DNA repair antibodies validated by MD Anderson

A series of publications (1, 2) in the past months has raised the never-ending debate on commercial antibodies and their validity in a given experiment. At tebu-bio, having sold antibodies for more than 40 years, we are well aware that not all antibodies fit all applications, and always strive to find the best antibody for a given experiment.

In collaboration with the MD Anderson Cancer Center, Rockland Inc. has developed and validated a highly specific toolkit to analyze PARP1 in a panel of control and siRNA knockdown cell lysates by multiple immunoassays. [Read more…]

Trapping PARP-DNA complexes – anti-cancer drug screening

PARPs (Poly ADP ribose polymerases) are found in the nucleus of the cell and they are involved in SSB repair (single-strand DNA breaks). PARP is known to bind damaged DNA through its N-terminal zinc finger domain. Subsequently it starts to synthesize a poly (ADP-ribose) chain which serves as a signal for other DNA-repairing enzymes.

SYNTHETIC_ LEATHALITY

Fig. 1: Principle of synthetic lethality

PARP inhibitors are considered to be promising  candidates as anti cancer drugs (recently Olaparib, the first drug directed against PARP1, has been approved by the European commission). One of the reasons is that some tumors are more dependent on PARP than regular cells. These cancer cells are mutated in BRCA1 or BRCA2 – both genes which are involved in key DNA damage repair mechanisms. In healthy cells PARPs can function as a kind of back-up system and let the cells survive even without functional BRCA gene products. When PARPs are inhibited the cells do not possess any functional SSB repair mechanism anymore and are bound to die (Fig. 1; take a look at PARPs as cancer drug targets – first EC-approved drug to learn more about the concept of “synthetic lethality”). [Read more…]

See now how BRCA2 works to repair DNA

How BRCA2 works to repair damaged DNA.

Picture showing how BRCA2 proteins (red) help Red51 (yellow) form short filaments on strands of broken DNA (blue). This structure enables it to look for matching strands. (2)

A great discovery related to BRCA2 molecular mechanisms has been recently published in the October 2014 edition of Nature Structural and Molecular Biology. (1)

Funded by the Medical Research Council and the Wellcome Trust, a team of researchers have described for the first time the structure and the mechanism of action of the BRCA2 breast cancer tumor suppressor by electron microscopy.

Interestingly, the authors found that BRCA2 proteins work in pairs to facilitates Rad51-mediated homologous-recombination to repair DNA damaged. (see picture below)

A better understanding of the DNA repair processes will definitely help researchers in designing potent therapeutical approaches targeting molecular events involved in DNA integrity.

Want to know more about this BRAC2 picture and interactions with Rad51?

Here are the sources of this post:

  1. Shahid et al. ‘Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor.’ Nature Structural and Molecular Biology, 5 October 2014. DOI:10.1038/nsmb.2899.

    BRCA2 representation

    BRCA2 representation. Study led by Professor Xiaodong Zhang from the Department of Medicine at Imperial College London and Dr Stephen West at the London Research Institute.

  2. First pictures of BRCA2 protein show how it works to repair DNA by Sam Wong

Feel free to leave a comment concerning the recent discoveries that impress you the most.

EndoG and ATM cell cycle checkpoint in Leukemogenesis

In a recent publication, Gole B. and Baumann C. demonstrate the role of the apoptotic nuclease EndoG in Mixed-Lineage Leukemia breakpoint cluster region (MLLbcr) destabilisation leading to MLL gene rearrangements and leukemogenesis. (1)

In this study, various engineered stable knockdown cell lines were used as in vitro cellular models.  Two of them are developed by tebu-bio: SilenciX® HeLa/shATM and HeLa/shATR. They have been selected as stable ATM and ATR KD cellular models to investigate the role of ATM (Ataxia Telangiectasia, Mutated) and ATR (ATM and Rad3-related) cell cycle checkpoint pathways in these MLL rearrangements.

The authors also describe a possible cytotoxic-induced model involving EndoG and ATM / RFN20 / H2B cascade in MLLbcr breakage.

About SilenciX stable KD HeLa cells

SilenciX are gene-specific knock-down (KD) engineered HeLa cell lines. The silencing technology used is not integrative, reducing thus potential off-target effects. These KD cellular models are ideal for a broad range of  signal transduction and drug discovery studies (Loss-of-function model, Synthetic lethality…). To date, 100+ HeLa SilenciX cell lines have been designed (customization on other cell types being possible). Recently, BRCA1-KD, BRCA2-KD and p53-KD SilenciX HeLa cells were used to show that  PARP inhibitors are less synthetically lethal in hypoxic conditions. (2)

SilenciX® is a registered trademark of tebu-bio; technology licensed from the Atomic Energy and Alternative Energies Commission (CEA). (3)CEA logo

SilenciX publications

(1) Gole B., Baumann C. et al. “Endonuclease G initiates DNA rearrangements at the MLL breakpoint cluster upon replication stress” (2014) Oncogene, pp-1-11. DOI:10.1038/onc.2014.268.

(2) Mennesson E. et al.SilenciX®, novel stable knock-down cellular models to screen new molecular targets through the synthetic lethality approach”  (2014) (“Experimental and Molecular Therapeutics” poster session – AACR 2014, San Diego – Abstract n° 3733.

(3) Further SilenciX publications and information can be found here.

Typical silencing results with HeLa and custom cell lines

Custom SilenciX MCR5 and MCF7 cell lines by tebu-bio

XPA-KD, KIN17-KD and XPC-KD Custom SilenciX MCR5 and MCF7 cell lines by tebu-bio.

HeLa stable KD cell lines by tebu-bio

Stable silencing of tebu-bio SilenciX HeLa stable KD cell lines (DNAPKcs-KD, KIN17-KD, XPA-KD, XPC-KD, ERCC1-KD and XPF-KD).

I hope these data will bring you new opportunities for your research programs. Share this post or leave any comments concerning the tools you are using to perform efficient and robust gene KD below!

 

PARP and Top1 inhibitors most beneficial in endonuclease-deficient cancer cells

The synergistic combination of Poly(ADP-ribose) polymerases (PARP) and Topoisomerase I (Top1) inhibitors most beneficial on endonuclease-deficient cancer cells in cancer therapies ?

This is one of the elements discussed in the recent publication by Dr Benu Brata (National Institutes of Health (USA) and Indian Association for the Cultivation of Science (India) in Nucleic Acids Research. (1)

[Read more…]