Study of activation cross sections of double neutron capture reaction on 193Ir for the reactor production route of radiotherapeutic 195mPt.

The radiotherapeutic 195mPt is among the most effective Auger electron emitters of the currently studied radionuclides that have a potential theranostic application in nuclear medicine. Production of 195mPt through double neuron capture of enriched 193Ir followed by ß--decay to the radioisotope of interest carried out at the research reactor IBR-2 is described. Because of the high radiation background, radiochemical purification procedure of 195mPt from bulk of iridium was needed to be developed and is detailed here as well. For the first time, cross section and resonance integral for the reaction 194Ir(n,γ)195mIr were determined. Resonance neutrons contribution was established to exceed that of thermal neutrons, and resonance integral for the reaction 194Ir(n,γ)195mIr is calculated to be 2900 b. Specific activity of 195mPt was estimated to reach a value of 38.7 GBq/(g Pt) at IBR-2 by the end of bombardment (EOB).

HyPer as a tool to determine the reductive activity in cellular compartments.

A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate for repurposing HyPer1, initially designed as a fluorescent probe for H2O2 levels, as a tool to measure the reductive power in various cellular compartments. The response of HyPer1 depends on kinetics between thiol oxidation and reduction in its OxyR sensing domain. Here, we focused on the reduction half-reaction of HyPer1. We showed that HyPer1 primarily relies on Trx/TrxR-mediated reduction in the cytosol and nucleus, characterized by a second order rate constant of 5.8 × 102 M-1s-1. On the other hand, within the mitochondria, HyPer1 is predominantly reduced by glutathione (GSH). The GSH-mediated reduction rate constant is 1.8 M-1s-1. Using human leukemia K-562 cells after a brief oxidative exposure, we quantified the compartmentalized Trx/TrxR and GSH-dependent reductive activity using HyPer1. Notably, the recovery period for mitochondrial HyPer1 was twice as long compared to cytosolic and nuclear HyPer1. After exploring various human cells, we revealed a potent cytosolic Trx/TrxR pathway, particularly pronounced in cancer cell lines such as K-562 and HeLa. In conclusion, our study demonstrates that HyPer1 can be harnessed as a robust tool for assessing compartmentalized reduction activity in cells following oxidative stress.

Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation.

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells.

The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 µm using PLIM and showed a gradient in the intracellular O2 level towards their center.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Phosphorus deficiency induces sexual reproduction in the dinoflagellate Prorocentrum cordatum.

Nitrogen (N) and phosphorus (P) are essential elements whose availability promotes successful growth of phytoplankton and governs aquatic primary productivity. In this study, we investigated the effect of N and/or P deficiency on the sexual reproduction of Prorocentrum cordatum, the dinoflagellate with the haplontic life cycle which causes harmful algal blooms worldwide. In P. cordatum cultures, N and the combined N and P deficiency led to the arrest of the cell cycle in the G0/G1 phases and attenuation of cell culture growth. We observed, that P, but not N deficiency triggered the transition in the life cycle of P. cordatum from vegetative to the sexual stage. This resulted in a sharp increase in percentage of cells with relative nuclear DNA content 2C (zygotes) and the appearance of cells with relative nuclear DNA content 4C (dividing zygotes). Subsequent supplementation with phosphate stimulated meiosis and led to a noticeable increase in the 4C cell number (dividing zygotes). Additionally, we performed transcriptomic data analysis and identified putative phosphate transporters and enzymes involved in the phosphate uptake and regulation of its metabolism by P. cordatum. These include high- and low-affinity inorganic phosphate transporters, atypical alkaline phosphatase, purple acid phosphatases and SPX domain-containing proteins.

Microenvironmental Impact on InP/ZnS-Based Quantum Dots in In Vitro Models and in Living Cells: Spectrally- and Time-Resolved Luminescence Analysis.

Quantum dots (QDs) have attracted great attention as tools for theranostics that combine the possibility of simultaneous biological target visualization and medicine delivery. Here, we address whether core/shell InP/ZnS QDs (InP-QDs) may be an alternative to toxic Cd-based QDs. We analyze InP-QD photophysical characteristics in cell culture medium, salt solutions, and directly in the cells. It was demonstrated that InP-QDs were internalized into endolysosomes in HeLa and A549 cells with dynamics similar to Cd-based QDs of the same design, but the two cell lines accumulated them with different efficiencies. InP-QDs were reliably detected in the endosomes despite their low quantum yields. Cell culture medium efficiently decreased the InP-QD photoluminescence lifetime by 50%, acidic pH (4.0) had a moderate effect (20-25% reduction), and quenching by salt solutions typical of intra-endosomal medium composition resulted in a decrease of about 10-15%. The single-vesicle fluorescence-lifetime imaging microscopy analysis of QDs inside and outside the cells shows that the scatter between endosomes in the same cell can be significant, which indicates the complex impact of the abovementioned factors on the state of InP-QDs. The PI test and MTT test demonstrate that InP-QDs are toxic for both cell lines at concentrations higher than 20 nM. Possible reasons for InP-QD toxicity are discussed.

