Serum TK1 protein and C-reactive protein correlate to treatment response and predict survival in dogs with hematologic malignancies.

Thymidine kinase 1 (TK1), involved in DNA precursor synthesis, is used as a serum biomarker in cancer diagnostics in both human and veterinary medicine. We investigated the utility of serum TK1 protein (TK1p) and TK1 activity (TK1a) determinations for prognosis and monitoring of canine hematological malignancies. The combination of TK1p or TK1a with canine C-reactive protein (CRP) determinations was also investigated. Serum samples from 51 client-owned dogs with naive hematological malignancies and from 149 healthy subjects were included. Serum TK1p levels were determined using a prototype TK1-ELISA, TK1a using the [3H]-dThd phosphorylation assay, and CRP using an immunoturbidimetric assay. Mean TK1p in sera from dogs with tumors was significantly higher than from healthy dogs (mean ± SD = 3.9 ± 5.9 vs. 0.45 ± 0.15 ng/mL). Similarly, TK1a in hematological malignancies was significantly higher than in healthy dogs (mean ± SD = 15.1 ± 31.3 vs. 0.96 ± 0.33 pmol/min/mL). The receiver-operating characteristic indicated that a combination of TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone for the prognosis of hematological malignancies. Median pretreatment TK1p and TK1a levels were significantly higher than in dogs in remission and correlated with clinical outcome. Kaplan-Meier curve analysis showed that naive dogs with high TK1p, TK1a, and CRP had significantly shorter survival. This study present two new polyclonal antibodies used in an ELISA system to determine TK1p. The study also show that combining TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone. Monitoring patients in the study while undergoing chemotherapy, suggests that the TK1 + CRP combination could be useful in a biomarker panel, possibly aiding the prognosis and therapy monitoring of hematological malignancies in dogs.

Impact of preoperative breast MRI on 10-year survival of patients included in the Swedish randomized multicentre POMB trial.

BACKGROUND: The value of preoperative breast MRI as an adjunct technique regarding its effect on re-excision rates has been a subject of discussion. No survival data regarding preoperative breast MRI are available from randomized studies. METHODS: Ten-year follow-up of the POMB randomized multicentre study was analysed, evaluating MRI and its effect on disease-free survival (DFS) and overall survival (OS). Patients with newly diagnosed breast cancer were randomized to either preoperative MRI or conventional imaging. Kaplan-Meier plots were used to analyse DFS and OS, and Cox regression to estimate hazard ratios (HRs). RESULTS: A total of 440 patients, aged 56 years or less, with newly diagnosed breast cancer were randomized to either preoperative MRI (220) or conventional imaging (220; control). Median follow-up for each group was 10 years. DFS rates were 85.5 and 80.0 per cent for the MRI and control groups respectively (P = 0.099). The risk of relapse or death was 46 per cent higher in the control group (HR 1.46, 95 per cent c.i. 0.93 to 2.29). OS rates after 10 years were 90.9 and 88.6 per cent in the MRI and control groups respectively (P = 0.427). The risk of death was 27 per cent higher in the control group (HR 1.27, 0.71 to 2.29). Locoregional, distant, and contralateral recurrence outcomes combined were increased in the control group (P = 0.048). A subgroup analysis of patients with breast cancer stages I-III showed that preoperative MRI improved DFS compared with conventional imaging, but this did not reach statistical significance (P = 0.057). CONCLUSION: After 10 years of follow-up, preoperative breast MRI as an adjunct to conventional imaging resulted in slightly, but non-significantly, improved DFS and OS. Registration number: NCT01859936 (http://www.clinicaltrials.gov).

Risk factors for pancreatitis following endoscopic retrograde cholangiopancreatography.

The aim of this study was to assess whether sex, age, ASA grade, previous history of acute pancreatitis, diabetes, hyperlipidaemia, hypercalcaemia, kidney disease and liver cirrhosis influence the risk for developing post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). A total of 15 800 ERCP procedures retrieved from the Swedish National Quality Register for Gallstone Surgery and ERCP (GallRiks) for 2006-2014 were identified and cross-checked with the National Patient Register. Women, patients aged less than 65 years, patients with hyperlipidaemia and those with a previous history of acute pancreatitis had a significantly increased risk of PEP, whereas patients with diabetes had a significantly decreased risk. Risk of pancreatitis following ERCP.

