Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.     

Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide ⁶⁸Ga (T_1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule Z_HER2:S1 targeting HER2. Affibody molecules were labeled with ⁶⁸Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1, as well as that of their ¹¹¹In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for ⁶⁸Ga-DOTA-Z_HER2:S1 (17.9±0.7%IA/g) was significantly higher than for both ⁶⁸Ga-NODAGA-Z_HER2:S1 (16.13±0.67%IA/g) and ⁶⁸Ga-NOTA-Z_HER2:S1 (13±3%IA/g) at 2 h after injection. ⁶⁸Ga-NODAGA-Z_HER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both ⁶⁸Ga-DOTA-Z_HER2:S1 (28±4) and ⁶⁸Ga-NOTA-Z_HER2:S1 (42±11). The tumor-to-liver ratio was also higher for ⁶⁸Ga-NODAGA-Z_HER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for ⁶⁸Ga than for ¹¹¹In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with ⁶⁸Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for ⁶⁸Ga and ¹¹¹In.     

The Prognostic Significance of Metabolic Response Heterogeneity in Metastatic Colorectal Cancer

Background

Tumoral heterogeneity is a major determinant of resistance in solid tumors. FDG-PET/CT can identify early during chemotherapy non-responsive lesions within the whole body tumor load. This prospective multicentric proof-of-concept study explores intra-individual metabolic response (mR) heterogeneity as a treatment efficacy biomarker in chemorefractory metastatic colorectal cancer (mCRC).

Methods

Standardized FDG-PET/CT was performed at baseline and after the first cycle of combined sorafenib (600mg/day for 21 days, then 800mg/day) and capecitabine (1700 mg/m²/day administered D1-14 every 21 days). MR assessment was categorized according to the proportion of metabolically non-responding (non-mR) lesions (stable FDG uptake with SUVmax decrease <15%) among all measurable lesions.

Results

Ninety-two patients were included. The median overall survival (OS) and progression-free survival (PFS) were 8.2 months (95% CI: 6.8–10.5) and 4.2 months (95% CI: 3.4–4.8) respectively. In the 79 assessable patients, early PET-CT showed no metabolically refractory lesion in 47%, a heterogeneous mR with at least one non-mR lesion in 32%, and a consistent non-mR or early disease progression in 21%. On exploratory analysis, patients without any non-mR lesion showed a significantly longer PFS (HR 0.34; 95% CI: 0.21–0.56, P-value <0.001) and OS (HR 0.58; 95% CI: 0.36–0.92, P-value 0.02) compared to the other patients. The proportion of non-mR lesions within the tumor load did not impact PFS/OS.

Conclusion

The presence of at least one metabolically refractory lesion is associated with a poorer outcome in advanced mCRC patients treated with combined sorafenib-capecitabine. Early detection of treatment-induced mR heterogeneity may represent an important predictive efficacy biomarker in mCRC.

Trial Registration

ClinicalTrials.gov NCT01290926     

Reversible cell cycle inhibition and premature aging features imposed by conditional expression of p16Ink4a

The cyclin‐dependent kinase (Cdk) inhibitor p16^Ink4a (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit‐amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de‐induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16‐mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible.     

Testing assumptions of central place foraging theory: a study of Adélie penguins Pygoscelis adeliae in the Ross Sea

We investigated central place foraging (CPF) in the context of optimal foraging theory in Adélie penguins Pygoscelis adeliae of the southern Ross Sea by using satellite tracking and time‐depth recorders to explore foraging at two spatio‐temporal scales: within the day‐to‐day (sub‐mesoscale: single foraging trip, 10s of km²) and the entire breeding season (mesoscale: trips by multiple individuals across the collective foraging area, 100s of km²). Specifically, we examine whether three basic assumptions of the Orians–Pearson CPF model, shown to occur in other CPF species, are met: 1) within a patch, the rate of prey acquisition declines with time spent in that patch; 2) food is distributed in discrete patches and is not available between those patches; and 3) CPF species have knowledge of the potential (or average, at least) feeding rate within their universe of patches, and use this knowledge to determine their foraging strategy when planning or engaging in a foraging trip. We found that prey consumption rates did not decline with time spent in patches, and penguins foraged to some degree most of the time when at sea. Food availability, as measured by foraging dive rate, appeared to be predictable within the same day at the same location, but predictability broke down after 2 d at distances > 10 km away. We conclude that the assumptions of the Orians–Pearson CPF model are not a good fit to the circumstances of Ross Sea penguins, which clearly are central place foragers.     

Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.