A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.

The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples.

Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens.

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells.

Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase.

Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells.

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics.

NAD bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD precursor dihydronicotinamide riboside (NRH) rapidly increased NAD levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression.