Stamatia Pouliliou

Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Lactate dehydrogenase 5 isoenzyme overexpression defines resistance of prostate cancer to radiotherapy.

Background:Radiotherapy provides high-cure rates in prostate cancer. Despite its overall slow clinical growth, high proliferation rates documented in a subset of tumours relate to poor radiotherapy outcome. This study examines the role of anaerobic metabolism in prostate cancer growth and resistance to radiotherapy.Methods:Biopsy samples from 83 patients with prostate cancer undergoing radical hypofractionated and accelerated radiotherapy were analysed for MIB1 proliferation index and for lactate dehydrogenase isoenzyme LDH5, a marker of tumour anaerobic metabolism. Ninety-five surgical samples were in parallel analysed. Correlation with histopathological variables, PSA and radiotherapy outcome was assessed. Dose–response experiments were performed in PC3 and DU145 cancer cell lines.Results:High MIB1 index (noted in 25% of cases) was directly related to Gleason score (P<0.0001), T3-stage (P=0.0008) and PSA levels (P=0.03). High LDH5 (noted in 65% of cases) was directly related to MIB1 index (P<0.0001), Gleason score (P=0.02) and T3-stage (P=0.001). High Gleason score, MIB1, LDH5 and PSA levels were significantly related to poor BRFS (P=0.007, 0.01, 0.03 and 0.01, respectively). High Gleason score (P=0.04), LDH5 (P=0.01) and PSA levels (P=0.003) were significantly related to local recurrence. MIB1 and T-stage did not affect local control. Silencing of LDHA gene in both prostate cancer cell lines resulted in significant radiosensitisation.Conclusions:LDH5 overexpression is significantly linked to highly proliferating prostate carcinomas and with biochemical failure and local relapse following radiotherapy. Hypoxia and LDHA targeting agents may prove useful to overcome radioresistance in a subgroup of prostate carcinomas with anaerobic metabolic predilection.

Gamma histone 2AX (γ-H2AX)as a predictive tool in radiation oncology.

Abstract Ionizing radiation cause DNA damage to cells, leading them to cell death via DNA double-strand breaks (DSBs) formation. DSBs formation is followed immediately by histone H2AX phosphorylation (γ-H2AX) and multitude repair factors accumulation. Here we present the methods and the bio-sampling for γ-H2AX detection, γ-H2AX formation in normal cells and animal tissues, in cancer cell lines/tissues and in clinical trials after radiation treatment, alone or in combination with other factors. The purpose of this review is to highlight the use of γ-H2AX, as a marker to assess DNA damage and repair.

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.

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.

Cytogenetic evaluation of pre-pregnancy smoking in maternal and newborn lymphocytes

OBJECTIVE To study cytogenetic damage in order to estimate the effect of pre-pregnancy smoking on pregnant women and their foetuses. STUDY DESIGN Lymphocyte cultures were obtained from peripheral blood of 20 women who quit smoking during pregnancy, and umbilical cord blood of their newborns at delivery. Cytogenetic analyses were performed for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) using the Fluorescence Plus Giemsa staining technique. Twenty non-smoking women and their newborns were evaluated as controls. CPT-11, a known antineoplastic, was used as a positive genotoxic agent in order to correlate non-smoking women with smoking women and reveal any underlying chromosome instability. Statistical evaluation of SCE frequencies, PRI and MI was based on independent samples t-test in order to estimate the effect of pre-pregnancy smoking on mothers and their newborns. RESULTS SCEs were induced in the cord blood lymphocytes of newborns whose mothers smoked before pregnancy when they were exposed to the mutagenic agent CPT-11 (p<0.01). A similar increase in SCEs was observed in both non-smoking and smoking mothers exposed to CPT-11. Newborns in both groups had significantly lower SCE levels than their mothers (p<0.01). CONCLUSION Pre-pregnancy smoking results in cytogenetic damage for both mothers and newborns, and is an important risk factor for cancer and/or other genetic-related diseases. Smoking cessation needs to occur well before conception in order to avoid the strong cytogenetic association between pre-pregnancy smoking by mothers and their newborns.

