Christos E. Zois

Radiation-induced autophagy in normal and cancer cells: towards novel cytoprotection and radio-sensitization policies?

Autophagy or Type II Programmed Cell Death (PCD) is a major intracellular pathway for the degradation and recycling of proteins, ribosomes and entire organelles. The role of this pathway in the anti-tumor effect of radiotherapy and in radiation toxicity is obscure. A complicated machinery of genes and proteins is involved in the regulation of autophagy as a response to a variety of stress factors including hypoxia, nutrient deprivation, cytotoxic agents and radiotherapy. Continuously accumulating data suggest that autophagic response of cancer cells to radiotherapy is a major pathway which, in contrast to apoptosis that leads to death, may lead to either death or cellular survival. A variety of agents have been recognized that induce or block autophagy, directly interfering with the cytotoxic effect of radiotherapy. Simultaneous targeting of autophagy and apoptosis during radiotherapy seems to further augment the anti-tumor effect. Radiobiology research should focus on the differential effect of fractionation on the induction of autophagy in different tumors and on the manipulation of this with autophagy triggering agents. Whether manipulation of this pathway in normal tissues may be used to confer cytoportection deserves also thorough investigation. Moreover, the role of pretreatment autophagic indices in tumor cells in predicting radiotherapy and chemotherapy outcome should be examined in translational studies.

EVALUATION OF THE ALAMARBLUE ASSAY FOR ADHERENT CELL IRRADIATION EXPERIMENTS

The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8th day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays.

Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis

Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.

Establishment and validation of a method for multi-dose irradiation of cells in 96-well microplates.

Microplates are useful tools in chemistry, biotechnology and molecular biology. In radiobiology research, these can be also applied to assess the effect of a certain radiation dose delivered to the whole microplate, to test radio-sensitivity, radio-sensitization or radio-protection. Whether different radiation doses can be accurately applied to a single 96-well plate to further facilitate and accelerated research by one hand and spare funds on the other, is a question dealt in the current paper. Following repeated ion-chamber, TLD and radiotherapy planning dosimetry we established a method for multi-dose irradiation of cell cultures within a 96-well plate, which allows an accurate delivery of desired doses in sequential columns of the microplate. Up to eight different dose levels can be tested in one microplate. This method results in fast and reliable estimation of radiation dose-response curves.

Lung autophagic response following exposure of mice to whole body irradiation, with and without amifostine

PURPOSE The effect of ionizing irradiation on the autophagic response of normal tissues is largely unexplored. Abnormal autophagic function may interfere the protein quality control leading to cell degeneration and dysfunction. This study investigates its effect on the autophagic machinery of normal mouse lung. METHODS AND MATERIALS Mice were exposed to 6 Gy of whole body γ-radiation and sacrificed at various time points. The expression of MAP1LC3A/LC3A/Atg8, beclin-1, p62/sequestosome-1 and of the Bnip3 proteins was analyzed. RESULTS Following irradiation, the LC3A-I and LC3A-II protein levels increased significantly at 72 h and 7 days. Strikingly, LC3A-II protein was increased (5.6-fold at 7 days; p<0.001) only in the cytosolic fraction, but remained unchanged in the membrane fraction. The p62 protein, was significantly increased in both supernatant and pellet fraction (p<0.001), suggesting an autophagosome turnover deregulation. These findings contrast the patterns of starvation-induced autophagy up-regulation. Beclin-1 levels remained unchanged. The Bnip3 protein was significantly increased at 8 h, but it sharply decreased at 72 h (p<0.05). Administration of amifostine (200 mg/kg), 30 min before irradiation, reversed all the LC3A and p62 findings on blots, suggesting restoration of the normal autophagic function. The LC3A and Beclin1 mRNA levels significantly declined following irradiation (p<0.01), whereas Bnip3 levels increased. CONCLUSIONS It is suggested that irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. Whether this is due to defective maturation or to aberrant degradation of the autophagosomes requires further investigation.

LC3 immunostaining pitfalls

Sir: We read with interest the article by Choi et al. on the expression of the autophagosome-related proteins LC3A and LC3B in a large series of breast cancer cases. Although the authors failed to find any overall association with prognosis, LC3A and LC3B were directly related to high-grade tumours and a triple-negative pattern. The authors are to be congratulated on producing such a thorough investigation, and their findings are, by and large, in accordance with a previously published study of ours, in which cytoplasmic LC3A overexpression was associated with nodal involvement, but not with patient survival. However, a tumour-specific pattern, so-called ‘stone-like’ structures, which was identified in a variety of epithelial carcinomas by our group, including breast, endometrial, colon, lung, skin and bladder carcinomas, has not been recognized in the study of Choi et al. The LC3A-positive stone-like structures, which we consider to be a hallmark of epithelial cancer tissues, are readily recognized as large, rounded, densely stained material, typically enclosed within an LC3A-expressing cytoplasmic vacuole (Figure 1A,B). Choi et al. have offered an explanation for their negative result by proposing probable differences in the specificity of the LC3A antibody used in their study. Indeed, the choice of a suitable antibody is of crucial importance in autophagy research, particularly in demonstrating immunohistochemically the stonelike autophagic pattern. Our experience suggests that the anti-LC3A antibody AP1805a (Abgent, San Diego, CA, USA) provides more clear and specific immunostaining than other tested commercially available antibodies, including the anti-LC3A antibody EP1528Y (Abcam, Cambridge, UK) used by Choi’s group. In a pilot study, this latter antibody produced specific staining in only a minority of breast cancer cases, and the staining was invariably of weak intensity. This finding is in accordance with that reported by Choi et al., who found strong and extensive (>30% of cells) staining in only 6% of the cases examined; a strikingly high proportion of tumours, up to 93.7%, were LC3A-negative. In a subsequent study, and in an attempt to increase the proportion of LC3A-positive cells, the authors considered as positive even cases with weak staining intensity and a very low percentage of positive cells. These staining patterns obtained with the EP1528Y antibody are in direct contrast to those obtained with the AP1805a antibody, which showed strong and extensive (>50% of cells) positivity in >30% of breast carcinomas. By use of the AP1805a antibody, the LC3A-positive ‘stonelike’ structures were readily recognized (Figure 1A,B) and they were related to an unfavourable prognosis. Similar results have been reported recently by Spowart et al., who, after using the AP1805a antibody, not only demonstrated stone-like structures in ovarian carcinomas but also associated their increased number with a poor outcome. It is therefore mandatory for authors to pay extra attention when they choose LC3 antibodies for autophagy research. Vigorous validation is necessary, because there is uncertainty regarding which form of

Normal tissue radioprotection by amifostine via Warburg-type effects

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.