Measurement Of Intracellular ATP Research Articles

Abstract 619: Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses

Abstract Dysregulation of the Mitogen-Activated Protein Kinase (MAPK) pathway is a frequent event in many cancers. The RAF family of kinases, which includes ARAF, BRAF, and CRAF (RAF1), plays a key role in MAPK signaling. Of the three isoforms, only BRAF is currently targeted by approved RAF inhibitors. These inhibitors were designed to target BRAF-mutant variants that act as oncogenic monomers. However, BRAF-monomer inhibitors can induce activation of wild-type RAF by the formation of dimers, resulting in hyperproliferative lesions of BRAF wild-type cells. Additionally, BRAF-monomer inhibitors do not target RAF-dimers, although this is a potential vulnerability of many MAPK-driven cancers. Novel inhibitors are in development to circumvent these limitations. Further insights into the precise targeting and mechanism of action of RAF inhibitors can help in determining the most promising avenues for drug development. We performed extensive cellular profiling experiments with three approved and five clinical stage inhibitors of RAF to gain more insight into the differences and cellular targeting of these agents. The inhibitors were tested in cell viability assays on a panel of 130 human cancer cell lines from diverse tissue origins. Cell lines were exposed to dose ranges of each inhibitor spanning four logarithmic orders, and viability was determined by measurement of intracellular ATP. Effects of compound on cell viability were related to genomic, transcriptomic, and proteomic status using bioinformatics, with the aim to identify predictive biomarkers for drug response, and to determine selective targeting of MAPK-driven cell lines. Additionally, the primary mechanism of cellular inhibitor activity was determined by relating cell line responses to gene dependency data from large CRISPR knockout screens. Our results showed striking similarity in the overall inhibitory profile of BRAF-monomer inhibitors and the vemurafenib-derivative and paradox breaker plixorafenib, which all selectively targeted BRAF-mutant cell lines. However, whereas vemurafenib increased cell viability in RAS-mutant cell lines at high drug concentrations, this effect was greatly reduced for plixorafenib. Western blot analysis of phosphorylated MEK and ERK levels in RAS-mutant cell lines treated with BRAF-monomer inhibitors confirmed MAPK pathway activation as cause of the increased cell viability. In addition, RAF-dimer inhibitors potently targeted BRAF-mutant cell lines, as well as several KRAS- and NRAS-mutant cell lines. Lastly, correlation analyses of cell line profiling data with CRISPR knockout data revealed the preferential targeting of RAF1-dependent cell lines by the dual RAF/MEK inhibitor avutometinib. The results of our analyses shed light on the intricacies of cellular targeting by RAF inhibitors and provide insights to guide the development of new RAF inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Sjoerd B. van der Leeuw, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Nicole Willemsen-Seegers, Guido J. Zaman. Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 619.

Olfactory Mucosal Cells of Individuals with Alzheimer’s Disease Display Alterations Similar to the Alzheimer Brain

AbstractBackgroundA sense of smell is orchestrated in the first place by the cells of the olfactory mucosa (OM), located in the rooftop of the nasal cavity. Impairments in olfaction are implicated in aging but also in Alzheimer’s disease (AD). Here, we describe the observed alterations in cultures of OM cells derived from individuals with late‐onset AD or amnestic form of mild cognitive impairment (MCI) when compared to OM cells derived from their cognitively healthy control subjects.MethodPrimary cultures of OM cells were harvested from biopsies of OM derived from cognitively healthy individuals and individuals with late‐onset AD or amnestic MCI and cultured in vitro. First, the cell types present in cultures of OM cells and genes differentially expressed between the OM cells derived from control subjects and individuals AD or MCI OM were assessed by single cell RNA‐sequencing (scRNA‐seq). Next, a total RNA‐seq was applied to a larger cohort of samples. The results of total and scRNA‐seq were further validated with various functional in vitro assays including inductively coupled plasma mass spectrometry (ICP‐MS) and measurements of intracellular ATP levels.ResultThe scRNA‐seq unveiled a total of five distinct cell populations in OM cell cultures. A total of 240 previously AD‐related genes were found to be differentially expressed between the AD and control OM cell cultures. Similarly, the MCI OM cells harbored 224 differentially expressed genes when compared to OM cells derived from their control subjects. Common for both AD and MCI OM cells were the alterations in genes related to mitochondrial function when compared to controls. Additionally, the ICP‐MS revealed increased levels of zinc, calcium and sodium in the AD OM cells in comparison to the OM cells derived from cognitively healthy subjects.ConclusionThe results implicate the role of olfactory mucosa as a site displaying early alterations related to Alzheimer’s disease.