Combined Cytotoxic Effect of Inhibitors of Proteostasis on Human Colon Cancer Cells.

Despite significant progress in the diagnosis and treatment of colorectal cancer, drug resistance continues to be a major limitation of therapy. In this regard, studies aimed at creating combination therapy are gaining popularity. One of the most promising adjuvants are inhibitors of the proteostasis system, chaperone machinery, and autophagy. The main HSP regulator, HSF1, is overactivated in cancer cells and autophagy sustains the survival of malignant cells. In this work, we focused on the selection of combination therapy for the treatment of rectal cancer cells obtained from patients after tumor biopsy without prior treatment. We characterized the migration, proliferation, and chaperone status in the resulting lines and also found them to be resistant to a number of drugs widely used in the clinic. However, these cells were sensitive to the autophagy inhibitor, chloroquine. For combination therapy, we used an HSF1 activity inhibitor discovered earlier in our laboratory, the cardenolide CL-43, which has already been proven as an auxiliary component of combined therapy in established cell lines. CL-43 effectively suppressed HSF1 activity and Hsp70 expression in all investigated cells. We tested the autophagy inhibitor, chloroquine, in combination with CL-43. Our results indicate that the use of an inhibitor of HSF1 activity in combination with an autophagy inhibitor results in effective cancer cell death, therefore, this therapeutic approach may be a promising treatment regimen for certain patients.

Resistance to H2O2-induced oxidative stress in human cells of different phenotypes.

Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H2O2) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H2O2 concentrations under conditions of H2O2-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells - all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes - mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H2O2, we showed that at low oxidative loads (<50 µM of H2O2) the gradient depended on extracellular H2O2 concentration. At high loads (>50 µM of H2O2), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H2O2 gradient on the oxidative load in human cells. At high H2O2 concentrations, the cytoplasmic H2O2 level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H2O2-detoxification processes is inherent to a period of early human development.

Combination of a Chaperone Synthesis Inducer and an Inhibitor of GAPDH Aggregation for Rehabilitation after Traumatic Brain Injury: A Pilot Study.

The recovery period after traumatic brain injury (TBI) is often complicated by secondary damage that may last for days or even months after trauma. Two proteins, Hsp70 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were recently described as modulating post-traumatic processes, and in this study, we test them as targets for combination therapy using an inhibitor of GAPDH aggregation (derivative of hydrocortisone RX624) and an inducer of Hsp70 synthesis (the pyrrolylazine derivative PQ-29). The protective effect of the combination on C6 rat glioblastoma cells treated with the cerebrospinal fluid of traumatized animals resulted in an increase in the cell index and in a reduced level of apoptosis. Using a rat weight drop model of TBI, we found that the combined use of both drugs prevented memory impairment and motor deficits, as well as a reduction of neurons and accumulation of GAPDH aggregates in brain tissue. In conclusion, we developed and tested a new approach to the treatment of TBI based on influencing distinct molecular mechanisms in brain cells.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Effects of IGFBP3 knockdown on human endometrial mesenchymal stromal cells stress-induced senescence.

Insulin-like growth factor binding protein 3 (IGFBP3) is known for its pleiotropic ability to regulate various cellular processes such as proliferation, apoptosis, differentiation etc. It has recently been shown that IGFBP3 is part of the secretome of senescent human endometrial mesenchymal stromal cells (MESCs) (Griukova et al., 2019) that takes part in paracrine propagation of senescence-like phenotype in MESCs (Vassilieva et al., 2020); however, mechanisms of pro-senescent IGFBP3 action in MESCs remain still unexplored. This study is aimed at elucidating the role of IGFBP3 upregulation in senescent MESCs. IGFBP3 knockdown in MESCs committed to H2O2-induced senescence led to partial abrogation of p21/Rb axis, to elevated ERK phosphorylation and to increase in SA-ß-gal activity. Additionally, MESCs derived from various donors were found to demonstrate different IGFBP3 regulation during stress-induced senescence. Obtained results suggest ambiguous role of IGFBP3 in stress-induced senescence of MESCs.

