RESUMEN
The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.
Asunto(s)
Factor Inhibidor de Leucemia , Animales , Células CHO , Cricetulus , Glicosilación , Humanos , Factor Inhibidor de Leucemia/biosíntesis , Factor Inhibidor de Leucemia/química , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
In our previous study, the Kelch domain-containing 7B (KLHDC7B) was revealed to be hypermethylated at the promoter but upregulated in breast cancer. In this study, we identified a long non-coding RNA, ST8SIA6-AS1 (STAR1), whose expression was significantly associated with KLHDC7B in breast cancer (R2 = 0.3466, P < 0.01). Involvement of the two genes in tumorigenesis was examined via monitoring their effect on cellular as well as molecular events after each gene dysregulation in cultured mammary cell lines. Apoptosis of MCF-7 decreased by 49.5% and increased by 33.1%, while proliferation noted increase and decrease by up- and downregulation of KLHDC7B, respectively, suggesting its oncogenic property. STAR1, however, suppressed cell migration and increased apoptosis. Network analysis identified many target genes that appeared to have similar regulation, especially in relation to the interferon signaling pathway. Concordantly, expression of genes such as IFITs, STATs, and IL-29 in that pathway was affected by KLHDC7B and STAR1. Taken together, KLHDC7B and STAR1 are both overexpressed in breast cancer and significantly associated with gene modulation activity in the interferon signaling pathway during breast tumorigenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proliferación Celular/genética , ARN Largo no Codificante/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Interferones/genética , Interferones/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Células MCF-7 , ARN Largo no Codificante/genética , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Secretin receptor (SCTR), the G-protein coupled receptor (GPCR) for secretin, has been observed to be upregulated in a few tumor types while downregulated in others, promoting or suppressing the proliferation of tumor cells, respectively. However, little is known about the molecular regulatory mechanism of dysregulation in cancer. In the present study, an analysis of the biological pathways affected by methylation in breast cancer using the methylome databases revealed that GPCRs played a major part in the affected pathway. SCTR, one of the dysregulated GPCRs, showed hypermethylation (p<0.01) and downregulation (p<0.05) in breast cancer tissues. Pathway analysis after the downregulation of SCTR by siRNA in MCF-10A cells identified the G2/M stage checkpoint as the top-scored pathway. Cell cycle-related genes were all upregulated or downregulated suppressing cell proliferation. However, the overexpression of SCTR in MCF-7 cells led to a 35% increase of the cell proliferation index and 2.1-fold increase of cellular migration. Our findings indicate that SCTR suppresses the proliferation of normal breast cells, while the gene stimulates the proliferation and migration of cancer cells being downregulated by promoter methylation.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/biosíntesis , Proliferación Celular/genética , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/genética , Apoptosis/genética , Neoplasias de la Mama/patología , Metilación de ADN/genética , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7RESUMEN
Aberrant methylation of specific CpG sites at the promoter is widely responsible for genesis and development of various cancer types. Even though the microarray-based methylome analyzing techniques have contributed to the elucidation of the methylation change at the genome-wide level, the identification of key methylation markers or top regulatory networks appearing common in highly incident cancers through comparison analysis is still limited. In this study, we in silico performed the genome-wide methylation analysis on each 10 sets of normal and cancer pairs of five tissues: breast, colon, liver, lung, and stomach. The methylation array covers 27,578 CpG sites, corresponding to 14,495 genes, and significantly hypermethylated or hypomethylated genes in the cancer were collected (FDR adjusted p-value <0.05; methylation difference >0.3). Analysis of the dataset confirmed the methylation of previously known methylation markers and further identified novel methylation markers, such as GPX2, CLDN15, and KL. Cluster analysis using the methylome dataset resulted in a diagram with a bipartite mode distinguishing cancer cells from normal cells regardless of tissue types. The analysis further revealed that breast cancer was closest with lung cancer, whereas it was farthest from colon cancer. Pathway analysis identified that either the "cancer" related network or the "cancer" related bio-function appeared as the highest confidence in all the five cancers, whereas each cancer type represents its tissue-specific gene sets. Our results contribute toward understanding the essential abnormal epigenetic pathways involved in carcinogenesis. Further, the novel methylation markers could be applied to establish markers for cancer prognosis.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Neoplasias Gástricas/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Análisis por Conglomerados , Neoplasias del Colon/metabolismo , Islas de CpG/genética , Metilación de ADN/fisiología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/metabolismoRESUMEN
BACKGROUND: MTO1 and MRPL41 are nuclear-encoded mitochondrial genes encoding a mitochondrial tRNA-modifying enzyme and a mitochondrial ribosomal protein, respectively. Although both genes have been known to have potential roles in cancer, little is known about their molecular regulatory mechanism, particularly from an epigenetic approach. In this study, we aimed to address their epigenetic regulation through the estrogen receptor (ER) in breast cancer. METHODS: Digital differential display (DDD) was conducted to identify mammary gland-specific gene candidates including MTO1 and MRPL41. Promoter CpG methylation and expression in breast cancer cell lines and tissues were examined by methylation-specific PCR and real time RT-PCR. Effect of estradiol (E2), tamoxifen, and trichostatin A (TSA) on gene expression was examined in ER + and ER- breast cancer cell lines. Chromatin immunoprecipitation and luciferase reporter assay were performed to identify binding and influencing of the ER to the promoters. RESULTS: Examination of both cancer tissues and cell lines revealed that the two genes showed an opposite expression pattern according to ER status; higher expression of MTO1 and MRPL41 in ER- and ER+ cancer types, respectively, and their expression levels were inversely correlated with promoter methylation. Tamoxifen, E2, and TSA upregulated MTO1 expression only in ER+ cells with no significant changes in ER- cells. However, these chemicals upregulated MRPL41 expression only in ER- cells without significant changes in ER+ cells, except for tamoxifen that induced downregulation. Chromatin immunoprecipitation and luciferase reporter assay identified binding and influencing of the ER to the promoters and the binding profiles were differentially regulated in ER+ and ER- cells. CONCLUSIONS: These results indicate that different epigenetic status including promoter methylation and different responses through the ER are involved in the differential expression of MTO1 and MRPL41 in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Proteínas Mitocondriales/genética , Receptores de Estrógenos/metabolismo , Proteínas Ribosómicas/genética , Línea Celular Tumoral , Metilación de ADN , Estradiol/farmacología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas de Unión al ARN , Elementos de Respuesta , Tamoxifeno/farmacologíaRESUMEN
Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of breast cancers, and an increasing number of marker genes have been identified. However, few genes which show methylation change in accordance with the progression of breast cancer have been identified. To identify genes which consistently undergo promoter methylation alterations as the tumor develops from a benign to a malignant form, genome-wide methylation databases of breast cancer cell lines from stage I to stage IV were analyzed. Heatmap and cluster analysis revealed that the genome-wide methylation changes showed a good accordance with tumor progression. Seven out of 14,495 genes were found to be consistently increased alongside the promoter methylation level through the normal cell line to the cancer stage IV cell lines. NEFL, one of the in silico hypermethylated genes in cancer, showed hypermethylation and lower expression in the cancer cell line MDA-MB-231, as well as in cancer tissues (methylation, p<0.05; expression, p<0.01). The expression was restored by inducing demethylation of the promoter in MDA-MB-231 cells. Our findings may lend credence to the possibility of using tumor stage-specific alterations in methylation patterns as biomarkers for estimating prognosis and assessing treatment options for breast cancer.