Functional analysis of the mitochondrial alternative oxidase gene (aox1) from Aspergillus niger CGMCC 10142 and its effects on citric acid production.

In this work, we constructed the aox1 disruption strains 3-4 and 4-10, as well as the aox1 overexpression strains 72 and 102 in Aspergillus niger. The energy metabolism, EMP, TCA pathways, and flux were investigated for the citric acid (CA) overproduction via the aox1 overexpression among them. As expected, the overexpression of the aox1 gene enabled a higher growth rate than that of the rate of its parent strain in medium with respiratory chain inhibitors. In liquefied corn starch medium supplemented with 0.2μg/mL antimycin A, the CA production of the overexpression strain 102 reached up to 169.1g/L, whereas the highest value of the parent strain was 158.9g/L. For the perspective of the aox1 disruption strain 4-10, the yield of CA dropped to 125.6g/L, and the loose mycelial pellets forming in the medium also revealed that the fundamentally important role of AOX in A. niger lies in the resistance to oxidative stress under fully aerobic conditions. Based on real-time qPCR gene expression analysis and measurement of intracellular ATP and NADH levels, we came to a conclusion that the higher NADH oxidation rate resulting from the overexpression of the aox1 gene mainly contributed to rate-limited step's acceleration and strengthened metabolic flow via mycelia and led to the CA yield in these strains increased by 13.5 and 10.8%, respectively. Subsequently, it was found that overexpression strains had higher AOX relative content and more oxygen consumption at different fermentation stages, which fully confirmed the close relationship between aox1 gene and energy metabolism, and comprehensively revealed aox1 gene function through the combination with the above conclusions.

Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods

Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.

8-Chloro-Adenosine Inhibits Molecular Poor-Risk Acute Myeloid Leukemia (AML) and Leukemic Stem Cells (LSC) Growth and Synergizes with the BCL-2 Inhibitor Venetoclax (ABT-199)

Only a minority of AML patients are cured with currently available chemotherapy regimens. Thus, new drugs with novel mechanisms of action are urgently needed. In previous proof-of-concept studies, the halogenated ATP analog 8-chloro-adenosine (8-Cl-Ado) has shown preclinical activity against a variety of solid tumors and hematologic malignancies, favorable pharmacokinetic and pharmacodynamic profiles and feasibility in a first-in-man phase I clinical trial in chronic lymphocytic leukemia. However, little is known regarding the activity of this compound in AML. In contrast to other nucleoside analogs used for treatment of AML, 8-Cl-Ado is metabolized by adenosine kinase and its triphosphate derivative is incorporated into RNA and significantly inhibited RNA synthesis in AML cells (KG-1a and MV4-11 cell lines and primary bone marrow samples) in a dose-dependent manner (300 nM to 1 uM) after 24 h exposure as compared to vehicle-treated controls (p<0.05). Similarly, 20-80% inhibition of total RNA synthesis was observed in AML patient blasts, regardless of the harbored gene mutations or cytogenetic aberrations. In contrast, 24 h exposure to 8-Cl-Ado did not significantly inhibit DNA synthesis compared to vehicle at all concentrations tested (p>0.25 at all concentrations tested). 8-Cl-Ado was metabolized into its cytotoxic triphosphate, 8-Cl-ATP, which accumulated to intracellular concentrations of more than 600 uM after 12 h exposure to 10 uM 8-Cl-Ado. Accumulation of 8-Cl-ATP was associated with >20% reduction of endogenous ATP compared to control-treated cells (p<0.05). In regard to antileukemia activity, 8-Cl-Ado inhibited growth of several AML cell lines (MOLM-13, MOLM-14, KG-1a, MV4-11, OCI-AML3) with IC50s ranging from 0.2 uM to 1.4 uM after 72 h of treatment and that of primary AML blasts including those harboring the poor-risk FLT3-ITD mutation (IC50: 800 nM for FLT3-ITD-positive blasts). In an orthotopic mouse model where FLT3-ITD-positive MOLM-14 AML cells were xenografted, animals treated with 50 mg/kg/day through an implanted osmotic pump had >70% reduction in tumor mass compared to vehicle-treated controls after 16 days of treatment (p<0.05). Importantly, 24 h pre-treatment of human LSC-enriched CD34+CD38- blasts with 5 uM 8-Cl-Ado resulted in a significant inhibition of their colony forming ability compared to vehicle-treated controls (>50% reduction of colonies after 14 days, p<0.05). To study the effect of 8-Cl-Ado on primary AML in vivo, sub-lethally irradiated Rag-2/gamma(c) double-knockout immunodeficient mice were injected retro-orbital with ten million primary human AML blast cells pre-treated with 5 uM 8-Cl-Ado or vehicle control 24 h prior to i.v. injection. Significant longer survival was observed of mice engrafted with drug-treated cells compared to those engrafted with vehicle-treated controls (p<0.0004). Given the previously reported higher expression of the antiapoptotic BCL-2 protein and the implication that this had on treatment resistance in AML, combination strategy using 8-Cl-Ado together with the BCL-2 inhibitor venetoclax was investigated and showed strong synergistic, anti-proliferative effects in both AML cell lines and primary AML blasts carrying a variety of mutations and cytogenetic aberrations. Combination index value calculation and isobologram display demonstrated strong synergy between venetoclax and 8-Cl-Ado in all 4 AML cell lines and 2 primary AML samples investigated, at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90). We also show that, in KG-1a and MV4-11 cells, 8-Cl-Ado and venetoclax synergize in the inhibition of intracellular ATP pools. AML cell lines were treated for 6h with 10 uM 8-Cl-Ado, 250 nM venetoclax or both, before measurement of intracellular ATP. While single agent treatment decreased intracellular ATP levels in both cell lines by about 20% (8-Cl-Ado) to 40% (venetoclax), combination treatment lowered ATP levels by about 70% (KG-1a) to 90% (MV4-11). Taken together, 8-Cl-Ado is a promising agent with a unique RNA and ATP-targeting mechanism of action, excellent toxicity profile and encouraging preclinical anti-leukemia activity in AML and synergizes with the BCL-2 inhibitor venetoclax in vitro. Based on these results, we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory AML and planned further combination with BCL-2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Neuroprotective effects of polydatin against mitochondrial-dependent apoptosis in the rat cerebral cortex following ischemia/reperfusion injury.

The neuroprotective effect of polydatin (PD) against hemorrhagic shock-induced mitochondrial injury has been described previously, and mitochondrial dysfunction and apoptosis were reportedly involved in ischemic stroke. In the present study the neuroprotective effect of PD in preventing apoptosis was evaluated following induction of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats. PD (30 mg/kg) was administered by caudal vein injection 10 min prior to ischemia/reperfusion (I/R) injury. 24 h following I/R injury, ameliorated modified neurological severity scores (mNSS) and reduced infarct volume were observed in the PD treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V/propidium iodide assays demonstrated the anti-apoptotic effect of PD in the ischemic cortex. In addition, PD improved I/R injury-induced mitochondrial dysfunction, reflected by morphological observations and measurements of mitochondrial membrane potential and intracellular ATP measurement. Western blot analysis revealed an increase in B-cell lymphoma 2 apoptosis regulator (Bcl-2) expression, and a decrease in Bcl-2-associated protein X apoptosis regulator expression in the PD group in comparison with the vehicle treated group. PD treatment also prevented the release of cytochrome c from mitochondria into the cytoplasm, and blunted the activities of caspase-9 and caspase-3. Furthermore, PD treatment decreased the levels of reactive oxygen species in neurons isolated from the ischemic cortex. The findings of this study, therefore, suggest that PD has a dual effect, ameliorating both oxidative stress and mitochondria-dependent apoptosis, making it a promising new therapy for the treatment of ischemic stroke.

Developmental defect of cytochrome oxidase mutants of Streptomyces coelicolor A3(2).

