Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment. Oncogene (2010) 29, 463-468; doi:10.1038/onc.2009.327; published online 19 October 2009

Fabrication and Repair of Cartilage Defects with a Novel Acellular Cartilage Matrix Scaffold

The objectives of this study were to develop a three-dimensional acellular cartilage matrix (ACM) and investigate its possibility for use as a scaffold in cartilage tissue engineering. Bovine articular cartilage was decellularized sequentially with trypsin, nuclease solution, hypotonic buffer, and Triton x 100 solution; molded with freeze-drying process; and cross-linked by ultraviolet irradiation. Histological and biochemical analysis showed that the ACM was devoid of cells and still maintained the collagen and glycosaminoglycan components of cartilage. Scanning electronic microscopy and mercury intrusion porosimetry showed that the ACM had a sponge-like structure of high porosity. The ACM scaffold had good biocompatibility with cultured rabbit bone marrow mesenchymal stem cells with no indication of cytotoxicity both in contact and in extraction assays. The cartilage defects repair in rabbit knees with the mesenchymal stem cell-ACM constructs had a significant improvement of histological scores when compared to the control groups at 6 and 12 weeks. In summary, the ACM possessed the characteristics that afford it as a potential scaffold for cartilage tissue engineering.

Spatio-temporal analysis of DNA damage repair using the X-ray microbeam

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

Monitoring and repair of an impact damaged prestressed bridge

This paper details the monitoring and repair of an impact damaged prestressed concrete bridge. The repair was required following an impact from a low-loader carrying an excavator while passing underneath the bridge. The repair was carried out by preloading the bridge in the vicinity of the damage to relieve some prestressing. This preload was removed following the hardening and considerable strength gain of the repair material. The true behaviour of damaged prestressed concrete bridges during repair is difficult to estimate theoretically due to lack of benchmarking and inadequacy of assumed damage models. A network of strain gauges at locations of interest was thus installed during the entire period of repair. Effects of various activities were qualitatively and quantitatively observed. The interaction and rapid, model-free calibration of damaged and undamaged beams, including identification of damaged gauges were also probed. This full scale experiment is expected to be of interest and benefit to the practising engineer and the researcher alike.