Hamster Fibroblasts Research Articles

Sperm induction of somatic cell-cell fusion as a novel functional test.

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

Chemical characterization of Callingcard Vine (Entada polystachya (L.) DC. var. polystachya) aqueous seed extract and evaluation of its cytotoxic, genotoxic and mutagenic properties.

Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster fibroblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors d-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

Feldspar-Modified Methacrylic Composite for Fabrication of Prosthetic Teeth.

This study was aimed at investigating poly(methyl methacrylate) (PMMA), modified with a silanized feldspar filler at 10 wt.% and 30 wt.%, as a dental material system for the production of prosthetic teeth. Samples of this composite were subjected to a compressive strength test, three-layer methacrylic teeth were fabricated with the said materials, and their connection to a denture plate was examined. The biocompatibility of the materials was assessed via cytotoxicity tests on human gingival fibroblasts (HGFs) and Chinese hamster ovarian cells (CHO-K1). The addition of feldspar significantly improved the material's compressive strength, with neat PMMA reaching 107 MPa, and the addition of 30% feldspar raising it up to 159 MPa. As observed, composite teeth (cervical part made of neat PMMA, dentin with 10 wt.%, and enamel with 30 wt.% of feldspar) had good adhesion to the denture plate. Neither of the tested materials revealed any cytotoxic effects. In the case of hamster fibroblasts, increased cell viability was observed, with only morphological changes being noticed. Samples containing 10% or 30% of inorganic filler were determined to be safe for treated cells. The use of silanized feldspar to fabricate composite teeth increased their hardness, which is of significant clinical importance for the duration of use of non-retained dentures.

Synbiotic-fluoride synergism on enamel remineralization, cytotoxicity and genotoxicity

Objective(s)The objectives of the present study were to examine the – a) enamel remineralization potential of synbiotic-fluoride (SF) therapy using a multi-species bacterial pH-cycling model; and b) cytotoxic and genotoxic effects of SF therapy extracts. Materials and methodsThe SF therapy group comprised of 2% arginine (Arg), 0.2% NaF, and a probiotic Lactobacillus rhamnosus GG (LRG). The intervention groups studied were: 1) No treatment; 2) 2% Arg; 3) 0.2% NaF; 4) LRG; 5) 2% Arg+0.2% NaF; 6) 2% Arg+LRG; 7) 0.2% NaF+LRG; and 8) 2% Arg+0.2% NaF+LRG (SF therapy). The enamel remineralization potential of SF therapy was investigated under cariogenic biofilm challenge; while the cytotoxicity and genotoxicity of SF therapy extracts were examined on HGF-1 and Chinese hamster fibroblast V79, respectively. To determine the remineralization effect, the specimens were subjected to mineral density (MD) assessment using micro-CT, Ca/P molar ratio with SEM-EDX, and enamel fluoride uptake (EFU) estimates. The HGF-1 proliferation assessment was quantified using MTT/CCK-8 assays with qualitative analysis by nuclei staining Hoechst-based fluorescence imaging. The genotoxicity was determined by micronuclei formation test. ResultsMineral gain and %remineralization derived from MD assessment for the SF therapy were significantly higher than the other groups (p<0.05). The %ΔCa/P for the SF and 2% Arg+0.2% NaF were significantly higher than the other groups (p<0.05). The SF and 2% Arg+0.2% NaF groups had the highest EFU compared to the other groups (p<0.05). No significant difference in the %viable HGF-1 cells were observed between the treatment interventions and no treatment group (p>0.05). Compared to the EMS-positive control, the micronuclei formation for all the intervention groups was significantly lower (p<0.05), with no significant difference among the treatment groups (p>0.05). ConclusionThe SF therapy enhanced enamel remineralization with no biocompatibility concerns. Clinical significanceWith the enhanced enamel remineralization potential discerned in the present study, the SF therapy can be used as a promising caries-preventive agent targeted for high caries-risk individuals.

Metabolism and in vitro assessment of the mutagenic activity of urinary extracts from rats after inhalation exposure to 1-methylnaphthalene.