To study the link between energy metabolism and secondary metabolism/morphological development in Streptomyces, knockout mutants were generated with regard to the subunits of the cytochrome oxidase supercomplex (CcO) in Streptomyces coelicolor A3(2). All mutants exhibited an identical phenotype: viable but defective in antibiotic production and cell differentiation when grown in both complex and minimal media. The growth yield of the CcO mutant was about half of that of the WT strain on glucose medium while both strains grew similarly on maltose medium. Intracellular ATP measurement demonstrated that the CcO mutant exhibited high intracellular ATP level. A similar elevation of intracellular ATP level was observed with regard to the WT strain cultured in the presence of BCDA, a copper-chelating agent. Reverse transcriptase PCR analysis demonstrated that the transcription of ATP synthase operon is upregulated in the CcO mutant. Addition of carbonylcyanide m-chlorophenylhydrazone, an inhibitor of ATP synthesis, promoted antibiotic production and aerial mycelia formation in the CcO mutant and BCDA-treated WT cells. We hypothesize that the deficiency of CcO causes accumulation of intracellular ATP, and that the high ATP level inhibits the onset of development in S. coelicolor.

Assessment of NK Cell Metabolism.

There has been increasing recognition of the importance of metabolism on immune cell differentiation, homeostasis, and function. Recently, our lab and others have begun to investigate the metabolic requirements for NK cell differentiation and activation. Here, we describe approaches for the in vitro assessment of NK cell metabolism. We present methods for using inhibitors to alter cellular metabolism, measurement of intracellular ATP in NK cells, assessment of real-time glycolysis and oxidative phosphorylation by an extracellular flux assay from Seahorse Biosciences, and some basic protocols for stimulation of NK cells via cytokines and receptors.

Abstract 5462: Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase

Abstract ATP plays important roles in cellular processes in cancer cells, which are a heterogeneous population in tumors regarding their genetic background and oxygen and energy supplies. The fact that cancer cells undergo rapid proliferation suggests that they must possess mechanisms, which may not be present in normal cells, to meet their high energy needs. Studies by others revealed that extracellular ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues (1). We recently reported that extracellular ATP can be taken up directly by cancer cells by macropinocytosis (2). Based on these results, we hypothesized that the internalized extracellular ATP not only promotes cancer cell growth and survival, but also drug resistance. In this study, human lung cancer A549 and breast cancer MCF7 cells were used as cell models to determine the effects of extracellular ATP on drug resistance to tyrosine kinase inhibitors (TKIs) in cancer cells. Cancer cell viability and intracellular ATP measurement results showed that extracellular ATP substantially increased intracellular ATP levels and promoted cancer cell drug resistance to TKIs. Viability of TKI treated cancer cells was significantly increased in the presence of extracellular ATP, compared to cancer cells with TKI treatment alone. Administration of macropinocytosis inhibitors significantly reduced the drug resistance induced by extracellular ATP, suggesting that extracellular ATP promoted drug resistance by contributing to the intracellular ATP pool. Protein and protein phosphorylation analyses results suggested that the increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitor for the ATP-binding site of platelet-derived growth factor receptor (PDGFR) and enhancing Akt and MAPK signaling pathways. These findings identify, for the first time, new biological functions of extracellular ATP in tumors and may lead to novel anti-cancer strategies combating drug resistance in cancers. 1. Pellegatti P, et al. PLoS ONE 2008;3:e2599. 2. Qian Y, et al. Cancer Lett 2014;351:242-51. Citation Format: Xuan Wang, Yanrong Qian, Yunsheng Li, Xiaozhuo Chen. Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2015-5462

Comparison of several functional methods to evaluate the immune response on stable kidney transplant patients

