ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor

A technology of kinase domains and residues, applied in the fields of diagnosis and treatment, and cancer detection, can solve problems such as side effects and troubles

Active Publication Date: 2013-11-20
CELL SIGNALING TECHNOLOGY
View PDF45 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of this drug represents a significant advance over conventional therapies (chemotherapy and radiation) for CML and ALL, which are p...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor
  • ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor
  • ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0295] Identification of ALK kinase activity in solid tumors by global phosphopeptide profile

[0296] a. Distribution of human NSCLC cell lines

[0297] Kinase activation in 22 human NSCLC cell lines, including H2228, was examined using a recently described powerful technique for the isolation and mass spectrometric characterization of modified peptides from complex mixtures (the "IAP" technique, see Rush et al., supra). global phosphorylation profile. Using a phosphotyrosine-specific antibody (C ELL S IGNALING T ECHNOLOGY , I NC ., Beverly, MA, 2003 / 04 Cat. #9411) implemented the IAP technique to isolate and subsequently characterize phosphotyrosine-containing peptides from extracts of NSCLC cell lines.

[0298] Specifically, the IAP approach was used to facilitate the identification of tyrosine kinases responsible for protein phosphorylation in each NSCLC cell line. In particular, atypical or abnormal kinase activity is considered.

[0299] cell culture

[030...

Embodiment 2

[0323] Isolation and sequencing of three ALK fusion genes

[0324] a. Sequencing in Human NSCLC Cell Lines

[0325] Given the high phosphorylation levels of ALK kinase detected in the NSCLC cell line H2228, rapid amplification of the 5' end of the cDNA at the sequence encoding the kinase domain of ALK was performed in order to determine the presence of chimeric ALK transcripts.

[0326] Rapid amplification of complementary DNA ends

[0327] RNeasy Mini Kit (Qiagen) was used to extract RNA from H2228 cell line. DNA was extracted by using the DNeasy Tissue Kit (Qiagen). The cDNA ends were rapidly amplified with the aid of the 5' RACE system (Invitrogen), with primers ALK-GSP1 for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for nested PCR reactions.

[0328] 5'RACE

[0329] Figure 5 (Panel A) shows the detection of the EML4-ALK fusion gene (short variant) by 5' RACE and the detection of the PCR amplification product after 2 rounds. PCR products were purified with a PCR puri...

Embodiment 3

[0353] Inhibition of Growth of ALK Fusion-Expressing Mammalian Solid Tumors Using siRNA

[0354] To confirm that truncated / fusion forms of ALK are driving cell growth and survival in the NSCLC cell line H2228 and NSCLC tumor samples from patients CS010 / 11, CS045, and CS110, siRNA (relative to ALK) inhibition of these cells can be examined and tumor growth capacity.

[0355] ALK SMARTpool siRNA duplexes (proprietary target sequences - data not shown) can be purchased from, eg, Dharmacon Research, Inc. (Lafayette, CO). Non-specific SMARTpool siRNA was used as a control. Cells were transfected with siRNA by means of electroporation. Briefly, 2 × 10 7 Each cell (H2228) was pulsed once (20 ms; 275 V, K562 20 ms; 285 V), incubated at room temperature for 30 min, and then transferred to a T150 flask containing 30 ml RPMI-1640 / 10% FBS.

[0356] With CellTiter96AQ ueous One solution cell proliferation assay (Promega) was used to determine the number of viable cells. IC was calcu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an ALK (Anaplastic Lymphoma Kinase) for gene deletion and a mutant in a human solid tumor. According to the invention, novel second chromosome related gene deletion and translocation are authenticated in the existing human solid tumor such as non-small cell lung cancer (NSCLC), thus a fusion protein which is combined with part of ALKs and part of secondary proteins is caused. Therefore, the invention partially provides separated polynucleotides and carriers, a mutant ALK polypeptide disclosed by codes of the separated polynucleotides and carriers, a probe for detecting the mutant ALK polypeptides, separated mutant polypeptides, recombined polypeptides and a reagent for detecting fused and truncated polypeptides. Due to the authentication for the disclosed novel fusion protein, the existence of the mutant ALK polypeptides in a biological sample can be determined by using a novel method. The invention also provides a method for screening a compound for inhibiting the protein and a method for inhibiting cancers which are characterized by the mutant polynucleotides or polypeptides.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 200780021431.4 entitled "Gene Deficiency and Mutant ALK Kinase in Human Solid Tumors" with the filing date of April 13, 2007 [0002] related application [0003] This application claims priority to and benefit of currently pending USSN 60 / 792,364, filed April 14, 2006, the entire disclosure of which is hereby incorporated by reference. technical field [0004] The present invention generally relates to proteins and genes associated with cancer, and to the detection, diagnosis and treatment of cancer. Background technique [0005] Many cancers are characterized by disruptions in cellular signaling pathways, which lead to abnormal control of cellular processes, or to uncontrolled growth and proliferation of cells. These disruptions are often caused by changes in the activity of specific signaling proteins such as kinases. These cancers include solid tumors such as non-small cell lung cance...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/62C12N15/11C12N15/63C12N9/12C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02C07K19/00
Inventor 克拉里萨·里科瓦赫伯特·哈克劳拉·沙利文顾挺磊安东尼·波塞马托郭爱兰琼·麦克尼尔俞建
Owner CELL SIGNALING TECHNOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap