The differential activation of metabolic pathways in leukemic cells depending on their genotype and micro-environmental stress.

INTRODUCTION: Acute myeloid leukemia (AML) is characterized by a set of malignant proliferations leading to an accumulation of blasts in the bone marrow and blood. The prognosis is pejorative due to the molecular complexity and pathways implicated in leukemogenesis. OBJECTIVES: Our research was focused on comparing the metabolic profiles of leukemic cells in basal culture and deprivation conditions to investigate their behaviors under metabolic stress. METHODS: We performed untargeted metabolomics using 1H HRMAS-NMR. Five human leukemic cell lines-KG1, K562, HEL, HL60 and OCIAML3-were studied in the basal and nutrient deprivation states. A multivariate analysis of the metabolic profile was performed to find over- or under- expressed metabolites in the different cell lines, depending on the experimental conditions. RESULTS: In the basal state, each leukemic cell line exhibited a specific metabolic signature related to the diversity of AML subtypes represented and their phenotypes. When cultured in a serum-free medium, they showed quick metabolic adaptation and continued to proliferate and survive despite the lack of nutrients. Low apoptosis was observed. Increased phosphocholine and glutathione was a common feature of all the observed cell lines, with the maximum increase in these metabolites at 24 h of culture, suggesting the involvement of lipid metabolism and oxidative stress regulators in the survival mechanism developed by the leukemic cells. CONCLUSIONS: Our study provides new insights into the metabolic mechanisms in leukemogenesis and suggests a hierarchy of metabolic pathways activated within leukemic cells, some dependent on their genotypes and others conserved among the subtypes but commonly induced under micro-environmental stress.

Stemazole promotes survival and preserves stemness in human embryonic stem cells.

Human embryonic stem cells (hESCs) are extremely delicate, and survive poorly under suboptimal culture conditions, severely restricting long-term studies and practical applications. Thus, a protective agent that promotes stem cell survival is urgently needed. In this study, we evaluated the protective effects of stemazole in single-cell and starved hESC cultures. Colony formation was quantified by alkaline phosphatase and immunofluorescence staining, while apoptosis was assessed by flow cytometry and TUNEL assay. Expression of hESC and other stem cell markers was evaluated by western blot, RT-PCR, and qPCR. We found that stemazole enhanced clonal expansion from single cells in dose-dependent fashion and clearly decreased apoptosis from 54.1% to 25.2%. Furthermore, the drug reduced apoptosis from 43.6% to 8.4% over 15 h of starvation, with 66% of stemazole-treated cells remaining viable after 2 weeks of starvation. Importantly, starved cells protected with stemazole retained the same proliferation and differentiation properties as cells in normal culture. In conclusion, stemazole significantly promotes survival of stem cells in single-cell or starvation cultures without compromising stemness and pluripotency.

A survey of scientists' awareness of and attitudes to the use of human blood products and alternatives in human assisted reproductive technology.

Scientists working in assisted reproduction [members of Scientists in Reproductive Technology (SIRT) Australia, and subscribers of the online forums EmbryoMail and Quartec] were invited to complete an online questionnaire on the use of human blood products in assisted reproductive technologies (ART). A total of 260 started the questionnaire, with 208 (80%) completing it. A total of 62% of respondents had worked in human ART ≥8 years and 68% had post-graduate qualifications. The majority (82%) reported using products of animal or human origin, with 75% knowing why protein was added to culture media and 41% not worried by this. Almost half (49%) of respondents were unaware of regulations surrounding the use of human blood products in health care and 70% were unaware of adverse events involving human blood products in human ART. Most respondents (70%) indicated that they were not concerned about infections such as hepatitis, but agents such as prions were a cause for concern (57%). A total of 57% of respondents were unaware of alternatives, but 77% would use a suitable alternative. Using blood products in human ART is surrounded by a lack of awareness, often independent of respondents' qualifications or experience. A better understanding of these products and possible alternatives is required if informed decisions about their suitability are to be made.