The introduction of new immunosuppressive drugs in the last two decades has been associated with a significant decline in the prevalence of acute rejection and a huge improvement of graft survival. Monitoring blood levels of immunosuppressive drugs is the most common way to control drug doses in renal transplant patients. This approach is useful and widely used but doesn't give accurate information about the immune status of the patient. For this goal, there are many “in house” protocols which give more information, but cannot be standardized, limiting their applicability to compare results between different laboratories. In this study we compare three classical functional methods to evaluate the immune response: Mixed lymphocyte reaction (MLR), phytohemagglutinin stimulated peripheral blood lymphocytes (PBLs), and anti-CD3 monoclonal antibodies (mAbs) against PBL with the only FDA-labeled assay to measure the patient immune status: Cylex ImmuKnow® that measures the intracelullar ATP in CD4+ lymphocytes. We used n=111 stable renal transplant patients, all the patients with more than one year functioning grafts. We referred the results to a control population of healthy blood donors (n=125).Results: Measurement of intracellular ATP in CD4+ lymphocytes is able to differentiate immunosuppressed populations in renal transplant patients from health controls (242.30±21.62 vs. 386.43±25.12, p 0.0001). By contrary, there were no differences between controls and renal recipients when functional response was measured by MLR, PHA and anti-CD3 mAbs (2.48±0.45 vs. 2.37±0.41; 2.84±0.76 vs. 2.37±0.32; 2.32±0.34 vs. 1.89±0.38 respectively). In summary, our results show that the measurement of ATP in CD4+ lymphocytes gives more accurate information in comparison to the classical methods.

Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase

Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10–9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.

Measurements of intracellularATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F(0)F(1) ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating the glycolytic oscillation. Measurements of intracellular ATP provide a unique tool to understand the role of these membrane ATPases and how their activities are regulated. We have constructed a new nanobiosensor that can perform time-resolved measurements of intracellular ATP in intact cells. Measurements of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal behaviour of intracellular ATP in yeast strains lacking either of the two membrane bound ATPases have confirmed that F(0)F(1) ATPase and Pma1p contribute significantly to the ATP consumption in the cell and to the regulation of glycolytic oscillation. Furthermore, our measurements also demonstrate that ATPase activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose is used up, the ATP consumption stops immediately. Thus, glucose or some compound derived from glucose must be involved in controlling the activity of these two ATPases.

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

Time and Dose Dependence of Pluronic Bioactivity in Hyperthermia-Induced Tumor Cell Death

Pluronic block copolymers have been shown to sensitize cancer cells resulting in an increased activity of antineoplastic agents. In the current study we examined a new application of Pluronic bioactivity in potentiating hyperthermia-induced cancer cell injury. DHD/K12/TRb rat adenocarcinoma cells were exposed to low-grade hyperthermia at 43 degrees C with or without Pluronic P85 or Pluronic L61. A range of Pluronic doses, pre-exposure and heat exposure durations were investigated, and the test conditions were optimized. Treatment efficacy was assessed by measurement of intracellular ATP and mitochondrial dehydrogenase activity. Both P85 and L61 in synergy with heat reduced cell viability appreciably compared to either heat or Pluronic alone. Under optimal conditions, P85 (10 mg/ml, 240 mins) combined with 15 mins heat reduced intracellular ATP to 60.1 +/- 3.5% of control, while heat alone and P85 without heat caused a negligible decrease in ATP of 1.2% and 3.8%, respectively. Similarly, cells receiving 120 mins pre-exposure of L61 (0.3 mg/ml) showed reduction in intracellular ATP to 14.1 +/- 2.1% of control. Again, heat or L61 pre-exposure alone caused a minor decrease in levels of intracellular ATP (1.5% and 4.4%, respectively). Comparable results were observed when viability was assessed by mitochondrial enzyme activity. Survival studies confirmed that the loss of viability translates to a long-term reduction in proliferative activity, particularly for L61 treated cells. Based on these results, we conclude that Pluronic is effective in improving hyperthermic cancer treatment in vitro by potentiating heat-induced cytotoxicity in a concentration and time dependent manner.

Complex I and ATP Content Deficiency in Lymphocytes from Friedreich's Ataxia

Friedreich's ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart abnormalities. A deficiency in the protein frataxin causes this disease. Frataxin deficiency leads to progressive iron accumulation in mitochondria, excessive free radical production and dysfunction of respiratory chain complexes. The expansion (GAA) repeat in the first intron causes decreased frataxin expression by interfering with transcription. Activity of mitochondrial respiratory chain complex I (measured as NADH ferricyanide reductase) and intracellular ATP measurement was performed on lymphocyte of FRDA patients (n=12) and control subjects (n=25). Our findings showed that complex I activity and intracellular ATP were significantly reduced (P=0.001) in patients compared with controls and we found strong correlation between complex I activity and intracellular ATP content in FRDA patients (r=0.93; P<0.002). 8.6 and 9.0 kb deletion in mtDNA was detected in 9 patients out of 12 (75%) by multiplex polymerase chain reaction (PCR) and Southern blot analysis. This study suggested that a biochemical defect in complex I activity and ATP production, which may be due to iron accumulation in mitochondria, can be involved in age of onset of FRDA.

