Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: Inhibition of ERK/RSK signalling decreases constitutive activation of mTORC1 in melanoma. (A) Four melanoma cell lines were used in this study. While A375 and Colo829 cells harbour a B-Raf V600E mutation, WM852 and WM1361 cells carry an N-Ras mutation at Q61 (R or K). (B) Phosphorylation of endogenous RSK, ERK1/2, rpS6, S6K1 and 4E-BP1 was monitored in total extracts from serum-starved melanoma cell lines and normal human melanocytes treated or not with insulin (100 nM) for 30 min. Cell lysates were also immunoblotted for total protein levels (RSK1, ERK1, rpS6, S6K1, 4E-BP1 and β-actin). (C) Serum-starved melanoma cells were treated with the indicated inhibitors for 60 minutes. Immunoprecipitated S6K1 kinase activity was assayed as in Fig. 1. (D) Phosphorylation of endogenous rpS6 and S6K1, and total rpS6 protein level were monitored by immunoblotting.

Article Snippet: The human epidermal melanocytic (105-25N) cell line was purchased from Cell Applications (San Diego, CA) and grown in Melanocyte Growth Medium (135-500, Cell Applications).

Techniques: Inhibition, Activation Assay, Mutagenesis, Phospho-proteomics, Immunoprecipitation, Activity Assay, Western Blot

Journal:

Article Title: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons

doi: 10.1158/0008-5472.CAN-05-2303

Figure Lengend Snippet: TUNEL assay: (FITC) positive DNA was used to assess apoptosis of cells that had been treated with IFNs over 4–5 days 16h after plating. All three malignant cell lines were resistant to apoptosis induction by up to 500 U/ml of IFN-α2 or IFN-β (A–C). In renal carcinoma (A, B) and melanoma (C) cells pretreatment with 200 nM 5-AZA-dC (AZA), daily over 2–6 days before IFN treatment overcame resistance to apoptosis induction by 50 to 100 U/ml IFN-α2 or IFN-β, while causing little to moderate apoptosis alone (5–20% TUNEL + cells). Normal neonatal human melanocytes (NHEM) and normal kidney epithelial cells (NKE) did not become sensitive to apoptosis induction by IFN-β (100 U/ml) after pretreatment with 200 nM 5-AZA-dC (AZA) daily over 4 days, which alone caused little apoptosis (up to 10% TUNEL + cells) (D). Reduction in DNMT1 protein was confirmed in whole cell lysates isolated after 4 days of 5-AZA-dC at 200 nM and subjected to SDS-PAGE and western blot analysis (A-D). 5-AZA-dC markedly decreased free DNMT1 protein in ACHN, SK-RC-45, A375, NHEM, and NKE cells (A-D). TUNEL graphs show the means and standard deviations (error bars) of independent experiments.

Article Snippet: NHEM normal human neonatal melanocytes (Cambrex, Baltimore, MD) were cultured under the same incubator conditions but in melanocyte media (Cambrex, Baltimore, MD) according to the supplier’s recommendations.

Techniques: TUNEL Assay, Isolation, SDS Page, Western Blot

Journal:

Article Title: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons

doi: 10.1158/0008-5472.CAN-05-2303

Figure Lengend Snippet: A) To reactivate RASSF1A mRNA expression indicated cell lines were treated daily with 40 nM DNMT1 AS (AS) over 4–8 d (ACHN) or 6d (SK-RC-45). Mismatch control oligonucleotide (MM) or lipofectin transfection reagent alone (L) were used as control treatments. Alternatively RASSF1A mRNA expression was reactivated by daily 5-AZA-dC treatments at 200 nM over 2–6 (ACHN) or 4 d (SK-RC-45, A375). For RASSF1A RT-PCR 500 ng RNA transcribed into cDNA was amplified over 35 cycles with RASSF1A primers; for GAPDH detection 250 ng RNA, transcribed into cDNA were amplified over 20 cycles. HeLa cells served as positive controls (19, 21, 23, 24) and WM9 cells as well as normal melanocytes (NHEM) and kidney epithelial (NKE) cells were found to express RASSF1A mRNA at baseline. B): Approximately 100 ng of bisulfite modified DNA from ACHN cells was used for methylation specific PCR. Bisulfite modified DNA from HeLa cells was unmethylated control. Forty nM DNMT1 AS over 8 days led to partial demethylation of RASSF1A promoter CpG island in ACHN cells. C): ACHN cells were transfected daily with 40 nM DNMT1 antisense (DNMT1 AS) or mismatch control oligonucleotide (MM) over 8 days before treatment with IFNs over 48 hr. Floating and adherent cells were harvested for RASSF1A (mAB) and MST1 (pAB) immunoblotting. DNMT1 AS reactivated RASSF1A protein expression, which was further augmented by IFNs. Cleavage (activation) of MST1 by IFNs occurred only in RASSF1A expressing DNMT1 pretreated ACHN cells. Augmentation of RASSF1A protein expression by IFN was also observed in A375 cells pretreated with 5-AZA-dC (AZA) at 200 nM daily over 4 days, and without pretreatment in WM9 cells, known to be sensitive to apoptosis induction by IFNs (27). Similar results were obtained in independent experiments.

Article Snippet: NHEM normal human neonatal melanocytes (Cambrex, Baltimore, MD) were cultured under the same incubator conditions but in melanocyte media (Cambrex, Baltimore, MD) according to the supplier’s recommendations.

Techniques: Expressing, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Modification, Methylation, Western Blot, Activation Assay