Effect of preoperative injection of superparamagnetic iron oxide particles on rates of sentinel lymph node dissection in women undergoing surgery for ductal carcinoma in situ (SentiNot study).

BACKGROUND: One-fifth of patients with a preoperative diagnosis of ductal carcinoma in situ (DCIS) have invasive breast cancer (IBC) on definitive histology. Sentinel lymph node dissection (SLND) is performed in almost half of women having surgery for DCIS in Sweden. The aim of the present study was to try to minimize unnecessary SLND by injecting superparamagnetic iron oxide (SPIO) nanoparticles at the time of primary breast surgery, enabling SLND to be performed later, if IBC is found in the primary specimen. METHODS: Women with DCIS at high risk for the presence of invasion undergoing breast conservation, and patients with DCIS undergoing mastectomy were included. The primary outcome was whether this technique could reduce SLND. Secondary outcomes were number of SLNDs avoided, detection rate and procedure-related costs. RESULTS: This was a preplanned interim analysis of 189 procedures. IBC was found in 47 and a secondary SLND was performed in 41 women. Thus, 78·3 per cent of patients avoided SLND (P < 0·001). At reoperation, SPIO plus blue dye outperformed isotope and blue dye in detection of the sentinel node (40 of 40 versus 26 of 40 women; P < 0·001). Costs were reduced by a mean of 24·5 per cent in women without IBC (€3990 versus 5286; P < 0·001). CONCLUSION: Marking the sentinel node with SPIO in women having surgery for DCIS was effective at avoiding unnecessary SLND in this study. Registration number: ISRCTN18430240 (http://www.isrctn.com).

Doxorubicin effects on leukemia and breast cancer cells in culture on the TK1 protein levels using AroCell TK 210 ELISA: a tool for drug development.

In this study changes in thymidine kinase 1 (TK1) levels after 24 hours of Doxorubicin (Dox) exposure of CEM and MDA MB-231 cells were determined using the commercially available AroCell TK 210 ELISA test. In cell extracts, TK1 levels increased twofold with 1 µM Dox in both cell lines, while the TK1 levels in the culture media increased with 5 and 10 µM of Dox only in case of CEM cells. In conclusion, this study reveals that the TK 210 ELISA can measure changes in intra- and extracellular TK1 levels apparently related to the mechanism of cytotoxicity of anti-cancer agents.

Superparamagnetic iron oxide nanoparticles as the sole method for sentinel node biopsy detection in patients with breast cancer.

BACKGROUND: Sentinel node biopsy (SNB) using superparamagnetic iron oxide (SPIO) nanoparticles is a novel method in breast cancer. Several studies have verified the non-inferiority of SPIO compared with the standard use of radioisotope 99m Tc with or without blue dye. The aim of the MONOS study presented here was to evaluate the use of SPIO as a sole tracer and the efficacy of tracer injection in the preoperative setting. METHODS: This prospective cohort study was carried out in two hospitals, one using 99m Tc and the other SPIO. 99m Tc was injected in the morning of the day of surgery or the day before. SPIO was either injected before surgery in the outpatient clinic or 1 h before the operation. RESULTS: A total of 338 consecutive patients with breast cancer underwent 343 procedures; SPIO nanoparticles were used in 184 procedures and 99m Tc-labelled tracer in 159. Detection rates for SPIO and 99m Tc were 95·6 and 96·9 per cent respectively (P = 0·537). All nodes with SPIO uptake were coloured brown. Fewer nodes were retrieved with SPIO (mean 1·35 versus 1·89), regardless of whether blue dye was used (P < 0·001). Preoperative SPIO injection (58·7 per cent of procedures), a median of 16 (range 2-27) days before the procedure, was associated with a better tracer-specific detection rate (95·3 versus 86 per cent; P = 0·031) and retrieval of more nodes (mean 1·43 versus 1·03; P < 0·001) than perioperative administration. Skin staining was present in 39·9 per cent of patients, and was related to breast-conserving surgery and periareolar injection. CONCLUSION: The use of SPIO alone is a safe alternative, with results comparable to those of the standard dual technique using 99m Tc and blue dye. The efficacy of injection in the preoperative setting simplifies logistics and improves performance. Skin staining can be prevented by a deeper peritumoral injection.

A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease.

