PURPOSE: There have only been a few cytogenetic studies of hepatocellular carcinoma (HCC), and so far, no consistent specific chromosomal abnormalities have been described. Here, we have used comparative genomic hybridization (CGH), a powerful molecular cytogenetic technique for detecting changes of the copy number throughout the genome, to screen for genetic alterations in HCC cell lines. The CGH results were compared with those derived from G-banding and chromosome painting. MATERIALS AND METGODS: Conventional cytogenetic analyses were performed on five HCC cell lines, SNU-354, SNU-368, SNU-387, SNU-449 and SNU-475, using a G- banding staining technique. In CGH, equal amounts of differently labeled DNA from the cell lines, and normal reference DNA, were hybridized simultaneously to normal metaphase chromosomes. They were visualized by different fluorochromes, and the signal intensities quantified separately as gray levels along the single chromosomes. The over- and under-represented DNA segments were determined by computation of ratio images and average ratio profiles. To confirm the CGH results, florescence in situ hybridization (FISH), with chromosome specific painting, was performed using indirectly labeled chromosome specific paints. RESULTS: Complex unbalanced chromosomal aberrations, which could not be identified reliably by conventional cytogenetics in HCC cell lines, were successfully resolved by CGH analysis. CGH results were validated using FISH with chromosome specific probes. In HCC cell lines, gains in DNA copy number were more common than losses. The most prominent changes were gains of 1q12- qter (80% of cases), 1q41-qter (100%), 7 (80%), 8q12-qter (60%), 8q23-qter (80%) and 20q12-qter (60%). Recurrent losses were mapped on 4q13-qter (60%), 16q12-qter (60%), 16q21-qter (80%), 13q12-q14.2 (60%) and Yq11.2 (100%). All four male HCC cell lines showed loss or rearrangement of the Y chromosome. CONCLUSION: Conventional cytogenetics, CGH and FISH using painting probes, represent complementary approaches that, when employed in combination, could greatly facilitate the comprehensive analysis of chromosomal imbalances in HCC cell lines. Our results suggest the existence of an oncogene, or protooncogenes, on chromosome 1q41-qter, and the tumor suppressor genes on Yq11.2, that play a role in the development and/or progression of hepatocellular carcinogenesis.
Carcinogenesis ; Carcinoma, Hepatocellular* ; Cell Line* ; Chromosome Aberrations ; Chromosome Painting ; Chromosomes, Human, Pair 1 ; Comparative Genomic Hybridization* ; Cytogenetic Analysis ; Cytogenetics ; DNA ; Fluorescent Dyes ; Genes, Tumor Suppressor ; Genome ; Humans ; In Situ Hybridization ; Male ; Metaphase ; Oncogenes ; Paint ; Paintings ; Y Chromosome

Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 >beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.
Adult ; Chromatography, Agarose ; Female ; Heparin/chemistry ; Human ; Immunohistochemistry ; Isoenzymes/isolation & purification/*metabolism ; Lung/*enzymology/pathology ; Male ; Phospholipase C/isolation & purification/*metabolism

PURPOSE: There are only a few cytogenetic studies in gastric cancer and so far no consistent specific chromosomal abnormalities have been described. In this study, we have used comparative genomic hybridization (CGH), a powerful molecular cytogenetic technique for detecting changes of the copy number throughout the genome, to screen for genetic alterations in gastric cancer cell lines. The CGH results were compared with those derived from G-banding and chromosome painting. MATERIALS AND METHODS: Conventional cytogenetic analysis was performed on five human gastric cancer cell lines, AGS, SNU-1, SNU-16, SNU-620, and SNU-719, by a G-banding staining technique. In CGH, equal amounts of differently labeled DNA from the cell lines and normal reference DNA were hybridized simultaneously to normal metaphase chromosomes. They were visualized by different fluorochromes, and the signal intensities were quantitated separately as gray levels along the single chromosomes. The over- and under- represented DNA segments were determined by computation of ratio images and average ratio profiles. To confirm the CGH results, fluorescence in situ hybridization (FISH) with chromosome specific painting was performed using indirectly labeled chromosome specific paints. RESULTS: Complex unbalanced chromosomal aberrations that could not be identified reliably by conventional cytogenetics in gastric cancer cell lines were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome specific probes. In gastric cancer cell lines, gains of DNA copy number were more common than losses. Gains were detected on 1p, 1q, 2p, 3q, 6p, 7q, 10q, 11p, and 19q, and losses were observed on 4p, 4q, 5q, 12p, 12q, and 18q. Interestingly, all the five gastric cancer cell lines tested showed gain of DNA copy number on the chromosome 20, suggesting an existence of oncogene. CONCLUSION: Conventional cytogenetics, CGH, and FISH using painting probes represent complementary approaches that, when employed in combination, could greatly facilitate the comprehensive analysis of chromosomal imbalances in gastric cancer cell lines. Our results suggest the existence of an oncogene or oncogenes on chromosome 20 that play a role in the development and/or the progression of gastric carcinogenesis.
Carcinogenesis ; Cell Line* ; Chromosome Aberrations ; Chromosome Painting ; Chromosomes, Human, Pair 20 ; Comparative Genomic Hybridization* ; Cytogenetic Analysis ; Cytogenetics ; DNA ; Fluorescence ; Fluorescent Dyes ; Genome ; Humans ; In Situ Hybridization ; Metaphase ; Oncogenes ; Paint ; Paintings ; Stomach Neoplasms*

