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Application of telomere-binding protein daxx in preparation of tumor cell regulator

A telomere-binding protein and tumor cell technology, which is applied in the application field of telomere-binding protein DAXX in the preparation of tumor cell regulators, can solve problems such as unclear and limited, and achieve the effect of promoting activity

Active Publication Date: 2016-04-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, however, it has been reported that cancers with telomerase activity can escape telomerase-inhibitory therapies by activating ALT, allowing cancer cells to survive.
At present, the research on the specific mechanism of this telomerase-independent way of extending telomeres (ALT) is still very limited, especially whether it is related to the elongation mechanism activated by telomerase

Method used

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  • Application of telomere-binding protein daxx in preparation of tumor cell regulator
  • Application of telomere-binding protein daxx in preparation of tumor cell regulator
  • Application of telomere-binding protein daxx in preparation of tumor cell regulator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 Immunofluorescence experiment

[0055] 1. Experimental materials

[0056] Reagents: the rabbit polyanti-DAXX endogenous antibody used in this experiment (purchased from Santa Cruz Company, product number sc-7152), diluted 1:200 with 3% bovine serum albumin (BSA) when used; coilin antibody (purchased from Abcam Company, product number ab11822), diluted 1:2000 with 3% BSA when used; PML antibody (purchased from Santa Cruz Company, product number sc-966), diluted 1:100 with 3% BSA when used; HA antibody (purchased from sigma company, product number H3663), diluted 1:500 with 3% BSA when used; secondary antibody (goat anti-mouse, FITC-labeled, purchased from Invitrogen, product number A11017, goat anti-rabbit, TXRED-labeled, purchased from Lianke Bio company, product number LK-GAR5492), diluted 1:2000 with 3% BSA when used; PNA telomere probe (purchased from Panagene, product number F1009-5), working concentration is 10nM.

[0057] 2. Experimental method

...

Embodiment 2

[0072] Example 2 Tandem affinity purification (TAP) combined with liquid chromatography mass spectrometry experiment

[0073] 1. Experimental method

[0074] Firstly, using Invitrogen’s Gateway technology, the entry vector DAXXpDonor223 purchased from Harvard Medical School was connected to the destination vector pBabe-CMV-SFB-puro (a gift from the Laboratory of Biochemistry and Molecular Biology, Baylor College of Medicine, USA, SFB also contains streptavidin) through LR reaction. The pBabe-CMV-SFB-DAXX-puro virus vector was prepared from the avidin-bound tag (B tag), a peptide segment after proteolytic cleavage by ribonuclease A (S tag, Flag tag) (reaction conditions refer to Invitrogen The company’s product LRCLONASEIIENZYMEMIX product manual, product number 11791020), infected 293T cells by packaging retroviruses expressing DAXX-SFB, and screened with puromycin for 48 hours to select 293T monoclonal cell lines that can stably express DAXX-SFB. After culturing, the cells...

Embodiment 3

[0077] Example 3 Bimolecular fluorescence complementation experiment (Bimolecular fluorescence complementation, BiFC)

[0078] 1. Experimental method

[0079]First, using Invitrogen’s Gateway technology, the entry vector DKC1pDonor223 purchased from Harvard Medical School was connected to the destination vector pBabe-CMV-YFPn-neo (a gift from the Laboratory of Biochemistry and Molecular Biology, Baylor College of Medicine, USA) through LR reaction to prepare pBabe - CMV-YFPn-DKC1-neo virus vector, the virus was used to infect HTC75 cells and the bait (Bait) cells stably expressing DKC1-YFPn were selected with antibiotic G418. Then use Invitrogen’s Gateway technology in the same way to connect the entry vector DAXXpDonor223 purchased from Harvard Medical School to the destination vector pBabe-CMV-YFPc-puro (a gift from the Biochemistry and Molecular Biology Laboratory of Baylor College of Medicine) through LR reaction, The pBabe-CMV-YFPc-DAXX-puro virus vector was prepared, ...

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Abstract

The invention provides the application of telomere binding protein DAXX in preparing tumor cell regulator. The telomere-associated protein DAXX includes an ATRX binding domain, a histone binding domain, a telomerase binding domain, a Cajal body binding domain and a telomere binding domain. DAXX can be located on the telomeres of telomerase-positive tumor cells, and maintain the length of telomeres by regulating the assembly and transport of telomerase, thereby regulating the growth and reproduction of tumor cells. In addition, DAXX can also be located on the telomeres of tumor cells independent of the telomerase-independent telomere elongation mechanism (ALT), and regulate the length of telomeres by affecting the ALT mechanism. Therefore, DAXX has important telomere regulation functions in telomerase-positive tumor cells and ALT mechanism-existing tumor cells, and effective tumor cell regulators can be developed for the regulation mechanism of DAXX on tumor cells.

Description

technical field [0001] The present invention relates to the field of tumor cell regulation. More specifically, it relates to the application of telomere-binding protein DAXX in the preparation of tumor cell regulators. Background technique [0002] Telomere is a special structure composed of highly repetitive DNA sequence (TTAGGG) and some binding proteins at the end of eukaryotic chromosome. It protects chromosome ends from degradation and fusion to ensure chromosome integrity. Every time a normal cell divides, the telomeres gradually become shorter. When the telomeres shorten to a certain limit, the cells cannot continue to divide, and they begin to age and die. However, tumor cells have a telomere elongation mechanism that allows them to escape senescence and death caused by telomere shortening to maintain an immortal state. About 85% of cancers extend telomeres by activating telomerase, and another 15% of cancers extend telomeres through a telomere-independent mechani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61P35/00
Inventor 松阳洲黄军就唐梦帆
Owner SUN YAT SEN UNIV
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