Sven Skog

Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in vivo and in vitro

8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5–1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.

Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan™ assays

Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan fluorescence microscopy. The levels of TK1 increased 6-7h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified.

A Comparative Study: Immunohistochemical Detection of Cytosolic Thymidine Kinase and Proliferating Cell Nuclear Antigen in Breast Cancer

To explore the expression of cytosolic thymidine kinase 1 (TK1) as a cell proliferative marker in human breast cancers, immunohistochemistry was used to detect the expression of TK1 in 52 malignant breast lesions, 20 benign breast lesions, and 16 normal breast tissues. The results were compared to the expression of proliferating cell nuclear antigen (PCNA) in the same specimens. The TK1-labelling index (TK1-LI) and PCNA-labeling index (PCNA-LI) were significantly higher in malignant lesions than in nonmalignant lesions (p<0.0001 and p<0.0013, respectively). The TK1-LI (78.9%) in malignant lesions was higher compared to PCNA-LI (64.5%). No significant difference was found for TK1-LI and PCNA-LI between benign lesions and normal tissues. Concerning the tumor stages and the tumor grades, TK1-LI showed a significant correlation with the increased tumor stages (p=0.023) and tumor grades (p=0.009). However, PCNA-LI was neither significantly different in tumor stages (p=0.062) nor in tumor grades (p=0.073). We conclude that TK1 might be a more accurate marker than PCNA for estimation of cell proliferation and malignant potentials in breast carcinomas.

The proliferation marker thymidine kinase 1 level is high in normal kidney tubule cells compared to other normal and malignant renal cells.

The activity of the proliferation related enzyme thymidine kinase 1 (TK1) was reported to be 3-fold higher in extracts from normal kidney tissue as compare to renal carcinoma extracts [3]. To verify these unexpected results, determinations of the protein levels of TK1 in normal kidney and in samples from different types of renal cell carcinoma (RCC) were done with immunohistochemistry and Western blot analysis. Two anti-TK1 peptide antibodies reacting with different TK1 epitops were used. TK1 levels were high in tubule cells as compared to glomerulus cells and connective tissue cells, while an intermediary TK1 was observed in renal cell carcinoma (RCC) cells. Western blot analysis demonstrated high levels of TK1 in extract from normal kidney, and lower levels of TK1 in the RCC extracts. The specificity of TK1 staining was demonstrated in competition experiments with excess TK1 antigen. The high TK1 levels in normal kidney tubule cells suggest that they are in a form of activated G1-state. The relatively low TK1 level in RCC, representing TK1 expression in S-phase cells, is in accordance with the low overall proliferation rate of these tumors. These results suggest that cell cycle regulation of TK1 in normal tubule cells differ from that in other type of normal and malignant renal cells.

The Half-Life of Serum Thymidine Kinase 1 Concentration Is an Important Tool for Monitoring Surgical Response in Patients with Lung Cancer: A Meta-Analysis.

AIMS In this meta-analysis, we evaluated the usefulness of serum thymidine kinase 1 concentration (STK1c) for monitoring the outcome of extensive open surgery in patients with lung cancer. We also compared STK1c between a healthy population and patients with benign and malignant lung tumors to assess its potential value for early detection of lung cancer and for distinguishing between benign lung disease and malignant cancer. MATERIALS AND METHODS Related studies were retrieved from publications in PubMed, Cochrane, China National Knowledge Infrastructure, Wanfang databases, and Internet searches. Correlation was evaluated using weighted mean difference. Fixed or random effect models were selected for data analyses based on heterogeneity tested with the chi-square test. Publication bias was assessed using a funnel plot and Eggers test. RESULTS Twenty studies were selected for analysis, which showed that STK1c was significantly (p < 0.00001) reduced by 41.7% 1 month after extensive open surgery, approximately corresponding to an STK1c half-life of 1 month. STK1c levels were significantly higher in lung cancer patients than in healthy persons (p < 0.00001) or in patients with benign lung disease (p < 0.00001). There was also a significant difference in STK1c between patients with benign and malignant lung disease (p < 0.0001). CONCLUSIONS The half-life of STK1c may be an important tool in the clinical evaluation of surgical response in patients with lung cancer. STK1c may also be beneficial in the early detection of lung cancer.