Thymidine kinase (TK1) is an enzyme involved in DNA synthesis that leaks into the blood as a result of high cell turnover, particularly in the case of cancer. Serum TK1 activity has been used for prognosis and monitoring of leukemia and lymphoma patients for many years. Here, we describe the first clinical results with the newly developed TK 210 ELISA from AroCell AB. Sera from 124 breast cancer patients with known TNM classification along with sera from 53 healthy females were analyzed by TK 210 ELISA for TK1 protein and TK1 activity levels by the 3[H]-deoxythymidine (dThd) phosphorylation assay. The limit of detection for the TK 210 ELISA was 0.17 ng/ml, and 60 % of the sera from female blood donors were below this value. The median TK1 levels found in sera from breast cancer patients with T1 to T4 stage disease were 0.31, 0.46, 0.47, and 0.55 ng/ml, and these levels significantly differed from healthy controls. The median values of the biomarker CA 15-3 were also increased in patient sera from T1 to T4 patients (16, 34, 36, 40 U/ml, respectively). TK 210 ELISA showed significantly higher sensitivity for the T1 and T2 breast cancer patients compared to the TK activity assay. The combination of the TK1 ELISA and CA 15-3 biomarkers demonstrated a significant increase in sensitivity up to 15 % compared to each marker alone. This evaluation of the TK 210 ELISA strongly suggests that it can provide independent and complementary information for patients with breast cancer.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis.

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.

Autoresonant-spectrometric determination of the residual gas composition in the ALPHA experiment apparatus.

Knowledge of the residual gas composition in the ALPHA experiment apparatus is important in our studies of antihydrogen and nonneutral plasmas. A technique based on autoresonant ion extraction from an electrostatic potential well has been developed that enables the study of the vacuum in our trap. Computer simulations allow an interpretation of our measurements and provide the residual gas composition under operating conditions typical of those used in experiments to produce, trap, and study antihydrogen. The methods developed may also be applicable in a range of atomic and molecular trap experiments where Penning-Malmberg traps are used and where access is limited.

High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: implications for in vitro diagnostics.

Determination of serum thymidine kinase 1 (STK1) activity has been used as a proliferation marker for neoplastic diseases in both human and veterinary medicine. The purpose of this study was to determine STK1 activity and enzyme levels in different dog tumours. Serum samples from three dogs with leukaemia, five with lymphoma, 21 with solid tumours and 18 healthy dogs were analyzed for STK1 activity, using an optimized [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for STK1 protein levels using an immunoaffinity/western blot assay. STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean ± standard deviation [SD] = 65 ± 79, 1.1 ± 0.5, and 1.0 ± 0.4 pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26 kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean ± SD=33 ± 12, 30 ± 13, and 10 ± 5 ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [(3)H]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26 kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker.

Elevation of serum thymidine kinase 1 in a bacterial infection: canine pyometra.

Pyometra is a bacterial infection of the uterus that is common in dogs and is potentially life-threatening if delayed in diagnosis and/or treatment. Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis, and it is also present in serum from patients with malignant diseases. TK1 has been used as a cell proliferation biomarker for many years in human medicine and recently in dogs. However, little is known regarding serum TK1 levels in individuals with bacterial infection. The objective of this study was to determine the activity of serum TK1 in dogs with pyometra and compare it with hematologic and biochemical parameters, e.g., acute phase proteins and inflammatory mediators such as C-reactive protein and Prostaglandin F(2α). Serum and plasma TK1 activity of 40 healthy female dogs and 54 dogs with pyometra were analyzed using an optimized [(3)H]-thymidine phosphorylation assay. TK1 activities in serum or plasma were significantly higher in dogs with pyometra as compared with healthy female dogs (mean ± SD: 4.0 ± 7.3 pmol/min/mL in the pyometra group and 1.07 ± 0.34 pmol/min/mL in healthy control group). However, there was no difference in TK1 activity between systemic inflammatory response syndrome (SIRS) positive (n = 38) and SIRS negative (n = 16) pyometra cases. Furthermore, the plasma TK1 activity decreased in six and increased in one pyometra patients (n = 10), 24 h after ovariohysterectomy. No significant correlations (P > 0.05) were found between TK1 activity and hematological or other biochemical parameters. In conclusion, the TK1 activity was significantly elevated in dogs with pyometra. Further studies are needed to evaluate the mechanism and role of serum TK1 activity in bacterial infections and its possible diagnostic or prognostic value.

