Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

Background: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone ‘Norgestrel’ as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1 μM) showed this rise to be PGRMC1-dependent, primarily utilising calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μM) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro.

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

18-Fluorodeoxyglucose positron emission tomography/computed tomography in the management of aggressive non-Hodgkin's B-cell lymphoma

18-Fluorodeoxyglucose (FDG-PET/CT) is an established imaging modality that has been proven to be of benefit in the management of aggressive B-cell non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma and advanced stage follicular lymphoma. The combination of anatomic and functional imaging afforded by FDG-PET/CT has led to superior sensitivity and specificity in the primary staging, restaging, and assessment of response to treatment of hematological malignancies when compared to FDG-PET and CT alone. The use of FDG-PET/CT for post treatment surveillance imaging remains controversial, and further study is needed to ascertain whether this modality is cost effective and appropriate for use in this setting.

JNK regulates Fas receptor mediated apoptosis in prostate cancer cell lines

Prostate Cancer is a disease that primarily affects elderly men. The incidence of prostate cancer has been progressively increasing in the western world over the last two decades. Life expectancy and diet are believed to be the main factors contributing to this increase in prevalence. Prostate cancer is a slowly progressing disorder and patients often live for over 10 years after initially being diagnosed with prostate cancer. However, patients with hormone refractory prostate cancer have a poor prognosis and generally do not survive for longer than 2 or 3 years. Hormone refractory prostate cancer is responsible for over 200,000 deaths each year and current chemotherapeutic regimens are only useful as palliative agents. The long-term survival rate is poor and chemotherapy does not significantly increase this. Cell lines derived from hormone refractory tumours usually display elevated resistance to many cytotoxic drugs. The Fas receptor is a membrane bound protein capable of binding to a ligand called Fas ligand. Engagement of Fas receptor with Fas ligand results in clustering of Fas receptor on the plasma membrane of cells. A number of proteins responsible for initiating apoptosis are recruited to the plasma membrane and are activated in response to elevated local concentrations. This series of events initiates a proteolysis cascade and that culminates in the degradation of structural and enzymatic processes and the repackaging of cellular constituents within membrane bound vesicles that can be endocytosed and recycled by surrounding phagocytic cells. The Fas receptor is believed to be a key mechanism by which immune cells can destroy damaged cells. Consequently, resistance to Fas receptor mediated apoptosis often correlates with tumour progression. It has been reported that prostate cancer cell lines display elevated resistance to Fas receptor mediated apoptosis and this correlates with the stage of tumour from which the cell lines were isolated. JNK, a stress-activated protein kinase, has been implicated both with increased survival and increased apoptosis in prostate cancer. Elevated endogenous JNK activity has been demonstrated to correlate with prostate cancer progression. It has been shown that endogenous JNK activity increases the expression of anti-apoptotic proteins and can increase the resistance of prostate cancer cell lines to chemotherapy. In addition, elevated endogenous JNK activity is required for improved proliferation and transformation of a number of epithelial tumours. However, prolonged JNK activation in response to cytotoxic stimuli can increase the sensitivity of cells to apoptosis. Prolonged JNK activity appears to induce the expression of a separate set of genes responsible for promoting apoptosis. Our group has recently shown that activation of JNK by chemotherapeutic drugs can sensitise DU 145 prostate carcinoma cells to Fas receptor mediated apoptosis. In order toidentify novel targets for treating hormone refractory prostate cancer we have investigated the role of JNK in Fas receptor mediated apoptosis. We have demonstrated that prolonged JNK activation is defective in DU 145 cells in response to Fas receptor activation alone. Co-administering anisomycin, a JNK agonist, greatly enhances the ability of DU 145 cells to undergo apoptosis by increasing the rate of Caspase 8 cleavage. We also investigated the role of endogenous JNK activity in Fas receptor mediated.