alcam promoter  (New England Biolabs)

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: Transcription initiation sites in the ALCAM gene . A) Schematic diagram of the 5'RACE approach. Positions of primers AL1, AL2 and AL3 are shown. B) Ethidium bromide stained agarose gel showing DNA marker (lane 1), and products of 5'RACE reaction using primers AAP and AL2 (lane 2), UAP and AL3 (lane 3). Lane 4 is negative PCR control (BL). C) Sequence of a 5' UTR clone of ALCAM showing multiple sites of RNA synthesis (arrows.) The most common site identified is shown underlined. Sequence of AL3 primer is double-underlined.

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Staining, Agarose Gel Electrophoresis, Marker, Sequencing

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: ALCAM promoter activity in melanoma tumor cells . A) Schematic diagram of DNA constructs used for reporter gene assays depicting truncations of the ALCAM promoter cloned upstream of a promoter-less luciferase (Luc) gene. Histogram shows the relative luciferase activity for each construct in melanoma cell lines. B) Schematic diagram as described above, and activity of the p1200ALCAMLuc construct containing a wild-type (filled circle) or mutant (open circle) consensus binding sequence for NF-κB in the ALCAM promoter.

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Activity Assay, Construct, Clone Assay, Luciferase, Mutagenesis, Binding Assay, Sequencing

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: NF-κB binding to the ALCAM promoter and activity of the ALCAM promoter in breast cancer cell lines . A) p65 binding and trans-activation of the ALCAM promoter. (i) EMSA showing mobility of the -1140 NF-κB probe in the absence of nuclear extract (lane 1). Complexes B1, B2 and B3 form in the presence of LOX melanoma cell nuclear extracts (lane 2), subsequent lanes contain unlabelled wild-type NF-κB probe (lane 3), unlabelled mutant NF-κB probe (lane 4) and anti-p65 antibody (lane 5). (ii) ChIP assay showing PCR products for the -1140 ALCAM NF-κB motif and flanking DNA sequence amplified from chromatin of C8161.9 (lane1), LOX (lane 2) and Meljuso (lane3) cells precipitated with anti-p65 antibodies. Absence of PCR product in immunoprecipitation with non-immune IgG (lane 4), lane 5 (input DNA). (iii) Activity of p1200ALCAMLuc in C8161.9 and LOX cells over-expressing p65 or control vector. B) Schematic diagram of ALCAM reporter constructs and histogram showing relative luciferase activity for each in breast cancer cells with high (HCC70, MDA-MB-231, T47D), medium (BT549, CAMA-1) and low (HCC 1500, MCF-7,-SK-BR-3) ALCAM expression.

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Binding Assay, Activity Assay, Activation Assay, Mutagenesis, Sequencing, Amplification, Immunoprecipitation, Expressing, Plasmid Preparation, Construct, Luciferase

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: Promoter methylation regulates ALCAM expression in tumor cells . A) Sequence of the proximal ALCAM promoter showing multiple CpG islands (in bold) and methylation-specific primer targets (underlined). Ethidium bromide stained agarose gel showing PCR products for unmethylated (U) and methylated (M) genomic DNA. B) CpG methylation profile of the ALCAM promoter in the interval -62 and -409 relative to the cap site as determined by pyrosequencing for the indicated breast (MDA-MB-435, MCF-7, HCC-1428, HCC-1008) and melanoma (FEMX-1, MelJuso, LOX, C8161.9) tumor cells. ALCAM protein detected by western blot analysis is shown for each cell line. Note that the maximum value on the %CpG methylatyion axis (y) for the ALCAM-negative tumor cells MDA-MB-435 and FEMX1 are 75% and 100% respectively, and 10% for cells with endogenous ALCAM expression.

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Methylation, Expressing, Sequencing, Staining, Agarose Gel Electrophoresis, CpG Methylation Assay, Western Blot

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: 5-aza-deoxydytidine activates ALCAM expression in tumor cells . A) top panel; Histogram showing baseline methylation at each CpG sites in the ALCAM promoter in MDA-MB-435 cells (blank columns) and the percent methylation following treatment with 5 μM 5-aza-deoxycytidine (filled columns). Lower panel; Profile of DNA demethylation across the proximal ALCAM promoter achieved by treating MDA-MB-435 cells with 5 μM 5-aza-deoxycytidine. B) Dose-dependent re-activation of ALCAM mRNA and protein expression in MDA-MB-435 cells treated with a concentration range (0-10 μM) of 5-aza-deoxycytidine.

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Expressing, Methylation, Activation Assay, Concentration Assay

Journal: Molecular Cancer

Article Title: Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

doi: 10.1186/1476-4598-9-266

Figure Lengend Snippet: ALCAM clusters tumor cells circulating through the lung . A) Ectopic expression of ALCAM in MDA-MB-435 tumor cells. Left panel ; Western blot analysis of ALCAM in MDA-MB-435 clones transfected with an empty vector (lane 1) or, an ALCAM-GFP vector (lane 2). Protein loading was verified by probing the same western blot filter for the house keeping protein EF1-α. Right panel ; Monolayer of MDA-MB-435-ALCAM-GFP cells were examined by live-cell microscopy to reveal expression of ALCAM at sites of cell-cell contact. B) Medium power image show virtual absence of control MDA-MB-435 cells, except for a single cell (arrow) while the ALCAM-expressing MDA-MB-435 clones form clusters (brown stain) after 90 minute perfusion in rat lungs. C) Number of tumor cells retained in rat lungs in experiments using ALCAM-positive (n = 7), or ALCAM-negative MDA-MB-435 cells (n = 4) pre-treated with non-immune IgG or monoclonal anti-ALCAM antibody. D) Number of tumor cell clusters retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7). E) Number of single tumor cells retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7).

Article Snippet: Cells (8 × 10 4 ) were seeded in 24-well tissue culture plates and co-transfected with ALCAM promoter luciferase plasmids (800 ng) and pcDNA3.1/His/LacZ (100 ng) (Invitrogen) plasmid DNA using lipofectamine 2000.

Techniques: Expressing, Western Blot, Clone Assay, Transfection, Plasmid Preparation, Microscopy, Staining