A Comparison of the Effects of Ocular Preservatives on Mammalian and Microbial ATP and Glutathione Levels

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO2), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H2O2). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5′-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H2O2 caused complete toxicity to conjunctival and corneal epithelial cells at ∼25 ppm, in contrast to NaClO2, where >100 ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO2 than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa, but the bacteria were extremely resistant to H2O2. NaClO2 caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from <10 ppm in Pseudomonas, 25–100 ppm in epithelial cells, to >500 ppm in fungal cells. The mechanisms of cytotoxicity of NaClO2, H2O2 and BAK all appeared to differ. NaClO2 was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO2 to the ocular cells, compared with BAK and H2O2, is in agreement with fewer reported adverse effects of application in the eye.

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement.

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement. We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.

Immunomodulatory consequences of oral administration of Lactobacillus rhamnosus strain GG in healthy volunteers.

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either 'self' or 'non-self' preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-gamma, TNF-alpha) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-alpha, IL-6 and in part IFN-gamma cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole 'non-self' intestinal flora was higher than by 'self' intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 μm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

Influence of Uremia on Cell Viability and Cytokine Release of Human Peritoneal Mesothelial Cells

Introduction: There is still no evidence whether human peritoneal mesothelial cells (HPMC) from patients with end-stage renal failure are altered in cell viability or show a different pattern of the release of proinflammatory cytokines. Also the serum of patients with uremia may contain substances stimulating the cytokine release of HPMC. Study Design: The IL-1β-induced IL-6/IL-8 release of HPMC from healthy donors and from patients with end-stage renal disease (ESRD) were measured before the start of chronic peritoneal dialysis (PD) and during PD therapy. Additionally the influence of uremic and non-uremic serum on IL-6 and IL-8 release of normal HPMC was studied. Cell viability was assessed by MTT assay and by the measurement of intracellular ATP (chemoluminescence assay). HPMC were obtained from the following patient groups: (1) non-uremic control patients (n = 7); (2) patients with ESRD undergoing PD catheter implantation for the first time (n = 7), and (3) patients on PD undergoing catheter exchange for noninfectious reasons (n = 6). Pooled human serum from PD patients and normal controls were used for stimulation experiments. HPMC from different donors were grown to confluence (second passage) and then stimulated with IL-1β (1,000 pg/ml in M199) for 24 h. IL-6 and IL-8 concentrations were measured in the supernatant by ELISA. Additionally uremic and non-uremic sera were incubated with HPMC from normal donors for 24 h with a subsequent 24-hour IL-1β stimulation. Mesothelial cell protein mass was determined by the Bradford reagent. Results: Non-uremic patients and ESRD patients did not differ with regard to the global cell viability of HPMC according to MTT assay activity or the intracellular ATP concentration. However, HPMC from uremic patients produced more IL-8 on IL-1β stimulation than the non-uremic controls (group 2, 53.5 ± 15.7 pg/µg; group 3, 70.5 ± 27.3 pg/µg vs. group 1, 24.0 ± 11.8 pg/µg). HPMC from patients on chronic PD additionally released significantly more IL-6 (30.5 ± 13.8 pg/µg) on IL-1β stimulation than uremic patients before the onset of PD (6.2 ± 2.6 pg/µg; p < 0.01). Incubation of normal HPMC with the serum from uremic donors produced an enhanced stimulated IL-8 release compared to the exposition with normal control serum (50.6 ± 6.1 vs. 20.8 ± 2.9 pg/µg; p < 0.01). Conclusion: HPMC from uremic patients more readily release IL-8 on stimulation with IL-1β. On chronic PD treatment IL-6 release was further enhanced. Not further classified serum components in uremia also enhance IL-6 and IL-8 release of HPMC.