Desensitization of neuromedin B receptors (NMB-R) on native and NMB-R-transfected cells involves down-regulation and internalization.

Abstract Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and β-catenin at the cell–cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of β-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2′-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.

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Modulation of cathepsin D activity in retinal pigment epithelial cells

Biochemical Journal ◽

10.1042/bj3240935 ◽

1997 ◽

Vol 324 (3) ◽

pp. 935-940 ◽

Cited By ~ 36

Author(s):

Piroska E. RAKOCZY ◽

C. May LAI ◽

Michael BAINES ◽

Salvatore DI GRANDI ◽

J. Helen FITTON ◽

...

Keyword(s):

Cathepsin D ◽

Enzyme Assay ◽

Retinal Pigment Epithelial ◽

Retinal Pigment ◽

Pcr Amplification ◽

Down Regulation ◽

Transfected Cells ◽

Rpe Cells ◽

Pigment Epithelial ◽

Outer Segments

This project used retinal pigment epithelial (RPE) cells to investigate the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digestive function of these cells. RPE cells were transfected with a pHβApr-1-neo vector construct carrying the full-length sequence of the translated region of human cathepsin D in sense and antisense directions. Transfected cells were characterized for the presence and expression of the transgene by PCR amplification using transgene-specific primers. Total aspartic proteinase activity present in transformed RPE cells was measured by an enzyme assay using haemoglobin as substrate. Flow cytometry was used to quantify phagocytosis of fluorescein isothiocyanate-labelled rod outer segments (ROS), and lysosomal digestion of ROS was monitored by immunofluorescence. A 435 bp fragment was present in RPE cells carrying the cathepsin D transgene in sense and antisense orientations after PCR amplification. Expression of both 52 kDa procathepsin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cells transfected with antisense cathepsin D. No other forms of cathepsin D were detected in the transfected cells, suggesting that, if pseudo-cathepsin D exists in RPE cells in vivo, it requires the presence of unknown specific regulatory elements. The up- and down-regulation of cathepsin D expression was further confirmed by enzyme assay. Transfected cells retained their phagocytosing ability after ROS challenge and maintained their ability to process ROS. The processing of ROS was significantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demonstrate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.

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Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin

Journal of Cell Science ◽

10.1242/jcs.108.10.3317 ◽

1995 ◽

Vol 108 (10) ◽

pp. 3317-3329 ◽

Cited By ~ 9

Author(s):

C. Albiges-Rizo ◽

P. Frachet ◽

M.R. Block

Keyword(s):

Antisense Rna ◽

Marked Decrease ◽

Cell Spreading ◽

Cdna Fragment ◽

Down Regulation ◽

Transfected Cells ◽

Cell Matrix ◽

Beta 1 Integrin ◽

Kinetics Of

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5′ cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.

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Epigenetic Silencing of Mir-203 Contributes to IL2Rb Overexpression and Malignant Transformation in Cutaneous T-Cell Lymphoma

Blood ◽

10.1182/blood.v124.21.3553.3553 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3553-3553

Author(s):

Ulrik Ralfkiaer ◽

Martin Jansson ◽

Katharina Kopp ◽

Andreas Willerslev-Olsen ◽

Fazila Asmar ◽

...

Keyword(s):