1-Methylnaphthalene (1-MN) is composed of 2 benzene rings and belongs to polycyclic aromatic hydrocarbons. The metabolism of 1-MN in laboratory animals and bacteria leads to the formation of 1-naphthoic acid (1-NA). In this study the distribution of 1-NA in lung, liver, spleen, kidney and urinary excretion of 1-NA in rats after single and repeated inhalation exposure to 1-MN vapors were investigated. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cytochrome were measured of the rats. Genotoxic effects were evaluated with the in vitro micronucleus test on V79 hamster fibroblasts. The concentrations of 1-NA in the tissues of rats after single and repeated exposure to 1-MN were dependent on the exposure dose. High levels of 1-NA were found in kidneys of animals after the single and repeated exposure to 1-MN. With an increase of 1-MN dose, an increase in the activity of cytochrome P450 (CYP1A1 and CYP1A2) was observed in the liver of rats. Compared to control animals, significantly higher ALT activity was noted in serum of rats exposed to 1-MN. The micronuclei frequency in V79 cells exposed to 1-MN (in the range of analyzable concentrations; i.e., 5-25 μg/ml) did not differ significantly from the vehicle control, whereas urine extracts from rats exposed to 1-MN induced a significant increase in the frequency of micronuclei compared to urine extracts from the group of control animals. Metabolism of 1-MN in rats after the inhalation exposure leading to 1-NA was mainly observed during the first day after the end of exposure. It is likely that 1-MN metabolites present in rat urine can induce the increased micronuclei frequency as was shown in V79 cells. Int J Occup Med Environ Health. 2022;35(6):731-46.

Investigation of the Properties of Linen Fibers and Dressings.

In antiquity, flax was used as a dressing for healing wounds. Currently, work is underway on the genetic modification of flax fibers to improve their properties. Genetic modifications have resulted in an increased content of antioxidants and more favorable mechanical properties. The works published so far have presented independent tests of fibers and dressings after appropriate technological treatments in cell cultures. This study aimed to compare the properties of the fibers and the dressing produced in cell cultures—hamster fibroblasts—V79. The research material was traditional NIKE fibers; genetically modified M, B, and MB fibers; and linen dressings obtained from these fibers. The extract from 48-h incubation of 40 mg of fiber in the culture medium, which was desolved into 10, 20, and 30 mg, was administered to the cell culture. On the other hand, a linen dressing was placed on cells with an area of 0.5 cm2, 1 cm2, 1.5 cm2, and 2 cm2. Cells with fiber or dressing were incubated for 48 h, and then, biological tests were performed, including cell viability (in propidium iodide staining), cell proliferation (in the SRB assay), evaluation of the intracellular free radical level (in the DCF-DA assay), genotoxicity (in the comet assay), assessment of the apoptotic and necrotic cells (in staining anexin-V and iodide propidium), the course of the cell cycle, and the scratch test. The correlation between apoptosis and genotoxicity and the levels of free radicals and genotoxicity were determined for the tested linen fibers and fabrics. The tests presented that the fibers are characterized by the ability to eliminate damaged cells in the elimination phase. However, the obtained fabrics gain different properties during the technological processing of the fibers into linen dressings. Linen fabrics have better regenerative properties for cells than fibers. The linseed dressing made of MB fiber has the most favorable regenerative properties.

Возможность применения ионно-трековых матриц на основе полиэфиров для создания искусственной соединительной ткани

Abstract-The cultivation of Chinese hamster fibroblasts (line V79) on ion-track membranes of various polyesters has been tested. Light and electron microscopy were used to take photographs of polyester membranes with fibroblasts grown on them, which indicated the attachment and proliferation of cells on ion-track adhesive surfaces. The greatest survival of fibroblasts was observed on ion-track membranes with a pore diameter of up to 0.5 μ. It was shown that the chemical composition of the track membrane affects the survival and proliferation rate of Chinese hamster fibroblasts. Polyethylene terephtalate and polyethylene naphtalate membranes provided the highest percentage of grown colonies and the greatest proliferation rate of cells. Track-etched polyester membranes can be used to create adhesive surfaces for the cultivation of fibroblasts and the production of artificial connective tissue. Key words: ion-track membranes and technologies, tissue engineering, fibroblasts, burn therapy

