hcc1937 brca1 mutated tumor cells  (ATCC)

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Influence of BRCA1 on IR-induced phosphorylation and localization of ATM. (A) HCC1937 (BRCA1-deficient) cells were transiently transfected with a human expression vector, carrying (BRCA1wt) or not carrying (vector) a wild-type BRCA1 gene. Following exposure to indicated doses of ionizing irradiation, total and phosphorylated ATM was assessed by immunoblot after immunoprecipitation, and total and phosphorylated SMC1 and total BRCA1 were assessed by immunoblot of whole-cell extracts. (B) Subcellular localization of BRCA1, phophorylated ATM, and H2AXγ in BRCA1-deficient or -proficient cells. Cells were fixed 15 min after 0 Gy (IR-) or 5 Gy (IR+) of γ irradiation, then labeled with antibodies or DAPI as indicated.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Transfection, Expressing, Plasmid Preparation, Irradiation, Western Blot, Immunoprecipitation, Labeling

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Interdependencies of IR-induced foci formation and pbosphorylation of NBS1 and BRCA1. (A,B) Subcellular localization of NBS1 and BRCA1 in HCC1937 (BRCA1-deficient) cells transfected with a transient expression vector carrying BRCA1 gene (BRCA1wt) or an empty vector (vector; A) and NBS1-LB1 (NBS1-deficient) cells stably transfected with a retrovirus vector carrying p95/Nbs1 gene (p95) or an empty vector (vec) were analyzed by immunofluorescence microscopy (B). Cells were fixed 15 min after 0 Gy (IR-) or 5 Gy (IR+) of γ irradiation, then labeled with anti-NBS1 (shown in red) or anti-BRCA1 (shown in green). DNA was visualized by staining with DAPI (shown in blue). (C) HCC1937 (BRCA1-deficient) cells were transiently transfected with a human expression vector, pcDNA3 carrying (BRCA1wt) or not carrying (vector) wild-type BRCA1 gene. Following exposure to indicated doses of ionizing irradiation, cellular extracts were subjected to immunoblotting. Cell extracts were blotted with antibody to phosphorylated Ser 343 of NBS1, then reblotted with anti-NBS1 antibody. (D) Cellular extracts of NBS1-LB1 cells stably transfected with a retrovirus vector carrying p95/NBS1 gene [p95(NBS1)] or an empty vector (vector) exposed to indicated doses of ionizing irradiation were separated on 5% Tris-acetate gel and blotted with anti-BRCA1 antibody.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Transfection, Expressing, Plasmid Preparation, Stable Transfection, Immunofluorescence, Microscopy, Irradiation, Labeling, Staining, Western Blot

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Targeted modification of the mouse Smc1 gene and effects on radiation responses. (A) Schematic of Smc1 knock-in procedure. (panel i) Genomic structure of the mouse Smc1 gene on chromosome X. The whole genomic Smc1 gene consists of 26 exons shown as boxes. Targeted region into which to insert the knock-in vector is shown as a thick bar. (panel ii) Targeting construct of the knock-in vector, pSmc1KI–Neotk, targeted locus, and targeted locus following cre-mediated recombination. Neomycin resistance and thymidine kinase genes (Neo-tk) flanked by loxP sites (shown as black triangles) are inserted into an intron between exon 18 and 19. In pSmc1KI–Neotk, two nucleotide exchanges to change serine to alanine at amino acid positions 957 and 966 are made in exon 19 (shown as a dark box labeled as 19*). Homologus recombination of the pSmc1KI–Neotk is depicted by the large “X”s. Positions of the flanking probe for genotyping and BstEII sites are indicated. (B) Wild-type (Smc1WT) or Smc1S957AS966A immortalized mouse fibroblast cells were exposed to 0 Gy (IR-) or 10 Gy (IR+) of ionizing irradiation and cellular extracts were prepared 30 min later. Whole-cell lysates were subjected to immunoblotting with antibodies as indicated. (C) Thirty minutes after exposure to 0 (-) or 10 (+) Gy of ionizing irradiation (IR), whole-cell lysates were prepared from wild-type or mutant knock-in fibroblasts were immunoblotted with anti-mouse NBS1 or BRCA1 antibodies. Duplicate samples of cell lysates from irradiated knock-in cells were treated with protein phosphatase (PPase+) prior to electrophoresis.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Modification, Knock-In, Plasmid Preparation, Construct, Labeling, Irradiation, Western Blot, Mutagenesis, Electrophoresis

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: SMC1 phosphorylation is not required for IR-induced formation of foci containing phospho-ATM, H2AXγ, NBS1, 53BP1, phosphorylated CHK2, or BRCA1. Wild-type (Smc1WT) or Smc1 phosphorylation mutant knock-in (Smc1S957AS966A) fibroblast cells were fixed with 4% paraformaldehyde 30 min after 0 Gy (-) 10 Gy (+) of ionizing irradiation (IR), then subjected to immunofluorescence microscopy. (A) Staining with antibodies recognizing phosphorylated ATM or SMC1. (B) Staining with antibodies recognizing H2AXγ, NBS1, 53BP1, phosphorylated CHK2, BRCA1, and phosphorylated SMC1. For costaining of phosphorylated SMC1 (red) with BRCA1 (green), rabbit polyclonal anti-Ser957p antibody was used.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Mutagenesis, Knock-In, Irradiation, Immunofluorescence, Microscopy, Staining

Journal:

Article Title: Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

doi: 10.1101/gad.1200304

Figure Lengend Snippet: Proposed model for an IR-induced signaling pathway. Chromatin structure changes caused by DNA breakage or other mechanisms leads to intermolecular autophosphorylation of ATM dimers, resulting in release of phosphorylated and active ATM monomers. If DNA-strand breaks are present, several proteins, including NBS1 and BRCA1, are recruited to the sites of the breaks independent of the ATM activation process. After activation, monomeric ATM can phosphorylate nucleoplasmic substrates, like p53, and if NBS1 and BRCA1 have localized to DNA breaks, activated ATM is recruited to the break. At the DNA break, activated ATM can phosphorylate substrates, including SMC1. The phosphorylation of SMC1 reduces chromosomal breakage and enhances cell survival.

Article Snippet: HCC1937 (BRCA1-mutated) tumor cells derived from a human ductal carcinoma was cultured in VitaCell (RPMI 1640 with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, 90%; ATCC) supplemented with 10% FBS.

Techniques: Activation Assay