Efficacy and safety of antifungal additives in Optisol-GS corneal storage medium.

IMPORTANCE: Optisol-GS, the most common corneal storage medium in the United States, contains antibacterial but no antifungal supplementation. Most postkeratoplasty endophthalmitis and keratitis cases are now of a fungal origin. OBJECTIVE: To assess the efficacy and safety of voriconazole and amphotericin B in reducing Candida species contamination of Optisol-GS under normal storage conditions. DESIGN, SETTING, AND PARTICIPANTS: In vitro laboratory study using 15 pairs of research-grade donor corneas and 20-mL vials of Optisol-GS. INTERVENTIONS: Twenty vials of Optisol-GS were supplemented with either voriconazole at 1×, 10×, 25×, or 50× minimum inhibitory concentration (MIC) or amphotericin B at 0.25×, 0.5×, 1×, or 10× MIC. Known concentrations of Candida albicans and Candida glabrata were each added to a set of vials. Safety studies were performed by separating 15 pairs of donor corneas into unsupplemented Optisol-GS or Optisol-GS plus an antifungal. MAIN OUTCOMES AND MEASURES: Efficacy outcomes were viable fungal colony counts determined from samples taken on days 2, 7, and 14 immediately after removal from refrigeration and after warming to room temperature for 2 hours. Safety outcomes included percentage of intact epithelium and endothelial cell density on days 0, 7, and 14, as well as percentage of nonviable endothelial cells by vital dye staining on day 14. RESULTS: Growth of C albicans and C glabrata was observed in all voriconazole-supplemented vials. In contrast, there was no growth of either organism in amphotericin B-supplemented vials, except at 0.25× and 0.5× MIC on day 2, when viable counts of C glabrata were reduced by 99% and 96%, respectively. Compared with paired controls, with the exception of Optisol-GS plus amphotericin B at 10× MIC, donor corneas in supplemented Optisol-GS appeared to have no difference in endothelial cell density reduction, percentage of intact epithelium, or percentage of nonviable endothelial cells. CONCLUSIONS AND RELEVANCE: The addition of amphotericin B to Optisol-GS may significantly improve activity against contamination with Candida species, the primary cause of fungal infection after corneal transplantation. This study found significant endothelial toxic effects at the maximal concentration of amphotericin B.

Posaconazole in human serum: a greater pharmacodynamic effect than predicted by the non-protein-bound serum concentration.

It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested.

BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells.

Identifying small molecules that suppress apoptosis is promising for the therapy of brain diseases. We recently showed that autocrine bone morphogenetic protein (BMP) signaling involves the effects of cholesterol myristate present in traditional Chinese medicine on mesenchymal stem cells. The present study evaluated the effects of cholesterol myristate on the apoptosis and BMP signaling of PC12 cells. PC12 cells transfected by the inhibitor of differentiation (Id1) promoter reporter construct target gene of BMP4 signaling; cholesterol myristate increases the activity of Id1 promoter. However, structurally related steroids such as cholesterol, ß-sitosterol and cholesten-3-one, lack of the myristate, did not affect the activity of Id1 promoter, suggesting that myristate is essential for the effect of cholesterol myristate. These effects depend on BMP signaling. Apoptosis analysis indicated that cholesterol myristate inhibited the apoptosis of PC12 cells induced in serum-free condition. Cholesterol myristate significantly increases the expression of BMP4, BMPRIA, p-Smad1/5/8, Id1 and its antiapoptotic target gene Bcl-xL in PC12 cells treated in serum-free condition. Moreover, BMP antagonist reduced the anti-apoptotic effect of cholesterol myristate. Thus, this study is to provide evidence that BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells. Our findings are therefore of considerable therapeutic significance and provide the potential of newly exploiting cholesterol myristate and clinically in brain disease therapies.

Dose-dependent protective effect of connexin43 mimetic peptide against neurodegeneration in an ex vivo model of epileptiform lesion.

