Microdosimetry modeling with auger emitters in generalized cell geometry.

A microdosimetry model was developed for the prediction of cell viability for irregular non-spherical cells that were irradiated by low energy, short range auger electrons. Measured cell survival rates for LNCaP prostate cancer were compared to the computational results for the radioisotopes177Lu and161Tb (conjugated to PSMA). The cell geometries used for the computations were derived directly from the cell culture images. A general computational approach was developed to handle arbitrary cell geometries, based on distance probability distribution functions (PDFs) derived from basic image processing. The radiation calculations were done per coarse grained PDF bin to reduce computation time, rather than on a pixel/voxel basis. The radiation dose point kernels over the full electron spectrum were derived using Monte Carlo simulations for energies below 50 eV to account for the propagation of auger electrons over length scales at and below a cellular radius. The relative importance of short range auger electrons were evaluated between the two nuclide types. The microdosimetry results were consistent with the cell viability measurements, and it was found that161Tb was more efficient than177Lu primarily due to the short range auger electrons. We foresee that imaging based microdosimetry can be used to evaluate the relative therapeutic effect between various nuclide candidates.

The efficacy of CHK1 inhibitors is not altered by hypoxia, but is enhanced after reoxygenation.

Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia). The influence of less severe hypoxia on the efficacy of CHK1 inhibitors, administered either as single agents or in combination with other treatments, remains to be investigated. Here, we have assayed the effects of the CHK1 inhibitors, AZD7762 and UCN-01, during various hypoxic conditions and after reoxygenation in the absence and presence of ionizing radiation. Treatment with CHK1 inhibitors during acute or prolonged hypoxia (< 0.03%, 0.2%, and 1% O2; 3 h or 20-24 h) gave similar effects on cell survival as treatment with these inhibitors during normoxia. CHK1 inhibitors combined with ionizing radiation showed similar radiosensitization in hypoxic and normoxic cells. However, when the inhibitors were administered after reoxygenation following prolonged hypoxia (< 0.03% and 0.2%; 20-24 h), we observed decreased cell survival and stronger induction of the DNA damage marker, γH2AX, in S-phase cells. This was accompanied by enhanced phosphorylation of the single-stranded DNA-binding replication protein A. These results suggest that the cytotoxic effects of CHK1 inhibitors are enhanced after reoxygenation following prolonged hypoxia, most likely due to the increased replication-associated DNA damage. Combining CHK1 inhibitors with other treatments that cause increased reoxygenation, such as fractionated radiotherapy, might therefore be beneficial.

The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells.

BACKGROUND AND PURPOSE: The radiation-induced G2 checkpoint helps facilitate DNA repair before cell division. However, recent work has revealed that human cells often escape the G2 checkpoint with unrepaired DNA breaks. The purpose was to explore whether G2 checkpoint activation occurs according to a threshold level of DNA damage. MATERIALS AND METHODS: G2 checkpoint activation was assayed at 75-90 min and 24-48 h after X-ray irradiation of BJ diploid fibroblasts and U2OS osteosarcoma cells. Multiparameter flow cytometry with pacific blue barcoding, and flow cytometry-based sorting of phospho-H3 positive cells to microscope slides, were used to examine the DNA damage marker γ-H2AX in individual mitotic cells that had escaped the G2 checkpoint. RESULTS: For all radiation doses and times tested, the number of γ-H2AX foci varied between individual mitotic cells. At 75 min the median levels of γ-H2AX in mitotic cells increased with higher radiation doses. At 24-48 h, following a prolonged G2 checkpoint, cells were more resistant to checkpoint re-activation by a second dose of radiation. CONCLUSION: Our results suggest that different amounts of DNA damage are needed to activate the G2 checkpoint in individual cells. Such single cell variation in checkpoint activation may potentially contribute to radiation-induced genomic instability.

Expression of cytosolic 5' nucleotidase does not correlate with expression of oxidative metabolism marker: myoglobine in human skeletal muscles.