A sensitive and kinetically defined radiochemical assay for canine and human serum thymidine kinase 1 (TK1) to monitor canine malignant lymphoma.

Thymidine kinase 1 (TK1) is a cell cycle regulated enzyme with maximum expression during the S phase. Serum TK1 (S-TK1) is a unique biomarker for cell proliferation. Here, an optimized [(3)H]-thymidine (dThd) phosphorylation assay is described, which is as sensitive as the commercially available TK-REA and TK-Liaison assays for measurement of S-TK1 activity in dogs and humans. Serum samples from dogs (35 healthy, 32 with lymphoma, 2 with leukemia, and 35 with solid tumors) and humans (18 healthy, 9 with chronic lymphocytic leukemia, 10 with myelodysplastic syndrome) were analyzed using the [(3)H]-dThd assay. Mean S-TK1 activities were 1.11 ± 0.46 pmol/min/mL in healthy dogs and 1.15 ± 0.32 pmol/min/mL in healthy humans. S-TK1 activities in dogs with hematological malignancies were 24.2 ± 47.9 pmol/min/mL, and the receiver operating characteristic curve showed an area under the curve of 0.88. With a cut-off value of 1.9 pmol/min/mL (mean value ± 2 SD), the sensitivity was 0.94 and the specificity was 0.68. Very similar results were obtained with human samples (healthy and lymphoma cases). S-TK1 activities measured during chemotherapy of six dogs with lymphoma were drastically reduced. In one case, S-TK1 activity increased prior to relapse. S-TK1 levels in dogs with solid tumors did not differ from the healthy group. S-TK1 activities correlated with those determined with the TK-REA and TK-Liaison assays (r=0.92 and r=0.96, respectively). In conclusion, this optimized [(3)H]-dThd assay is fast, sensitive and economical for measuring S-TK1 activity and should increase its clinical use as biomarker.

The effects of isoflurane anesthesia and mechanical ventilation on renal function during endotoxemia.

BACKGROUND: Isoflurane is a common anesthetic agent used in human surgery and in animal models of sepsis. It has been suggested to have beneficial anti-inflammatory properties and to protect kidney function. Here, we investigated the effect of isoflurane on the development of kidney injury and dysfunction during 48-h endotoxemia in sheep. METHODS: Before the experiments, the sheep (n=16) were surgically equipped with transit-time flowprobes around the renal, femoral and superior mesenteric artery. The animals were randomized to either be anesthetized with isoflurane and mechanically ventilated or to remain conscious while they received intravenous Escherichia coli lipopolysaccharide (LPS) for 48 h (25 ng/kg/min). In two animals in each group, the LPS was excluded to investigate any effect of isoflurane per se over time. RESULTS: Endotoxemia caused cardiovascular changes typical for hyperdynamic sepsis and, although renal hyperemia occurred, impaired renal function in both groups. Compared with conscious animals, isoflurane significantly (P<0.05) reduced urine output, renal creatinine clearance, fractional sodium excretion and renal blood flow during endotoxemia. Furthermore, the plasma concentrations of urea and creatinine increased more in the anesthetized animals. Isoflurane anesthesia also enhanced neutrophil activity and accumulation in the kidney during endotoxemia. N-acetyl-ß-D-glucosaminidase was significantly increased, with no inter-group difference as an indication of tubular injury. CONCLUSIONS: The results of the current study suggest that isoflurane anesthesia (minimum alveolar concentration 1.0) with mechanical ventilation aggravates renal dysfunction during 48 h of endotoxemia and does not significantly reduce the inflammatory response or signs of tubular damage.

Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases.

As cell proliferation is one of the hallmarks of cancer, various types of proliferation markers are used as important tools in diagnosis, prognosis, treatment decision-making and follow-up in clinical oncology. The S phase-specific protein thymidine kinase 1 (TK1) can be used in immunohistochemistry for RNA/protein expression in tissue specimens and for activity or protein/peptide levels in serum from patients. TK1 has been used mainly in haematologic malignancies in humans, but also found beneficial in canine malignancies. As the protein sequence homology is high between humans and dogs, findings in canine models will have a high comparative value in further human research as well. In the present review, we will focus on the recent results concerning TK1's S phase-correlated expression, increased serum levels of TK1 in patients with malignancies and the relevance for veterinary and comparative oncology. Finally, the benefit of recently developed specific anti-TK1 antibodies suitable for immunologic assay is discussed.