Gaucher's disease (GD) is the most common inherited lysosomal storage disease, manifested by generalized accumulation of glucocerebroside in macrophages of the reticuloendothelial system due to a deficient lysosomal beta-glucocerebrosidase (GC). It is inherited by an autosomal recessive pattern in which three clinical phenotypes have been described based on the presence and severity of neurologic involvement. GD is treated possible by GC enzyme replacement therapy, allogeneic bone marrow transplantation (BMT), and gene therapy. We here report the exprience of successful allogeneic BMT in a 16-year-old female patient with GD type III which was demostrated markedly increased Gaucher cells in bone marrow and absence of GC activity in peripheral blood monocytes by FACS using 5'- pentafluorobenzoylaminofluorescein-di-beta-D-glucoside (PFBFDGlu) as substrate. Donor marrow engraftment was confirmed by chromosome analysis using microsatellite and by bone marrow examination. Assay of GC activity using FACS revealed normal level of enzyme activity. She remains alive and well after 12 months of BMT.
Adolescent ; Bone Marrow Examination ; Bone Marrow Transplantation* ; Bone Marrow* ; Enzyme Replacement Therapy ; Female ; Gaucher Disease* ; Genetic Therapy ; Glucosylceramidase ; Humans ; Lysosomal Storage Diseases ; Macrophages ; Microsatellite Repeats ; Monocytes ; Mononuclear Phagocyte System ; Phenotype ; Tissue Donors

PURPOSE: NOS2 induce NO production and NO activate TGF-beta. The TGF-beta is a inhibitor of NOS2. If this negative feedback mechanism operating in radiation pneumonitis model, NOS2 inhibitor may play a role in TGF-beta suppression. We planned this study to evaluate the expression patterns of NO, NOS2 and TGF-beta in vivo radiation pneumonitis model. MATERIALS AND METHODS: Sixty sprague-Dawley rat were irradiated 5 Gy or 20 Gy. They were sacrificed 3, 7, 14, 28 and 56 days after irradiation. During sacrifice, we performed broncho-alveolar lavage (BAL). The BAL fluids were centrifuged and supernatents were used for measure NO and TGF-beta, and the cells were used for RT-PCR. RESULTS: After 5 Gy of radiation, NO in BAL fluid increased at 28 days in both lung and TGF-beta in left lung at 56 days. NO increased in BAL fluid at 28 days in both lung after irradiation and TGF-beta in right lung at 28-56 days after 20 Gy of radiation. After 5 Gy of radiation, NOS2 expression was increased in right lung at 14 days, in both lung at 28 days and in left lung at 56 days. TGF-beta expression was reduced in both lung at 28 days and increased in left lung at 56 days. CONCLUSIONS: The proposed feedback mechanism of NO, NOS2 and TGF-beta was operated in vivo radiation pneumonitis model. At 56 days, however, NOS2 and TGF-beta expressed concurrently in left lung after 5 Gy and in both lung after 20 Gy of radiation.
Animals ; Lung* ; Radiation Effects* ; Radiation Pneumonitis ; Rats* ; Rats, Sprague-Dawley ; Therapeutic Irrigation ; Transforming Growth Factor beta*

To determine the precise chromosomal localization of tyrosine related protein-1 and -2 (TRP-1 and TRP-2) genes by fluorescence in situ hybridization, we used DNAs isolated from human bacterial artificial chromosome clones. They contain genomic sequences with approximately 120 kb inserts for TRP-1 and TRP-2. The TRP-1 and TRP-2 genes were assigned to human chromosome bands 9p23 and 13q32.1, respectively. These results confirmed the previously mapped location for the TRP-1 gene and more precisely located the TRP-2 gene, which had previously been mapped to chromosome 13q31-q32.
Chromosomes, Human, Pair 13/genetics* ; Chromosomes, Human, Pair 9/genetics* ; Gene Library ; Human ; In Situ Hybridization, Fluorescence ; Intramolecular Oxidoreductases/genetics* ; Proteins/genetics*