Our previous studies had shown that cytosolic 5'nucleotidase-I (cN-I) is expressed in several tissues in pigeons, including brain and several different skeletal muscles. We observed that cN-I mRNA levels varied among different pigeon muscles. Initial quantification of the differences revealed that about 5-10 times more of cN-I transcript was present in red, oxidative muscles (breast muscle and gastrocnemius) than in white ones, composed of glycolytic fibers (biceps brachii). We had found this observation very intriguing and decided to compare human skeletal muscles distribution of cN-I with the type of oxygen metabolism. Our screen involved 60 samples of several human muscles and we assayed the correlation between the amount of transcripts of cN-I and myoglobine, which we took as a measure of oxidative-slow twitch fibers. Our question was whether in humans, cN-I presence in skeletal muscles was related to their fiber composition. If that was the case, then cN-I expression could serve as a tool to assess the percentage of oxidative fibers in any given human muscle sample, where myoglobine expression could not be readily measured. After quantification of expression of both genes, we concluded that there was no correlation between expression of cN-I and fiber type. Therefore, contrary to the pigeon muscles, cN-I did not reflect the ratio of oxidative fibers to the total mass of the muscle sample in humans. That difference indicated that there were certain mechanisms that differentially regulated the expression of cN-I in muscle tissues of mammals and lower vertebrates.

Adenosine as a metabolic regulator of tissue function: production of adenosine by cytoplasmic 5'-nucleotidases.

Adenosine is a product of complete dephosphorylation of adenine nucleotides which takes place in various compartments of the cell. This nucleoside is a significant signal molecule engaged in regulation of physiology and modulation of the function of numerous cell types (i.e. neurons, platelets, neutrophils, mast cells and smooth muscle cells in bronchi and vasculature, myocytes etc.). As part a of purinergic signaling system, adenosine mediates neurotransmission, conduction, secretion, vasodilation, proliferation and cell death. Most of the effects of adenosine help to protect cells and tissues during stress conditions such as ischemia or anoxia. Adenosine receptors and nucleoside transporters are targets for potential drugs in many pathophysiological situations. The adenosine-producing system in vertebrates involves a cascade dephosphorylating ATP and ending with 5'-nucleotidase (EC 3.1.3.5) localized either on the membrane or inside the cell. In this paper the cytoplasmic variants of 5'-nucleotidase are broadly characterized as well as their clinical relevance. The role of AMP-selective 5'-nucleotidase (cN-I) in the heart, skeletal muscle and brain is highlighted. cN-I action is crucial during ischemia and important for the efficacy of some nucleoside-based drugs and in the regulation of the substrate pool for nucleic acids synthesis. Inhibitors used in studying the roles of cytoplasmic and membrane-bound 5'-nucleotidases are also described.

Induction of hypoxia-inducible factor-1alpha overexpression by cobalt chloride enhances cellular resistance to photodynamic therapy.

Although photodynamic therapy (PDT) has been approved by regulatory agencies worldwide for the treatment of several oncologic and non-oncologic conditions, PDT-induced tissue hypoxia as a result of vascular damage and photochemical oxygen consumption limits the efficacy of this modality. This may largely be due to hypoxia-mediated angiogenesis via hypoxia-inducible factor-1alpha (HIF-1alpha), a major transcription factor involved in angiogenesis, hematopoiesis and anaerobic energy metabolism. We hypothesized that hypoxia-induced HIF-1alpha overexpression may also lead to tumor cells resistant to PDT by favouring tumor cell proliferation. Human esophageal normal Het-1A and tumor KYSE-70 and KYSE-450 cell lines were used in the present study. High-expression of HIF-1alpha induced in vitro by cobalt chloride (CoCl(2))-mediated chemical hypoxia mimic was clearly seen in the Het-1A cell line. In addition, cells treated with CoCl(2) were more resistant to 5-aminolevulinic acid (ALA)-mediated PDT than those without CoCl(2) treatment. The photosensitivity of the cells to ALA-PDT decreased with increasing HIF-1alpha expression by enhancing CoCl(2) concentrations. Moreover, transfection of the cells with anti-HIF-1alpha short interfering RNA (siRNA) knocked down the HIF-1alpha expression and restored the photosensitivity of the cells to ALA-PDT. However, the induction of HIF-1alpha expression by CoCl(2) was not indicated in both KYSE-70 and KYSE-450 cell lines, and no difference in cell survival was found after ALA-PDT in the presence and absence of CoCl(2). We thus conclude that high-expression of HIF-1alpha induced by CoCl(2) plays an important role in the resistance of the Het-1A cells to ALA-PDT. The present finding suggests that hypoxia-induced HIF-1alpha overexpression attenuates PDT efficacy through probably not only angiogenesis, but also cellular resistance to the modality. PDT in combination with anti-HIF-1alpha treatment may thus enhance the PDT efficacy.

Targeting PBR by hexaminolevulinate-mediated photodynamic therapy induces apoptosis through translocation of apoptosis-inducing factor in human leukemia cells.

Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.

Isolation and characterization of pigeon breast muscle cytosolic 5'-nucleotidase-I (cN-I).

5'-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5'-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5'-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.