Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines

Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/b-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing b-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations.publishedVersio

Porous layer open tubular (PLOT) protein separation and trypsin open tubular (TOT) columns

Porous layer open tubular (PLOT) columns and immobilized trypsin open tubular (TOT) columns of 10 μm inner diameter (i.d.) were made and characterized for fast intact protein analysis and protein digestion, respectively. A 0.1 mg/mL standard protein solution of cytochrome C, myoglobin, and carbonic anhydrase was used to investigate the PLOT column performance and on-column trypsination was investigated using 5 and 0.3 mg/mL cytochrome C solutions. Mobile phase combination of A (H2O/FA/TFA, 100/0.1/0.05, v/v/v) and B (ACN/H2O/FA/TFA, 90/10/0.1/0.05, v/v/v/v) were used for protein separation on PLOT columns and mobile phase combination of A (50 mM NH4OAc pH 8.75) and B (ACN) were used for protein digestion on the TOT columns. The optimal combination of flow rate and gradient time for separation of intact proteins on the poly(styrene-divinylbenzene) (PS-DVB) PLOT column was explored in the range of 20 to 80 nL/min, and 60 nL/min was found as the efficient flow rate regarding peak shape and peak width at a gradient time of 40 min. The mass spectrometric (MS) signal intensity was found to depend on flow rate and the temperature in the investigated range from 25 to 80℃. Gradient steepness on different column lengths, and column i.d.s, was also examined. Long columns provided better performance at longer gradient times. At elevated flow rates the pressure increases, thus 60 ℃ was found as the optimal temperature at 60 nL/min. The optimal gradient time on a 3 m long column at a flow rate of 60 nL/min was 15 min. Due to difficulties with mass spectrometric detection of many intact proteins the possibility of an on-line digestion using a TOT column was explored. Unfortunately, no digestion was observed with continuous flow at 150 nL/min. Different stop flow times from 20 to 120 s, and 15 min at different flow rates from 20 to 340 nL/min at temperatures 25 and 37℃, different amount of ACN within a range of 5 to 90% at 25℃, and 37℃ and column length in the range of 27 to 50 cm was examined to obtain digestion, and a 2 min stop flow time at 37℃ were found as optimal digestion time and temperature, respectively. Shorter columns gave better digestion and a column length of 30 cm column was found to be optimal. Proteins mostly elute from a reversed phase column within a range of 40 to 90% of ACN. No difference in protein digestion on the TOT column was found in the range 40 to 90% of ACN, at both 25 and 37℃

Integrated enzyme reactor and high resolving chromatography in ‘‘sub-chip’’ dimensions for sensitive protein mass spectrometry

Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using “sub-chip” columns (10–20 µm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 µm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 µm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3–5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage

Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols[S]

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000–80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1–21% and 2–20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics. This research was originally published in the Journal of Lipid Research. Roberg-Larsen H, Lund K, Vehus T, Solberg N, Vesterdal C, Misaghian D, Olsen PA, Krauss S, Wilson SR, Lundanes E.. Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols. J. Lipid Res. 2014 Jul;55(7):1531-6. © the American Society for Biochemistry and Molecular Biology

Altered DNA base excision repair profile in brain tissue and blood in Alzheimer’s disease

Background Alzheimer’s disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia globally. AD is associated with increased oxidative stress, resulting from imbalance in production and clearance of reactive oxygen species (ROS). ROS can damage DNA and other macromolecules, leading to genome instability and disrupted cellular functions. Base excision repair (BER) plays a major role in repairing oxidative DNA lesions. Here, we compared the expression of BER components APE1, OGG1, PARP1 and Polβ in blood and postmortem brain tissue from patients with AD, mild cognitive impairment (MCI) and healthy controls (HC). Results BER mRNA levels were correlated to clinical signs and cerebrospinal fluid biomarkers for AD. Notably, the expression of BER genes was higher in brain tissue than in blood samples. Polβ mRNA and protein levels were significantly higher in the cerebellum than in the other brain regions, more so in AD patients than in HC. Blood mRNA levels of OGG1 was low and PARP1 high in MCI and AD. Conclusions These findings reflect the oxidative stress-generating energy-consumption in the brain and the importance of BER in repairing these damage events. The data suggest that alteration in BER gene expression is an event preceding AD. The results link DNA repair in brain and blood to the etiology of AD at the molecular level and can potentially serve in establishing novel biomarkers, particularly in the AD prodromal phase