Thymidine kinase 1 is a potential marker for prognosis and monitoring the response to treatment of patients with breast, lung, and esophageal cancer and non-Hodgkin's lymphoma.

Thymidine kinase 1 (TK1) is converting thymidine to thymidine monophosphate, and is related to DNA replication and cell proliferation. The use of the TK1 protein levels as a proliferation marker in malignancies is here summarized. TK1 protein in serum (STK1p) and TK1 expression in tissues were determined by a chemoluminescent dot blot assay and by immunohistochemistry staining, respectively. The expression of TK1 in tumor tissues correlated to pathological stages and clinical grades of carcinomas (ca) of esophagus, lung and in premalignancy of breast ductal ca. STK1p could monitor the out-come of tumor therapy by being correlated to remission [breast ca, non-Hodgkin's lymphoma], relapse [breast ca] and to survival [non-Hodgkin's lymphoma] of patients. In a health screening study of 12,641 persons, STK1p seemed to predict the risk of development of neoplasia related diseases at early stage.

Infection by Helicobacter pylori expressing the BabA adhesin is influenced by the secretor phenotype.

Helicobacter pylori (Hp) infects half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. Our aim was to evaluate the significance of secretor and Lewis status in infection and in vitro adherence by Hp expressing BabA adhesin. We enrolled 304 Hp-infected individuals from Northern Portugal. Gastric biopsies, blood and saliva were collected. Polymerase chain reaction (PCR) and immunofluorescence were used to detect BabA+ Hp in gastric biopsies. In vitro adherence by a BabA expressing Hp strain to gastric biopsies was performed. Secretor status was identified by Ulex, a lectin that recognizes secretor-dependent glycan structures in saliva and in gastric mucosa, and by Lewis(a/b) antibodies, and indirectly by identification of an inactivating mutation in the FUT2 gene (G428A). BabA status of infecting Hp was associated with CagA and VacAs1 (p < 0.05), intercellular localization of Hp (p < 0.01) and the presence of intestinal metaplasia (p < 0.05) and degenerative alterations (p < 0.005) in the biopsies. BabA was associated (p < 0.05) with Ulex staining of gastric biopsies and, although not significantly, to absence of homozygosity for FUT2 G428A inactivating polymorphism. In vitro Hp adherence was higher in cases wild-type or heterozygous for FUT2 G428A mutation (p < 0.0001), cases staining for Ulex (p < 0.0001) and a(-)b+ and a(-)b(-) secretor phenotypes (p < 0.001). In conclusion, BabA+ Hp infection/adhesion is secretor-dependent and associated with the severity of gastric lesions.

Parenchymal lesions in pharmacoresistant temporal lobe epilepsy: dual and multiple pathology.

OBJECTIVES: Dual pathology is reported in 5-30% of temporal lobe resections performed in pharmacoresistant epilepsy. Dual pathology may be of importance for surgical planning and also for the understanding of the pathogenesis of epilepsy. We describe the frequency of dual or multiple pathology, i.e. more than one histopathological diagnosis, in adults with temporal lobe resections. MATERIAL AND METHODS: Surgical specimens from 33 consecutive patients with resections including mesial as well as neocortical temporal structures were reviewed. All histopathological findings were recorded. Post-mortem specimens from 11 control subjects were also reviewed. RESULTS: Dual or multiple pathology was found in almost half of the epilepsy patients (48%). Hippocampal sclerosis was found in 25 patients (76%), malformations of cortical development in 15 (46%), of which 12 (36%) were microdysgenesis, and low-grade tumours in seven (21%). Apart from mild gliosis, there were no histopathological changes in the control specimens. CONCLUSION: Dual or multiple pathology was a common finding in this group of adults with temporal lobe resections. In order to increase our understanding of how aetiological factors may combine in the development of seizures, we consider it relevant and important to report all histopathological findings in epilepsy surgery series.

Dose-response for radiation-induced apoptosis, residual 53BP1 foci and DNA-loop relaxation in human lymphocytes.

The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1-2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose-response with saturation at 2-3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose-responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2-3 Gy induced at least one residual 53BP1 focus per cell. The dose-responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2-3 Gy. The correlation between dose-responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.