Survival Fraction at 2 Gy and γH2AX Expression Kinetics in Peripheral Blood Lymphocytes From Cancer Patients: Relationship With Acute Radiation-Induced Toxicities

PURPOSE Predictive assays for acute radiation toxicities would be clinically relevant in radiation oncology. We prospectively examined the predictive role of the survival fraction at 2 Gy (SF2) and of γH2AX (double-strand break [DSB] DNA marker) expression kinetics in peripheral blood mononuclear cells (PBMCs) from cancer patients before radiation therapy. METHODS AND MATERIALS SF2 was measured with Trypan Blue assay in the PBMCs from 89 cancer patients undergoing radiation therapy at 4 hours (SF2[4h]) and 24 hours (SF2[24h]) after ex vivo irradiation. Using Western blot analysis and band densitometry, we further assessed the expression of γH2AX in PBMC DNA at 0 hours, 30 minutes, and 4 hours (33 patients) and 0 hour, 4 hours, and 24 hours (56 patients), following ex vivo irradiation with 2 Gy. Appropriate ratios were used to characterize each patient, and these were retrospectively correlated with early radiation therapy toxicity grade. RESULTS The SF2(4h) was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio(30min) (band density of irradiated/non-irradiated cells at 30 minutes) revealed, similarly, a significant inverse association (P=.0001). The DSB DNA repair rate from 30 minutes to 4 hours, calculated as the relative RγH2AX-ratio (γH2AX-ratio(4h)/γH2AX-ratio(30min)) showed a significant direct association with high toxicity grade (P=.01). CONCLUSIONS Our results suggest that SF2 is a significant radiation sensitivity index for patients undergoing radiation therapy. γH2AX Western blot densitometry analysis provided 2 important markers of normal tissue radiation sensitivity. Low γH2AX expression at 30 minutes was linked with high toxicity grade, suggesting that poor γH2AX repair activity within a time frame of 30 minutes after irradiation predicts for poor radiation tolerance. On the other hand, rapid γH2AX content restoration at 4 hours after irradiation, compatible with efficient DSB repair ability, predicts for increased radiation tolerance.

Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy

Background:The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation.Methods:A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1nu was evaluated.Results:Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours.Conclusions:The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.

Effect of maternal smoking during pregnancy on fetus: a cytogenetic perspective.

Abstract Objective: The examination of the genotoxic, cytostatic and cytotoxic effects of smoking during pregnancy. Method: Lymphocyte cultures of peripheral blood were received from 20 women who smoked during pregnancy as well as umbilical cord blood of their newborns. Fluorescence Plus Giemsa staining technique was used in order to perform cytogenetic analyses for three indices, Sister Chromatid Exchanges (SCEs), Proliferation Rate Index (PRI) and Mitotic Index (MI). To reveal any underlying chromosome instability, CPT-11 was used as a positive control. Results: Newborns whose mothers smoke during pregnancy had increased SCEs levels on their lymphocytes when they were exposed to the mutagenic agent CPT-11 (p < 0.01) compared with newborns lymphocytes exposed to the same agent with non-smoking mothers. Also, mothers smoking during pregnancy had increased SCE levels when their lymphocytes were exposed to CPT-11 (p < 0.01) compared with non smoking mothers whose lymphocytes were exposed to the same agent. In both groups newborns appeared as having decreased (p < 0.01) spontaneous SCEs levels compared with the corresponding SCE rates of their mothers. Decreases of PRIs and MIs are observed in mothers compared to their newborns. Conclusion: Smoking during pregnancy can promote cytogenetic damage in newborn’s DNA, causing chromosome instability. The clinical importance of this indirect damage lies in the fact that this type of damage can act synergistically with other environmental and/or chemical mutagenic substances possibly leading to carcinogenicity.

Overexpression of LC3A autophagy protein in follicular and diffuse large B-cell lymphomas

BACKGROUND AND OBJECTIVES Autophagy is a self-degradation mechanism induced under stress conditions in all eukaryotic cells. Its activity in human lymphomas has not been studied as yet. METHODS In this study, the autophagic activity of lymphoid cells was investigated in follicular lymphomas (FL; 48 cases), diffuse large B-cell lymphomas (DLBCL; 78 cases), and in reactive follicular hyperplasias (41 cases), using the light chain 3A (LC3A) antibody and a standard immunohistochemical technique. RESULTS In all cases, the pattern of LC3A reactivity was uniformly diffuse cytoplasmic, but expressed more frequently in FLs (68.8%) than in DLBCLs (41%) (p=0.02), and much more commonly in DLBCLs than in reactive lymph nodes (24.3%) (p<0.006). Interestingly, FLs expressing LC3A in >10% of lymphoid cells (high reactivity) were associated with the hypoxia-related protein HIF1α and the enzyme of anaerobic metabolism lactate dehydrogenase LDH5 (p=0.004 and p=0.003, respectively). Such associations, however, were not a feature in DLBCLs of increased LC3A activity. CONCLUSIONS LC3A expression in FLs is hypoxia-induced, whereas its expression in DLBCLs may be regulated by other molecular mechanisms. The current study provides a tool for further assessment of autophagic activity in translational and autophagy targeting therapy studies.