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TAZ/WWTR1 for diagnosis and treatment of cancer

A cancer, metastatic cancer technology, applied in molecular biology and genetics, cell biology, medicine

Inactive Publication Date: 2010-12-15
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Around 1 in 4 cases will die from their disease despite better diagnosis and routine screening

Method used

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  • TAZ/WWTR1 for diagnosis and treatment of cancer
  • TAZ/WWTR1 for diagnosis and treatment of cancer
  • TAZ/WWTR1 for diagnosis and treatment of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0523] Example 1: Cell Lines and Plasmids

[0524] Cell lines MCF10A, MCF7, MDA-MB-231, Hs578T, ZR-75-1 were purchased from the American Type Culture Collection and maintained in the recommended medium except for MCF10A cells, which were Cultures were performed in DMEM supplemented with 5% horse serum, 20 ng / ml EGF, 0.5 μg / ml hydrocortisone, 100 ng / ml cholera toxin, 10 μg / ml insulin and penicillin / streptomycin. BT-549 cells were cultured in RPMI supplemented with 10% FBS. MDA-MB-435S, T-47D cells were from Lo Ting Ling (Institute of Molecular and Cellular Biology) and maintained in DMEM supplemented with 10% FBS and 10 μg / ml insulin. BT-20, MDA-MB-453 and BT-474 were provided by Yoshiaki Ito (Institute of Molecular and Cellular Biology). BT-20 cells were maintained in MEM supplemented with 10% FBS. MDA-MB-453 cells were maintained in DMEM with 10% FBS supplemented with 10 μg / ml insulin. Amphotropic Phoenix packaging cells were kindly provided by G. Nolan (Stanford Universi...

Embodiment 2

[0526] Example 2: Purification of GST-tagged proteins

[0527] 1 liter of Escherichia coli BL21(DE3) carrying the pGEX-TAZ or pGEX-YAP construct was cultured in LB medium until OD 0.8-1, and was incubated with 0.1 mM IPTG (isopropyl-β-D-thio galactopyranoside) induced overnight. Cells were harvested by centrifugation and lysed by sonication in phosphate buffered saline (PBS). The lysate was spun at 10,000 g for 30 minutes at 40°C and the supernatant was mixed with 0.5 ml Glutathione-Sepharose 4B (Amersham Biosciences) for 2 hours at 4°C. Wash beads four times with PBS. Bound protein was eluted with 5 volumes of 10 mM reduced glutathione in 50 mM Tris, pH 8.

Embodiment 3

[0528] Example 3: Purification of His-tagged proteins

[0529] One liter of E. coli BL21(DE3) carrying the pET-TAZ fusion construct was induced as pGEX-TAZ. By breaking the buffer (100mM Hepes-KOH, pH7.4, 5mM MgCl 2 , 500 mM KCl, 0.1% Triton-X-100, 2 mM β-mercaptoethanol and a protease inhibitor cocktail from Roche Molecular Biochemicals) to lyse the bacteria. The lysates were spun and the supernatant collected and incubated with 1 ml Talon metal affinity resin (Clontech) for 2 hours at 4°C. The resin was washed four times with wash buffer (20 mM Hepes, pH 7.4, 200 mM KCl, 10% glycerol, 10 mM imidazole, and 2 mM β-mercaptoethanol), and washed with 5 volumes of elution buffer (20 mM Hepes, pH 7.4, 200 mM KCl, 10% glycerol, 250 mM imidazole and 2 mM β-mercaptoethanol) elution.

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Abstract

The invention provides an anti-TAZ agent for the treatment, prophylaxis or alleviation of cancer. We further provide a kit for detecting breast cancer in an individual or susceptibility of the individual to breast cancer comprising means for detection of TAZ expression in the individual or a sample taken from him or her as well as a method of detecting a cancer cell, the method comprising detecting modulation of expression, amount or activity of TAZ in the cell.

Description

field of invention [0001] The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. The present invention relates to the field of medicine. In particular, it relates to the treatment and diagnosis of diseases, especially breast cancer, and compositions for such use. Background of the invention [0002] TAZ (also known as WWTR1) is a 14-3-3 binding protein with a PDZ binding motif. TAZ / WWTR1 is known to regulate mesenchymal stem cell differentiation. [0003] TAZ / WWTR1 was first cloned by Kanai et al. (2000). Kanai showed the highest expression of TAZ RNA in human kidney, followed by heart, placenta and lung. Expression was detected in all tissues tested except thymus and peripheral blood leukocytes. Northern blot analysis of mouse tissues showed that the transcript was expressed at the highest level in kidney, lung, liver, and heart, but also in testis. Western blot analysis revealed expression of TAZ in several epithelial a...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/16G01N33/68A61K39/395C12N15/63C12N5/10C12N1/21A61P35/00
CPCC12N2310/14C12N2503/02G01N33/57415G01N33/6893C12N5/0693C07K16/18C12N15/113C12N2310/111A61P35/00A61K38/17A61K39/39558A61K48/00C12N5/10C12N15/1135C12N15/63
Inventor 洪万进曾秀薇
Owner AGENCY FOR SCI TECH & RES
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