Epileptic seizures typically result in delayed neuronal loss secondary to the initial damage and an up-regulation in connexin43 (Cx43). This study investigated the role of Cx43 gap junctions in lesion spread and cell loss following epileptiform activity. Epileptiform injury in hippocampal slice cultures was induced by 48 h exposure to 100 µM bicuculline methochloride (BMC). During the 24h recovery period following BMC treatment, lesion spread was observed in the CA1. A Cx43 mimetic peptide, applied during either the BMC treatment or recovery periods, produced concentration- and exposure time-dependent neuroprotection, as measured by propidium iodide uptake at the end of the recovery period. During the BMC period, peptide concentrations between 5 and 50 µM (sufficient to block hemichannels) had a protective effect while a substantial gap junction blockade with 500 µM peptide exacerbated the lesion. By contrast, all doses applied during the recovery period protected the CA1 region from further damage. The results indicate that while the slices are undergoing excessive neuronal firing and epileptic stress, gap junction communication appears to be essential for tissue survival but hemichannel opening may be damaging. Following epileptiform insult, however, gap junction communication plays a crucial role in the spread of neuronal damage. The findings from this study identify gap junction communication as a potential therapeutic target for epilepsy.

Downregulation of CREB-binding protein expression sensitizes endothelial cells to serum-deprived apoptosis: important role of nitric oxide.

Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis.

Integration site selection by HIV-based vectors in dividing and growth-arrested IMR-90 lung fibroblasts.

DNA integration is a defining step in the retroviral life cycle and the basis of stable gene transfer in retrovirus-based gene therapy. Previous studies of integration by HIV-based vectors have shown that integration is not random, but favored in active transcription units. Studies to date have focused on HIV integration in dividing cells, leaving open the question of whether integration target site selection might differ in nondividing cells. According to one idea, division of the host cell might be required for favored integration in transcription units, possibly as a result of chromatin remodeling during DNA replication. Here we have investigated this issue by comparing integration in dividing IMR-90 primary lung fibroblasts to integration in nondividing IMR-90 cells arrested in G1 by serum starvation and contact inhibition. We identified several differences in integration site selection in arrested versus dividing cells, including the frequency of integration in transcription units and in gene-rich regions. However, integration in nondividing cells was in fact more favored in transcription units, contrary to the idea that cell division was important for this bias. These data provide the first view of lentiviral integration in nondividing cells and help constrain models for the mechanism of favored integration in genes.

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis.

AKR-2B cells disintegrate after serum removal. After a delay of approximately 90 minutes, cell death began and reached after six hours a plateau of 40-50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die. The first change was the rapid appearance of membrane blebs. Membrane vesicles were rapidly extruded and reintegrated by the cell. This highly dynamic process of an affected cell stopped after 80+/-20 minutes with its death. Conductivity measurements showed that at that time the membrane was electrically permeable. By using fluorescence double staining with propidium iodide and Hoechst 33258, we show that membrane leakage leading to disintegration is accompanied, and for some cells preceded, by nuclear condensation. The energy state of the intact cells was monitored by measuring the intracellular ATP content which remained high (6 mM) throughout the entire time of investigation. Mitochondrial potential was determined by rhodamine 123 fluorescence in parallel to the measurement of membrane permeability via uptake of propidium iodide and lead to the detection of a cell population that exhibits a high mitochondrial potential and an uptake of propidium iodide indicating a membrane disruption of cells which still have a high energy charge. It is shown by electron microscopy that mitochondria were swollen and damaged in parallel to nuclear condensation. There was no DNA fragmentation as shown by two independent methods. Addition of the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone immediately after serum starvation lead to an almost complete survival of the cells up to 6 hours. A pronounced protection was still observed after 24 hours, suggesting an involvement of this type of protease in the onset of cell death after serum removal. Apparently, serum withdrawal activates a succession of initial events that are similar to those defined as 'apoptosis', i.e. nuclear condensation and membrane blebbing. These steps are, however, accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.