The human Flap Endonuclease 1 (a.k.a. hFEN1) is a structure-specific nuclease involved in DNA replication and repair. This nuclease is thought to be a potential therapeutic target for treating cancers (e.g. chemosensitization, synthetic lethality) hence, the need for discovering and characterizing new hFEN1 inhibitors.
The anti-cancer property of the Vitamin K3 (Vitk3) was again recently studied by Dr Meng-Er Huang’s team (Curie Institute, CNRS UMR3348, Paris).
By using in vitro pro-oxydative cellular models, Dr Huang and collaborators observed a cancer-selective cytotoxicity of vitk3 in Peroxyredoxin 1 (PRDX1 aka PRX1)-deficient cancer cell lines.
To further investigate the underlying molecular mechanisms, the authors used a stably PRX1-depleted HeLa cell line (PRX1-) (vs. Control (PRX1+)) under various experimental conditions. They notably treated PRX1- and PRX1+ cell lines by vitk3 and 6 other known anticancer molecules targeting different cellular events (microtubule (vinblastine & taxol); DNA (doxorubicin & daunorubicin); DNA transcription (actinomycin D) and cell cycle (5-FU)). [Read more…]
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)
(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.
Typical silencing results with HeLa and custom cell lines
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!
Deak AT et al. decipher the mechanisms by which mitochondria contribute to Ca2+ intracellular signaling. In their recent paper (1), they show that mitochondrial “Ca2+ buffering” close to the Endoplasmic Reticulum predominately shapes cytosolic Ca2+ micro-domains. To perform their studies, the authors used stable knock-down (KD) HeLa cellular models optimized for loss-of-function analysis.
Two proteins known to be essential for mitochondrial Ca2+ uptake (Mitochondrial Calcium Uniporter (MCU) or Uncoupling Protein 2 (UCP2)) have thus been stably silenced with sh-RNA engineered HeLa cell lines (MCU-KD and UCP2-KD HeLa cell lines). This shRNA technology has already been used in Drug Discovery approaches (ex. PARP inhibitors efficiency by Synthetic Lethality in Hypoxic conditions). (2)
Finally, the authors present two diagrams illustrating the role of mitochondrial Ca2+ uptake for Stromal Interacting Molecule 1 (STIM1) oligomerization and Store-Operated Ca2+ Entry (SOCE) maintainance in their HeLa cellular models.
Want to know more?
(1) Deak AT et al. “Inositol-1,4,5-trisphosphate (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake” (2014) J Cell Sci. 2014 May 7. DOI: 10.1242/?jcs.149807
(2) Mennesson E. et al. “SilenciX®, novel stable knock-down cellular models to screen new molecular targets through the synthetic lethality approach” (“Experimental and Molecular Therapeutics” poster session – AACR 2014, San Diego) Abstract n° 3733
Congratulations to the authors!
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)
Hypoxia has important effects on chemosensitivity of cancer cells and the synthetic lethal effects of drugs.
In a recent work presented during the “Experimental and Molecular Therapeutics” sessions at the AACR 2014, Claudine Kiéda’s and Nadia Normand’s teams showed that PARP inhibitors are less synthetically lethal in hypoxic conditions with increased IC50 and survival percentage at higher concentrations.
Synthetic Lethality (SL) is defined as when loss of two genes independently has no effect on viability, but simultaneous loss of both genes causes cell death.
In cancer research and drug discovery, SL is observed when the cancer mutation and the drug simultaneously inhibit two otherwise independent pathways, leading to cell death. The best known SL relationship is between BRCA1/2 mutation (tumors deficient in Homologous Recombination (HR) DNA Repair pathway) and PARP inhibitors (affecting the Base Excision Repair (BER) DNA Repair pathway).
In their work, Dr Kiéda and Dr Normand used already published in vitro stable BRCA1- and BRCA2-KD cell lines (SilenciX® technology) to measure the synthetic lethal efficiency of PARP inhibitors (Olaparib, Veliparib and Rucaparib) in both normoxic and hypoxic conditions. They demonstrate that the BRCA-KD SilenciX® cell lines are effective and convenient in vitro cellular models to design new cancer drug candidates through the SL approach in oxygen-controlled conditions to better mimick physioxia seen in solid tumors.
In a recent paper published in PNAS, Wang et al. show that the inhibition of the deubiquitinase USP9x by the small molecule WP-1130 is able to induce degradation of the transcription factor E-twenty-six related gene (ERG).
Meet Anne-Marie Renault , our Business Development SilenciX® representative at the AACR Annual Meeting 2014 in San Diego during the “Experimental and Molecular Therapeutics” poster session.