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1.
Int J Oncol ; 43(6): 2073-81, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24141649

RESUMO

Permanently growing cell lines can be invaluable because of their usefulness in a variety of experimental situations. We report the characteristics of seven cell lines designated, SNU-306, SNU-334, SNU-1528, SNU-1553, SNU-1581, SNU-1958 and SNU-2372, which were established from three primary carcinomas, two pleural effusion, one pericardial effusion and one ascitic fluid samples obtained from seven Korean breast carcinoma patients. The histopathology of the primary tumors and their in vitro growth characteristics are described. DNA fingerprinting analysis and genetic alterations in the p53 and EGFR genes were conducted. The expression levels of the ER-α, PR, C-erbB2, E-cadherin, COX-2, MDR and MXR genes were investigated and sensitivity to anticancer drugs was screened. Growth was as adherent cells (four cell lines), floating aggregates (one cell line) and both (two cell lines). All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis using 18 microsatellite markers. Estrogen receptor (ER) mRNA was highly expressed in five cell lines and low or undetectable in SNU-1958 and SNU-2372. Progesterone receptor (PR) mRNA was expressed only in the SNU-306. SNU-1958 and SNU-2372 were hormone receptor-negative and C-erbB2-negative (triple-negative). SNU-1528 had an in-frame deletion of 42 base pairs of p53 gene and showed over 20-fold resistance for taxol compared to the other cell lines. There were no mutation in the EGFR gene; COX-2 was expressed in four cell lines and MXR was expressed in two cell lines. These well-characterized seven breast cancer cell lines, which include two triple-negative cell lines, will be useful for the study of breast cancer biology.


Assuntos
Receptor alfa de Estrogênio/genética , Receptor ErbB-2/genética , Receptores de Progesterona/genética , Neoplasias de Mama Triplo Negativas/genética , Caderinas/biossíntese , Técnicas de Cultura de Células , Ciclo-Oxigenase 2/biossíntese , Impressões Digitais de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/biossíntese , Feminino , Genes MDR , Humanos , Proteínas de Membrana/genética , RNA Mensageiro/biossíntese , Receptor ErbB-2/biossíntese , Receptores de Progesterona/biossíntese , Células Tumorais Cultivadas
2.
Hepatology ; 56(5): 1838-48, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22711680

RESUMO

UNLABELLED: V-set and Ig domain-containing 4 (VSIG4, CRIg, or Z39Ig), a newly identified B7-related cosignaling molecule, is a complement receptor and a coinhibitory ligand that negatively regulates T-cell immunity. Despite its exclusive expression on liver Kupffer cells (KCs) that play key roles in liver tolerance, the physiological role of VSIG4 in liver tolerance remains undefined. Mice lacking VSIG4 had poor survival rates and severe liver pathology in a concanavalin A (ConA)-induced hepatitis (CIH) model, which could be prevented by adoptive transfer of VSIG4(+) KCs. The absence of VSIG4 rendered endogenous liver T- and natural killer T (NKT)-cells more responsive to antigen-specific stimulation and impaired tolerance induction in those cells against their cognate antigens. T-cell costimulation with VSIG4.Ig suppressed Th1-, Th2-, and Th17-type cytokine production and arrested the cell cycle at the G(0) /G(1) phase but did not induce apoptosis in vitro. VSIG4-mediated tolerance induction and cell-cycle arrest were further supported by down-regulation of G(1) phase-specific Cdk2, Cdk4, and Cdk6, and up-regulation of tolerance-inducing p27(KIP-1) in VSIG4.Ig-stimulated T-cells. Administration of soluble VSIG4.Ig to wildtype mice prevented CIH development and prolonged the survival of mice with established CIH. CONCLUSION: Collectively, our results suggest that VSIG4(+) KCs play a critical role in the induction and maintenance of liver T- and NKT-cell tolerance, and that modulation of the VSIG4 pathway using a VSIG4.Ig fusion protein may provide useful immunological therapies against immune-mediated liver injury including autoimmune hepatitis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Tolerância Imunológica , Células de Kupffer/imunologia , Fígado/imunologia , Fígado/lesões , Receptores de Complemento/imunologia , Linfócitos T/imunologia , Animais , Proliferação de Células , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Técnicas de Cocultura , Concanavalina A/toxicidade , Citocinas/metabolismo , Galactosilceramidas/imunologia , Hepatite/imunologia , Imunoglobulina G/imunologia , Células de Kupffer/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Ovalbumina/imunologia , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo
3.
Methods Mol Biol ; 791: 23-32, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21913069

RESUMO

DNA methylation patterns in CpG-rich regions of promoter, CpG islands, are concerned in regulation of gene expression in mammalian cells. Excessive methylation of CpG dinucleotides in promoter represses the gene expression. In cancer, especially, gene silencing is occurred through aberrant methylation in promoter of tumor suppressor genes. Methylation-specific PCR (MSP) is a method for analysis of DNA methylation patterns in CpG islands. For performing MSP, DNA is modified by and PCR performed with two primer pairs, which are detectable methylated and unmethylated DNA, respectively. MSP is a rapid measure for assession of the methylation status in CpG island.