Pyrrolylquinoxaline-2-One Derivative as a Potent Therapeutic Factor for Brain Trauma Rehabilitation.

Traumatic brain injury (TBI) often causes massive brain cell death accompanied by the accumulation of toxic factors in interstitial and cerebrospinal fluids. The persistence of the damaged brain area is not transient and may occur within days and weeks. Chaperone Hsp70 is known for its cytoprotective and antiapoptotic activity, and thus, a therapeutic approach based on chemically induced Hsp70 expression may become a promising approach to lower post-traumatic complications. To simulate the processes of secondary damage, we used an animal model of TBI and a cell model based on the cultivation of target cells in the presence of cerebrospinal fluid (CSF) from injured rats. Here we present a novel low molecular weight substance, PQ-29, which induces the synthesis of Hsp70 and empowers the resistance of rat C6 glioma cells to the cytotoxic effect of rat cerebrospinal fluid taken from rats subjected to TBI. In an animal model of TBI, PQ-29 elevated the Hsp70 level in brain cells and significantly slowed the process of the apoptosis in acceptor cells in response to cerebrospinal fluid action. The compound was also shown to rescue the motor function of traumatized rats, thus proving its potential application in rehabilitation therapy after TBI.

Fluorometric Na+ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

BACKGROUND/AIMS: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. METHODS: We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. RESULTS: It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. CONCLUSION: The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells.

Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.

Extracellular Hsp70 Reduces the Pro-Tumor Capacity of Monocytes/Macrophages Co-Cultivated with Cancer Cells.

Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial-mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.

Intracellular K+ and water content in human blood lymphocytes during transition from quiescence to proliferation.

Many evidence shows that K+ ions are required for cell proliferation, however, changes in intracellular K+ concentration during transition of cells from quiescence to cycling are insufficiently studied. Here, we show using flame emission assay that a long-term increase in cell K+ content per g cell protein is a mandatory factor for transition of quiescent human peripheral blood lymphocytes (PBL) to proliferation induced by phytohemagglutinin, phorbol ester with ionomycin, and anti-CD3 antibodies with interleukin-2 (IL-2). The long-term increase in K+ content is associated with IL-2-dependent stage of PBL activation and accompanies the growth of small lymphocytes and their transformation into blasts. Inhibition of PBL proliferation with drugs specific for different steps of G0/G1/S transit prevented both blast-transformation and an increase in K+ content per cell protein. Determination of the water content in cells by measuring the density of cells in the Percoll gradient showed that, unlike the K+ content, the concentration of K+ in cell water remains unchanged, since water and K+ change in parallel. Correlation of proliferation with high cell K+ and water content has been confirmed by the data obtained in comparative study of PBL and permanently cycling Jurkat cells. Our data suggest that K+ is important for successful proliferation as the main intracellular ion that participates in regulation of cell water content during cell transition from quiescence to proliferation. We concluded that high K+ content in cells and the associated high water content is a characteristic feature of proliferating cells.

Etoposide-Induced Apoptosis in Cancer Cells Can Be Reinforced by an Uncoupled Link between Hsp70 and Caspase-3.

The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein⁻protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70⁻Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.

Discovery and optimization of cardenolides inhibiting HSF1 activation in human colon HCT-116 cancer cells.

Combinational anticancer therapy demonstrates increased efficiency, as it targets different cell-survival mechanisms and allows the decrease of drug dosages that are often toxic to normal cells. Inhibitors of the heat shock response (HSR) are known to reduce the efficiency of proteostasis mechanisms in many cancerous cells, and therefore, may be employed as anti-tumor drug complements. However, the application of HSR inhibitors is limited by their cytotoxicity, and we suggested that milder inhibitors may be employed to sensitize cancer cells to a certain drug. We used a heat-shock element-luciferase reporter system and discovered a compound, CL-43, that inhibited the levels of heat shock proteins 40, 70 (Hsp70), and 90 kDa in HCT-116 cells and was not toxic for cells of several lines, including normal human fibroblasts. Consequently, CL-43 was found to reduce colony formation and motility of HCT-116 in the appropriate assays suggesting its possible application in the exploration of biology of metastasizing tumors. Importantly, CL-43 elevated the growth-inhibitory and cytotoxic activity of etoposide, cisplatin, and doxorubicin suggesting that the pro-drug has broad prospect for application in a variety of anti-tumor therapy schedules.

A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.