T Cell ◽

Tumor Suppressor ◽

Malignant Transformation ◽

Cell Lines ◽

Luciferase Reporter ◽

Melting Curve Analysis ◽

Down Regulation ◽

Transfected Cells ◽

Stat Signaling ◽

Reporter Assays

Abstract Introduction Cutaneous T-cell lymphomas (CTCL) are rare malignancies, which present in the skin and presumably arise from malignant transformation of T-cells normally destined to home to the cutaneous environment. MicroRNAs (miRs) regulate gene expression at the post transcriptional level. Many studies have shown that altered miR expression is a central event in lymphomagenesis, and that miRs have potential as both diagnostic and predictive tumor markers. In CTCL we have previously identified and validated a 3 miR classifier that distinguishes CTCL from BID with > 95% accuracy, based upon the up-regulation of miR-155 combined with the down-regulation of miR-203 and miR-205. In normal adult tissues, miR-203 is mainly associated with keratinocyte differentiation, acting to repress stemness, and to induce cell cycle arrest and differentiation. In cancer, miR-203 has been shown to hold tumor suppressor properties, and may be down-regulated by promoter hyper-methylation. The function and implications of miR-203 for CTCL has not previously been described. In this study we have investigated the regulation and function of miR-203 in primary CTCL biopsies and cell lines. Materials and Methods Twenty-one fresh frozen primary CTCL biopsies, IL-2 independent CTCL cell lines (MyLa2059 and PDB2B), and the IL-2 dependent CTCL cell lines (SeAx and SeZ4) were analyzed in this study. Promoter methylation was analyzed by methylation specific melting curve analysis. Cell lines were transfected by electroporation of miR-203 mimic or non-template-control (mirVana, Ambion). Proliferation was measured by 3H-Thymidine and apoptosis by MMT assays. MiR-203 mimic and mock transfected cells were examined by Affymetrix RNA expression arrays (GeneChip Human Gene 2.0 ST). IL2Rβ mRNA expression was confirmed by qPCR and IL2Rβ protein levels by flow cytometry as measured by CD122 (IL2Rβ-chain), compared to CD25 (IL2Rα-chain) and CD132 (IL2Rγ-chain). Cloning was done according to the manufacturers’ recommendation (In-Fusion, Clontech) and luciferase reporter assays were performed using the Dual-Glo system (Promega). Results We show that miR-203 is epigenetically silenced by DNA methylation in both CTCL cell lines and in 9 of 21 (43%) of primary CTCL samples, and that miR-203 can be up-regulated by the hypo-methylating agents 5-azacytidine and 5-aza-2-deoxycytidine in vitro. We also show, that forced miR-203 expression in CTCL cells targets known oncogenes such as p63, Survivin and CREB. Furthermore, it is shown that induction of miR-203 reduces cell viability and decreases proliferation. mRNA array analysis of miR-203 mimic and mock transfected cells lead to the identification of 19 significantly de-regulated genes (P<0.5/log fold change>2), including the as yet unrecognized miR-203 target molecule IL2Rb, which is essential for IL-2 induced JAK/STAT signaling. qPCR and FACS analysis confirmed this up-regulation both at the mRNA and protein level. The IL-2 dependent cell line SeAx showed significantly more profound down-regulation of IL2Rβ upon in miR203 transfected cell lines. Preliminary luciferase reporter assays confirm that IL2Rβ expression is regulated by miR-203, providing novel evidence that miR-203 may act in concert with IL-2/STAT in CTCL pathogenesis. These experiments are currently being validated. Conclusion We provide the first evidence that miR-203 acts as a tumor suppressor in CTCL. Furthermore we show that down-regulation of miR-203 leads to increased expression of an as yet unidentified target gene, IL2Rβ, which is directly involved in JAK/STAT signaling, that plays an essential role in the regulation of T-cell proliferation. Thus, we suggest that epigenetic miR-203 down-regulation and IL2Rβ up-regulation are important early and driving events in CTCL pathogenesis. Disclosures No relevant conflicts of interest to declare.

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LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21WAF1/Cip1 expression

Biochemistry and Cell Biology ◽

10.1139/o10-162 ◽

2011 ◽

Vol 89 (3) ◽

pp. 287-298 ◽

Cited By ~ 12

Author(s):

Wei Liu ◽

Qinsheng Dai ◽

Na Lu ◽

Libin Wei ◽

Jun Ha ◽

...

Keyword(s):

Cell Cycle ◽

Colorectal Carcinoma ◽

Cell Cycle Arrest ◽

Caspase 3 ◽

Down Regulation ◽

Transfected Cells ◽

Cycle Arrest ◽

Hct 116 ◽

Human Colorectal Carcinoma ◽

Hct 116 Cells

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21WAF1/Cip1 and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21WAF1/Cip1, as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21WAF1/Cip1 and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53–p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.

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miR-21 down-regulation promotes apoptosis and inhibits invasion and migration abilities of OVCAR3 cells

Clinical & Investigative Medicine ◽

10.25011/cim.v34i5.15671 ◽

2011 ◽

Vol 34 (5) ◽

pp. 281 ◽

Cited By ~ 21

Author(s):

Yanhui Lou ◽

Zhumei Cui ◽

Fuling Wang ◽

Xingsheng Yang ◽

Jinhua Qian

Keyword(s):

Cell Proliferation ◽

Migration And Invasion ◽

Control Group ◽

Control Groups ◽

Down Regulation ◽

Transfected Cells ◽

Invasion And Migration ◽

Scratch Wound ◽

Scratch Wound Assay ◽

And Migration

Purpose: To investigate the influence of miR-21 down-regulation on cell proliferation, apoptosis, invasion and migration of ovarian papillary adenocarcinoma cell lines (OVCAR3). Methods: Short-hairpin RNA (shRNA), specifically targeting miR-21, was constructed and transfected into OVCAR3 cells using the pSIREN-RetroQ linear vector (pSIREN-miR-21). The expression of miR-21 was detected with stem-loop real-time RT-PCR in OVCAR3 cells. Cell proliferation and apoptosis were monitored using the MTT assay and flow cytometry, respectively. Cell migration and invasion were assessed using the transwell migration and scratch-wound assay, respectively. Western-bloting was used for PDCD4 protein expression. Results: pSIREN-miR-21 suppressed miR-21 expression in OVCAR3 cells. miR-21 expression levels in pSIREN-miR-21 cells was 0.3 ± 0.1, which was significantly lower when compared with pSIREN-miR-21-Neg and control groups (P < 0.01). Cell inhibition rate in the pSIREN-miR-21 group was higher than the control group (29.4% vs 9.0%, P < 0.01), as was the percentage of apoptotic and necrotic cells. By transwell migration assay, the number of cells migrating in the pSIREN-miR-21 group was significantly lower than in the control group. In addition, fewer cells were observed in the wounded area of the pSIREN-miR-21 group following the scratch-wound assay. PDCD4 expression was increased in OVCAR-3 cells transfected by pSIREN-miR-21 compared with vector-control transfected cells. Moreover, the optical density of the transfected cells was significantly lower than the two control groups.

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Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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