PURPOSE: Because of difficulty of obtaining metaphase cells from tumor specimens, there are only a few cytogenetic studies in nasal NK/T-cell lymphomas, and so far no consistent specific chromosomal abnormalities have been described. In this study, we have used degenerate oligonucleotide primed PCR (DOP-PCR) and comparative genomic hybridization (CGH) to deter mine chromosomal alterations from 6 nasal NK/T-cell lymphoma tissues dissected from formalin- fixed paraffin-embedded slide sections. MATERIALS AND METHODS: For the isolation of tumor DNA, four 7-micrometer-thick tissue sections from each sample were dewaxed and rehydrated, and areas of high tumor cell content (more than 60%) were dissected and pooled into a tube. Normal DNA was prepared from the peripheral blood of a healthy volunteer. Tumor DNA was labeled with biotin-16-dUTP by DOP-PCR and normal DNA was labeled with digoxigenin-dUTP using a nick translation kit. In CGH, equal amounts of differently labeled DNA from the tumors and normal reference DNA were hybridized simul taneously to normal metaphase chromosomes. They were visualized by different fluordegrees Chromes, and the signal intensities were quantitated separately as gray levels for each chromosome. The over- and underrepresented DNA segments were determined by computation of image ratios and average ratio profiles. RESULTS: Our results show that gains of DNA copy number were more prevalence than DNA losses. The most commonly observed gains were mapped to chromosomal regions of 1p32.2 ter,19 and 20 in 4 of 6 cases (67%). The other frequent gains were found on chromosomes 12q in 3 of 6 cases. The most frequent loss was detected on 6q in 4 of 6 cases(67%), and less fre quently observed on 13q21.1 q34 and 13q14 q34. CONCLUSION: These genomic changes found in specific chromosomal regions are likely to harbor genes of importance in nasal NK/T-cell lymphomagenesis, therefore such cytogenetic mapping of genomic imbalance may be of value for further molecular delineation of NK/T-cell lymphoma.
Chromosome Aberrations ; Comparative Genomic Hybridization* ; Cytogenetics ; DNA ; Healthy Volunteers ; Lymphoma* ; Metaphase ; Polymerase Chain Reaction ; Prevalence

BACKGROUND: Phospholipase C (PLC) plays an important role in cellular signal transduction and is thought to be critical in cellular growth, differentiation and transformation of certain malignancies. Two second messengers produced from the enzymatic action of PLC are diacylglycerol(DAG) and lnositol 1, 4, 5-trisphosphate(IP3). These two second messengers are important in down stream signal activation of protein kinase C and intracelluar calcium elevation. In addition, functional domains of the PLC isozymes, such as Src homology 2(SH2) domain, Src homology 3(SH3) domain, and pleckstrin homology(PH) domain play crucial roles in protein translocation, lipid membrane modification and intracellular memrane trafficking which occur during various mitogenic processes. We have previously reported the presence of PLC-γ1, γ2, β1, β3, and δ1 isozymes in normal human lung tissue and tyrosine-kinase-independent activation of phospholipase C-γisozymes by tau protein and AHNAK. We had also found that the expression of AHNAK protein was markedly increased in various histologic types of lung cancer tissues as compared to the normal lungs. However, the report concerning expression of various PLC isozymes in lung cancers and other lung diseases is lacking. Therefore, in this study we examined the expression of PLC isozymes in the paired surgical specimens taken from lung cancer patients. METHODS: Surgically resected lung cancer tissue samples taken from thirty seven patients and their paired normal control lungs from the same patients. The expression of various PLC isozymes were studied. Western bolt analysis of the tissue extracts for the PLC isozymes and immunohistochemistry was performed on typical samples for localization of the isozyme. RESULTS: In 16 of 18 squamous cell carcinomas, the expression of PLC-γ1 was increased. PLC-γ1 was also found to be increased in all of 15 adenocarcinoma patients. In most of the non-small cell lung cancer tissues we had examined, expression of PLC-δ1 was decreased. However, the expression of PLC-δ1 was markedly increased in 3 adenocarcinomas and 3 squamous carcinomas. Although the numbers were small, in all 4 cases of small cell lung cancer tissues, the expression of PLC-δ1 was nearly absent. CONCLUSION: We found increased expression of PLC-γ1 isozyme in lung cancer tissues. Results of this study, taken together with our earlier findings of AHNAK protein-a putative PLD-γ, activator-over-expression, and the changes observed in PLC-δ1 in primary human lung cancers may provide a possible insight into the derranged calcium-inositol signaling pathways leading to the lung malignancies.
Adenocarcinoma ; Calcium ; Carcinogenesis ; Carcinoma, Non-Small-Cell Lung ; Carcinoma, Squamous Cell ; Humans* ; Immunohistochemistry ; Isoenzymes* ; Lung Diseases ; Lung Neoplasms* ; Lung* ; Membranes ; Phospholipases ; Protein Kinase C ; Protein Transport ; Rivers ; Second Messenger Systems ; Signal Transduction ; Small Cell Lung Carcinoma ; tau Proteins ; Tissue Extracts ; Type C Phospholipases