bone metastatic subclone EBC-1/bone was also selected in the same manner. EBC-1/brain was largest in the diameter, and showed the highest metastatic probability to the brain among these calls. We have studied the interaction to laminin (LN), type I collagen (COL), and fibronectin (FN) of EBC-1, EBC-1/brain and EBC-1/bone. Adhesion to FN was increased in both EBC-1/brain and EBC-1/bone compared with EBC-1. Adhesion to LN was identical in these 3 cells after 1 hour incubation. On ECMs at 24 hours incubation, there were striking differences of the percentage of stretched cells among these cells. EBC-1/brain was stretched on LN strongly, while, EBC-1 and EBCl/bone were almost never stretched. The cell proliferation on LN was fastest in EBC-1/brain. Brain has a unique environment that ECMs other than laminin (LN) rarely exists. These cellular interactions of EBC-1/brain to LN might affect the highly metastatic potential to the brain.
[6-•
Expression of a putative human mismatch repair gene ME01 in small cell lung cancer cell lines
•8]
L.R. Hansen, M. Spang-Thomsen, L.N. Petersen. Institute of
Molecular Pathology, Copenhagen, Denmark Recently a putative human mismatch repair (MMR) gene MED1 (methyl CpG-binding endonuclease) has been cloned and shown to interact with MLH1 as well as being involved in microsatellite stability. Microsatellite instability (MSI) is thought to reflect cellular mismatch repair (MMR) deficiency, tn small call lung cancer (SCLC) the reports on MSl are conflicting with frequencies ranging from 0-76%. Thus, the role of MMR in the development of SCLC is still controversial. To investigate the role of MMR and particular MED1 in SCLC we designed a MED1 probe and analyzed the gene expression in a panel of 21 SCLC cell lines. Cells were harvested when subconfluent, and DNA, mRNA, and total cellular RNA was extracted and analyzed by Southern and northern blotting. The RNA expression of the MED1 gene was normalized to the level of the GAPDH or 28S ribosomal expression. In preliminary studies no aberrant MED1 bands on the Southern blots were detected and we therefore conclude that MED1 contains no gross genetic alterations in these SCLC cell lines. Furthermore a MED1 expression of the expected size was detected in all SCLC cell lines and only with a minor variation among them. This is in contrast to the highly heterogeneously expression pattern we previously have shown for other MMR genes in these cell lines. Ongoing studies include microsatellite analysis and determination of the in vitro MMR capacity in the SCLC cell lines.
•
Down-regulation of KAI1 messenger RNA expression is not associated with loss of heterozygosity of the KAI1 gene region in lung adenocarcinoma
199
Profile of TCR Vb gene usage of cytotoxic T cells induced by intrapleural administration of streptococcal a preparation, O1(432, in malignant effusions
E. Miyahara, Y. Yamaguchi, J. Hihara, T. Toge. Research Institute for Radiation Biology and Medicine, Hiroshima University, Department of Surgical Oncology, Hiroshima, Japan T cell receptor (TCR) gene rearrangements were analyzed in tumor infiltrating lymphocytes (TIL) using the reverse transcription-polymerase chain reaction (RT-PCR) method to understand whether oligoclonal usage of TCRVb is present in TIL and whether the usage is involved in clinical response and action mechanism of the Iocoregional immunotherapy with a streptococcal preparation, OK432. Patients with malignant effusion were treated with the intrapleural administration of OK432 and clinical responses were assessed by cytological and chest X-ray examinations. Pleural exudate cells (PEC) were obtained before and after the administration of OK432, designated as pre- and OK432PEC, respectively, and subjected to TCR analysis. Both pre- and OK432-PEC showed highly diverse expressions of TCRVb gene usage and there was no relationship between clinical responses and TCRVb gene usage in pre- and OK432-PEC. The frequency of TCRVb20 gene usage in OK432-PEC was significantly higher than that in pre-PEC. Moreover, the overexpression of TCRVb20 gene usage was also induced in peripheral blood lymphocytes (PBL) and pre-PEC of patients by the in vitro stimulation with OK432, but not in healthy PBL. Single strand conformational polymorphism (SSCP) analysis revealed the clonotypes in these TCRVb20 genes. Autologous tumor-specific killing activity could be detected in OK432-PEC and was significantly reduced by the treatment with a TCRVb20-specific monoclonal antibody. It is suggested that the rearrangement of TCRVb20 gene usage may be highly involved in the autologous tumor-specific action of malignant effusions in the treatment with OK432.
K. Tagawa, Y. Takeshima, E. Hiyama, M. Yamasaki, K. Inai. Second
Department of Pathology, Hiroshima University School of Medicine, Hiroshima; Department of General Medicine, Hiroshima University School of Medicine, Hiroshima, Japan KAI1, a metastasis suppressor gene of prostate cancer, is located on human chromosome 1 l p l 1.2. Down-regulation of KAI1 mRNA during tumor progression and metastasis has been reported for several kinds of cancer, however the mechanism of this down-regulation still remains unknown. In our study, the aim is to ascertain the relationship between down-regulation of KAI1 mRNA expression and KAI1 gene alterations in lung cancer. Forty-nine cases of adenocarcinoma of the lung were studied by reverse-transcriptase polymerase chain reaction (RT-PCR) assay of KAI1 mRNA and by immunohistochemical detection of KAI1 protein. In addition, markers of the microsatellite loci, DllS1344 and D1 lS1326, were used to investigate loss of heterozygosity (LOH) and replication errors (RERs) of the KAI1 gene region. The RT-PCR assay showed that there was no correlation between KAI1 mRNA expression and either the age of the patients or tumor size. By contrast, KAI1 mRNA expression was significantly correlated with patient gender (P = 0.047), metastasis to the lymph nodes or other organs (P = 0.004), the histological grade of the tumor (P = 0.036) and the pathological stage (P = 0.049), Immunohistochemical staining showed that loss of KAI1 mRNA was associated with invasion of the stroma by KAI1 proteinnegative cancer cells in one case without metastasis. The numbers of informative cases by microsatellite analysis were 14 (28.6%) of 49 at D11S1344 and 27 (55.1%) of 49 at D11S1326; none of 49 adenocarcinomas showed LOH or RERs at these loci. These results suggest that down-regulation of KAI1 mRNA expression rarely related to the LOH or RERs of the KAI1 gene region in primary lung adenocarcinoma.
~--~
Role of extracellular matrix in the brain metastasis of non-small cell lung cancer; analysis in the newly developed brain metastatic model
T. Sakurai, T. Yoshimasu, N. Matsuura, I. Ota. Wakayama Medical
College, Wakayama; Osaka University, Osaka, Japan Extracellular matrices (ECMs) play important roles in the metastatic process. Lung cancer is highly metastatic to the brain. The mechanism of brain metastasis of lung cancer, however, has not been fully explained. One of the reasons is that the experimental model for brain metastasis of lung cancer has not been developed up to now. We have established an in vivo experimental model for brain metastasis of human non-small call lung cancer (NSCLC). The objective of this study was to investigate the roles of ECMs in brain metastasis of NSCLC using our brain metastatic model. A NSCLC cell line EBC-1 was injected into the nude mouse left ventricle. Two or three months after transplantation, brain metastases were developed. Using serial intraventricular passages of the brain metastasis, a highly brain metastatic subclone EBC-1/brain was selected. EBC-1 can also develop the bone metastasis after intraventricular injection. Highly bone metastatic subclone EBC-1/bone was also selected in the same manner. EBC-1/brain was largest in the diameter, and showed the highest metastatic probability to the brain among these calls. We have studied the interaction to laminin (LN), type I collagen (COL), and fibronectin (FN) of EBC-1, EBC-1/brain and EBC-1/bone. Adhesion to FN was increased in both EBC-1/brain and EBC-1/bone compared with EBC-1. Adhesion to LN was identical in these 3 cells after 1 hour incubation. On ECMs at 24 hours incubation, there were striking differences of the percentage of stretched cells among these cells. EBC-1/brain was stretched on LN strongly, while, EBC-1 and EBCl/bone were almost never stretched. The cell proliferation on LN was fastest in EBC-1/brain. Brain has a unique environment that ECMs other than laminin (LN) rarely exists. These cellular interactions of EBC-1/brain to LN might affect the highly metastatic potential to the brain.