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COP1 molecules and uses thereof

A molecular, versatile technology with applications in the field of cancer diagnosis and therapy that can address poorly defined problems as

Inactive Publication Date: 2007-11-28
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although COP1 is an important light-mediated developmental switch in plants, its role in mammalian cells is not well defined 20

Method used

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  • COP1 molecules and uses thereof
  • COP1 molecules and uses thereof
  • COP1 molecules and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1: Materials and methods

[0110] Expression Vectors, Recombinant Proteins and Antibodies

[0111] Flag-COP1 has been described earlier 33 . HA-COP1 was generated by PCR subcloning COP1 into pcDNA3.1+ (Invitrogen) and GST-COP1 was generated by subcloning COP1 into pGEX6P1 (Pharmacia). pcDNA3.1+53, pG13-Luc, p21-Luc, bax-Luc, NS-Luc and pCMV-MDM2 have been described earlier 21，22，35 .

[0112] Full-length COP1 was amplified from cDNA from HEK293T cells. HA-COP1 was subcloned by PCR into pcDNA3.1+ (Invitrogen) and GST-COP1 was generated by PCR and subcloned into pGEX6P1 (Pharmacia). COP1-Luc and COP1mut-Luc were generated by ligating oligonucleotides containing two copies of the p53 consensus site from the COP1 promoter, or a mutant containing the consensus site, into the pGL3-promoter (Promega).

[0113] All GST recombinant proteins were expressed in E. coli BL21(DE3) codon+ (Stratagene), sonicated with 1 mg / ml lysozyme, dissolved in PBS with 1% TritonX-100...

Embodiment 2

[0164] Example 2: p53 is a substrate of COP1

[0165] To gain insight into how COP1 regulates cellular processes, lysates from U2-OS cells stably expressing FLAG-tagged COP1 or empty vector were subjected to immunoprecipitation with anti-FLAG, bound proteins were eluted by FLAG peptide, and bound proteins were eluted by SDS - PAGE analysis, sequencing specific bands by mass spectrometry (Fig. 1A). Mass spectrometric analysis of the band at approximately 53 kDa revealed 5 matching peptides from the tumor suppressor protein p53. To confirm that this interaction was real, p53-null Soas-2 cells were transfected with p53 and Myc-COP1, immunoprecipitated with anti-p53 (DO-1), and immunoblotted with anti-Myc (Fig. 1B). COP1 was immunoprecipitated only in the presence of transfected p53, thus suggesting that COP1 can interact with p53. This interaction was also observed with transfected COP1 and endogenous p53 (Fig. 1C). Furthermore, there was an interaction between p53 and COP1 at...

Embodiment 3

[0173] To determine the effect of COP1 overexpression on p53-dependent transactivation, Saos-2 cells were transfected with p53 and COP1 or COP1ΔRING, and p21-luciferase (Luc) or bax-Luc (Fig. 2H, L). Addition of COP1 markedly reduces the ability of p53 to transactivate from the p21 and bax promoters. Furthermore, the transactivation capacity of endogenous p53, as well as the capacity induced by DNA damage, was abrogated in the same manner by overexpression of COP1 (Fig. 2M). To further assess the ability of COP1 to negatively regulate p53, we determined whether COP1 could inhibit p53-induced cell death in Saos-2 cells (Fig. 21). Transfection of p53 significantly increased the cell population in the SubG1 population, however, this was significantly inhibited by co-transfection of COP1, suggesting that COP1 inhibits p53-induced cell death. COP1 transfection alone had no profound effect on cell cycle distribution.

[0174] To reveal the role of COP1 in more physiological situat...

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Abstract

The invention provides diagnostic, prognostic, and therapeutic uses for detecting COP1 overexpression in a variety of cancers. The methods and uses can further include detecting p53 expression. The invention also provides reagents and kits for use in screening for test compounds that interfere with COP1 and p53 binding.

Description

field of invention [0001] The present invention relates to the field of cancer diagnosis and therapy. Background of the invention [0002] It is well established that p53 functions as a traditional tumor suppressor. 1 Biochemically, p53 functions as a stress-activated sequence-specific transcription factor that activates transcription from promoters bearing p53 consensus binding sites. 2 In addition, p53 also acts as a potent transcriptional repressor, adding another layer of gene regulation. 3 Thus, it protects cells from various stress signals, such as DNA damage, nucleotide depletion, and oncogene activation, to name a few, by inhibiting, among others, genes involved in angiogenesis, anti-apoptosis, and cell cycle progression. In addition, transcription of a cadre of genes involved in cell cycle arrest, apoptosis and DNA repair is activated. The physiological consequences of p53 primarily cause growth retardation or apoptosis, preventing cells from replicating a geneti...

Claims

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Application Information

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IPC IPC(8): G01N33/574
CPCG01N2500/02G01N33/57496G01N2333/9015G01N33/574G01N2800/52A61P35/00A61P35/02A61P35/04A61P43/00
Inventor 戴维·多南多萝西·弗伦奇维什瓦·迪克西特
Owner GENENTECH INC
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