ATDC/TRIM29 phosphorylation by ATM/MAPKAP kinase 2 mediates radioresistance in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by therapeutic resistance for which the basis is poorly understood. Here, we report that the DNA and p53-binding protein ATDC/TRIM29, which is highly expressed in PDAC, plays a critical role in DNA damage signaling and radioresistance in pancreatic cancer cells. Ataxia-telangiectasia group D-associated gene (ATDC) mediated resistance to ionizing radiation in vitro and in vivo in mouse xenograft assays. ATDC was phosphorylated directly by MAPKAP kinase 2 (MK2) at Ser550 in an ATM-dependent manner. Phosphorylation at Ser-550 by MK2 was required for the radioprotective function of ATDC. Our results identify a DNA repair pathway leading from MK2 and ATM to ATDC, suggesting its candidacy as a therapeutic target to radiosensitize PDAC and improve the efficacy of DNA-damaging treatment.

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci.

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein-ubiquitin interactions.

The roles of DNA polymerase ζ and the Y family DNA polymerases in promoting or preventing genome instability.

Cancer cells display numerous abnormal characteristics which are initiated and maintained by elevated mutation rates and genome instability. Chromosomal DNA is continuously surveyed for the presence of damage or blocked replication forks by the DNA Damage Response (DDR) network. The DDR is complex and includes activation of cell cycle checkpoints, DNA repair, gene transcription, and induction of apoptosis. Duplicating a damaged genome is associated with elevated risks to fork collapse and genome instability. Therefore, the DNA damage tolerance (DDT) pathway is also employed to enhance survival and involves the recruitment of translesion DNA synthesis (TLS) polymerases to sites of replication fork blockade or single stranded DNA gaps left after the completion of replication in order to restore DNA to its double stranded form before mitosis. TLS polymerases are specialized for inserting nucleotides opposite DNA adducts, abasic sites, or DNA crosslinks. By definition, the DDT pathway is not involved in the actual repair of damaged DNA, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and lessening the chance for genome instability. However this may be associated with increased mutagenesis. In this review, we will describe the specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability, the latter due to newly appreciated roles in DNA repair. The recently described role of the Fanconi anemia pathway in regulating Rev1 and Polζ-dependent TLS is also discussed in terms of their involvement in TLS, interstrand crosslink repair, and homologous recombination.

Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities.

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

The use of pepsin in receptor internalization assays.

For internalization experiments that use fluorescent antibody (Ab) staining to distinguish between inside versus outside cellular localization of various receptor targeting ligands, it is critical that there be efficient removal of all residual surface-bound fluorescent Ab. To achieve this, a fluorescent Ab removal technique is commonly employed in receptor internalization assays that utilizes low pH glycine-based buffers to wash off the residual non-internalized fluorescent Ab retained on cell surfaces. In this study, we highlight the shortcomings of this technique and propose an alternative in situ proteolytic approach that we found to be non-deleterious to the cells and significantly more effective in removing the residual fluorescence resulting from non-internalized surface-bound Ab.