Background: The Mre11/Rad50/Nbs1 (MRN) complex regulates DNA repair and signaling through the Ataxia Telangiectasia-Mutated (ATM) kinase.Results: ATM activation requires ATP binding by Rad50 and the coiled-coils but not ATP hydrolysis, zinc hook connection, or Mre11 nuclease function.Conclusion: The ATP-bound form of MRN with Rad50 catalytic domains engaged is the form that activates ATM.Significance: ATP-driven changes in MRN conformation control ATM signaling.

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes Ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. Here, we genetically separated DNA damage activation of ATM from oxidative activation using separation-of-function mutations. We found that deficiency in ATM activation by Mre11-Rad50-Nbs1 and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of ATM lacking oxidative activation generates widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicates ATM in the control of protein homeostasis.

Regulated phosphorylation by protein tyrosine kinases (PTKs) such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the Chronic Myeloid Leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogelimmobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semi-quantitative detection of peptide phosphorylation by recombinant cAbl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay were demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

Histone deacetylase inhibitors (HDACIs) are a class of antineoplastic agents previously demonstrating preclinical chemosensitizing activity against drug-resistant cancer cells and mouse xenografts. However, whereas clinical studies have shown efficacy against human hematologic malignancies, solid tumor trials have proved disappointing. We previously developed a novel HDACI, "OSU-HDAC42," and herein examine its activity against ovarian cancer cell lines and xenografts. OSU-HDAC42, (i) unlike most HDACIs, elicited a more than five-fold increase in G(2)-phase cells, at 2.5 microM, with G(2) arrest followed by apoptosis; (ii) at 1.0 microM, completely repressed messenger RNA expression of the cell cycle progression gene cdc2; (iii) at low doses (0.25-1.0 microM for 24 hours), induced tumor cell epithelial differentiation, as evidenced by morphology changes and a more than five-fold up-regulation of epithelium-specific cytokeratins; (iv) potently abrogated the growth of numerous ovarian cancer cells, with IC(50) values of 0.5 to 1.0 microM, whereas also remaining eight-fold less toxic (IC(50) of 8.6 microM) to normal ovarian surface epithelial cells; and (v) chemosensitizated platinum-resistant mouse xenografts to cisplatin. Compared with the clinically approved HDACI suberoylanilide hydroxamic acid (vorinostat), 1.0 microM OSU-HDAC42 was more biochemically potent (i.e., enzyme-inhibitory), as suggested by greater gene up-regulation and acetylation of both histone and nonhistone proteins. In p53-dysfunctional cells, however, OSU-HDAC42 was two- to eight-fold less inductive of p53-regulated genes, whereas also having a two-fold higher IC(50) than p53-functional cells, demonstrating some interaction with p53 tumor-suppressive cascades. These findings establish OSU-HDAC42 as a promising therapeutic agent for drug-resistant ovarian cancer and justify its further investigation.