Assuntos
Metilação de DNA/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Linhagem Celular , Ilhas de CpG/genética , Criopreservação , DNA/genética , DNA/isolamento & purificação , Metilação de DNA/efeitos dos fármacos , Primers do DNA/genética , Inclusão em Parafina , Sulfitos/farmacologia
4.
Cell Oncol (Dordr) ; 34(1): 45-54, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21290211

RESUMO

BACKGROUND: Six human lung cancer cell lines (SNU-371, SNU-963, SNU-1327, SNU-1330, SNU-2292 and SNU-2315) were newly established through primary cell cultures. These cell lines were derived from a pulmonary blastoma, a small cell lung cancer, three adenocarcinomas and a squamous cell carcinoma of the lung of six Korean lung cancer patients. METHODS: The histopathology of the primary tumors and their in vitro growth characteristics were described. DNA fingerprinting analysis and genetic alterations in the p53, ß-catenin, TGFßRII, K-ras and EGFR genes were conducted. mRNA expressions levels of E-cadherin, COX-2, MDR1, MXR, CGA, synatophysin and TTF1 genes were investigated and sensitivity to anticancer drugs was screened. RESULTS: Five cell lines grew as adherent cells and one cell line grew as floating aggregates. All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis. A significant polymorphism at codon 72 (Arg to Pro) of the p53 gene was found in one line (SNU-1327) and a mutation at codon 176 was found in SNU-2292. No mutations in the K-ras, ß-catenin and TGF-ßRII genes were observed. E-cadherin was not expressed in SNU-371 and COX-2 was overexpressed in SNU-1330, SNU-2292 and SNU-2315 cell lines. MDR1 was overexpressed in SNU-371 and SNU-2292 cell lines and MXR was overexpressed in SNU-1327 cell line. Interestingly, the SNU-371 cell line derived from a pulmonary blastoma and which overexpressed MDR1 displayed cross resistance for several anticancer drugs. Neuroendocrine markers, chromogranin A and synaptophysin, were overexpressed in the small cell lung cancer cell line, SNU-963 and thyroid transcription factor-1 was also over expressed in this cell line. Two mutations (p.Glu746_Ser752delinsVal and p.Glu746_Ala750del) in exon 19 of EGFR were found in SNU-1330 and SNU-2315 cell lines, respectively. CONCLUSION: These well-characterized lung cancer cell lines may be useful tools for investigations of the biological characteristics of lung cancers, particularly for investigations related to mutations of EGFR.


Assuntos
Antineoplásicos/farmacologia , Linhagem Celular Tumoral/patologia , Receptores ErbB/genética , Genes Neoplásicos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Forma Celular/efeitos dos fármacos , Impressões Digitais de DNA , Análise Mutacional de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Loci Gênicos/genética , Humanos , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo
5.
World J Gastroenterol ; 16(25): 3153-60, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20593500

RESUMO

AIM: To understand CD133 promoter hypermethylation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfite modification and by clonal sequencing analysis. The correlation between expression and promoter methylation of CD133 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. RESULTS: We measured CD133 expression levels in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low levels of CD133 expression in 34.4% of cell lines. To verify the relation between CD133 expression and methylation status of the CD133 gene promoter in colorectal carcinogenesis, CD133 gene promoter hypermethylation was analyzed in 32 cancer cell lines. Promoter hypermethylation was detected in 13 (40.6%) of the cell lines using methylation specific-PCR and confirmed by bisulfite sequencing analysis. Treatment of 11 of the cell lines with the demethylation agent 5-aza-2'-deoxycytidine recovered CD133 expression in most of them. CONCLUSION: Transcriptional repression of CD133 is caused by promoter hypermethylation of the CD133 CpG islands in some of colorectal cancer cell lines. The study may contribute to the understanding of the role of CD133 inactivation in the progression of colorectal cancers.


Assuntos
Antígenos CD/genética , Neoplasias Colorretais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Peptídeos/genética , Regiões Promotoras Genéticas , Antígeno AC133 , Antígenos CD/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Ilhas de CpG , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Análise de Sequência de DNA
6.
Carcinogenesis ; 31(6): 1003-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20176655

RESUMO

Thirteen human colorectal cancer (CRC) cell lines were established from 10 primary tumors and 3 metastatic tumors obtained from 13 Korean patients. Characteristics of the cell lines including morphology in vivo and in vitro; mutations of the K-ras, p53, APC and MMR genes and microsatellite instability (MSI) status in vitro were determined. Expression of drug-sensitivity genes including MDR1, MXR, MRP1 and COX2 was also analyzed. The cell lines were unique as judged by DNA fingerprinting using 16 short tandem repeats. Eleven of the cell lines grew as adherent populations and the remaining two as floating aggregates. None of the cell lines were contaminated with Mycoplasma or bacteria. All cell lines showed high viability with relatively long doubling times. Six cell lines contained mutations at K-ras. Seven cell lines displayed p53 gene missense, nonsense and frameshift mutations. MSI was found in three cell lines and two cell lines with an MSI-high phenotype-possessed hMLH1 mutations. Nine cell lines had an APC mutation. MRP1 was highly expressed in all cell lines, and high expression of MDR1, MXR and COX2 evident in eight, six and six cell lines, respectively. Embryonal stem cell markers (MELK, SOX4 and OCT4) were expressed in most of cell lines. The cancer stem cell biomarkers CD133, CD44 and Lgr5 were expressed in 12, 13 and 13 cell lines, respectively. The presently well-characterized CRC cell lines should be useful in investigations of the biological characteristics of CRC, particularly for investigations related to gene alterations associated with CRC and biology of cancer stem cells.


Assuntos
Biomarcadores/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Int J Cancer ; 124(6): 1258-62, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19089928

RESUMO

Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell-to-cell and cell-to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation-specific PCR (MSP) and bisulfate-modified DNA sequencing analysis. Methylated cells were treated with 5-aza-2'-deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70-90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5-aza-2'-deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down-regulated by aberrant promoter hypermethylation during the progression of colorectal cancer.


Assuntos
Proteínas ADAM/genética , Neoplasias Colorretais/genética , Regiões Promotoras Genéticas , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA , Primers do DNA , DNA de Neoplasias/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética
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