Abstract 1375: Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells

Abstract Introduction: PARP inhibitors (PARPi) effectively target BRCA mutated cancers by exploiting defects in homologous recombination repair (HRR) by synthetic lethality. PARPi cause replication stress, activating ATR, which signals to HRR and to S and G2/M cell cycle checkpoints via CHK1 and WEE1. Our aim was to determine if a CHK1 inhibitor (CHK1i) would be synthetically lethal with PARPi and to identify the underlying mechanism. Methods: Effects of the PARPi rucaparib and the CHK1i PF-477736 were investigated in V-C8 (BRCA2 mutant Chinese hamster fibroblasts) and V-C8.B2 (BRCA2 restored) cell lines. PARP activity was determined by immunoblot densitometry of the PAR product. CHK1, ATR and WEE1 activity was measured by CHK1S296, CHK1S345 and CDK1y15 phosphorylation, respectively, by Western blot. HRR activity was determined by γH2AX and RAD51 foci formation by immunofluorescence microscopy. Cytotoxicity was determined by colony formation assay and cell cycle analysis by flow cytometry using propidium iodide. Results: V-C8 cells had higher baseline PARP activity than V-C8.B2 cells (1.6 and 0.7 nmol/106 cells, respectively), and in both cell lines PARP activity was suppressed 99% by 1 µM rucaparib. ATR, CHK1 and WEE1 were activated by rucaparib (10 µM) in both V-C8 cells (2.1, 1.5 and 1.8-fold) and V-C8.B2 cells (1.7, 1.4 and 1.5-fold) respectively. Conversely, activation of CHK1 and WEE1 was completely inhibited by 50 nM PF-477736 in both cell lines, while upstream activation of ATR by PF-477736 was almost 2-fold in both cell lines. Rucaparib was 550x more cytotoxic to HRR-defective V-C8 cells compared to HRR-competent V-C8.B2 cells (LC50 &amp;lt;0.1 µM vs 8.8 µM; p&amp;lt;0.001 respectively). Combination treatment with PF-477736 (50 nM) sensitised V-C8.B2 cells to rucaparib cytotoxicity 4.9-fold but did not sensitise V-C8 cells. In V-C8.B2 cells, 10 µM rucaparib caused a 6-fold increase in γH2AX and a 4.8-fold increase in RAD51 foci in γH2AX positive cells, demonstrating both increased replication stress and HRR activity in response to DNA damage. Combination treatment with PF-477736 (50 nM) increased γH2AX foci 2-fold compared to rucaparib single agent and inhibited the rucaparib induced RAD51 foci formation entirely, showing complete inhibition of HRR in V-C8.B2 γH2AX positive cells. Conclusion: The CHK1i PF-477736 sensitised HRR competent, but not HRR defective V-C8 cells to rucaparib suggesting that the primary mechanism was through PF-477736 abrogation of HRR mediated by its direct inhibition of CDK1. Cell cycle analysis is ongoing to determine the impact of rucaparib plus PF-477736 modulation of CHK1 and WEE1 activities on S and G2/M checkpoint control. Citation Format: Hannah L. Smith, Lisa Prendergast, Nicola J. Curtin. Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1375.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Differential Innate Immune Responses Elicited by Nipah Virus and Cedar Virus Correlate with Disparate In Vivo Pathogenesis in Hamsters.

Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens of infected hamsters, a neutralizing antibody was produced in some hamsters within 8 days post-challenge, and no conspicuous signs of disease occurred. CedPV replicated to a similar magnitude as NiV-Bangladesh in type I IFN-deficient BHK-21 Syrian hamster fibroblasts but replicated 4 logs lower in type I IFN-competent primary Syrian hamster and human pulmonary endothelial cells, a principal target of henipaviruses. The coinfection of these cells with CedPV and NiV failed to rescue CedPV titers and did not diminish NiV titers, suggesting the replication machinery is virus-specific. Type I IFN response transcripts Ifna7, Ddx58, Stat1, Stat2, Ccl5, Cxcl10, Isg20, Irf7, and Iigp1 were all significantly elevated in CedPV-infected hamster endothelial cells, whereas Ifna7 and Iigp1 expression were significantly repressed during NiV infection. These results are consistent with the hypothesis that CedPV’s inability to counter the host type I IFN response may, in part, contribute to its lack of pathogenicity. Because NiV causes a fatal disease in Syrian hamsters with similarities to human disease, this model will provide valuable information about the pathogenic mechanisms of henipaviruses.

Thermal expansion of substrate may affect adhesion of Chinese hamster fibroblasts to surfaces during freezing

Despite success in cryopreservation of cells in suspension, cryopreservation of cells in monolayers is still challenging. One of the major problems is detachment of the cells from the substrate which occurs during cryopreservation. We hypothesized that this detachment may be due to a mismatch in the coefficient of linear thermal expansion αL between glass and the frozen cell layer which manifests as residual stress and stress relaxation. This mismatch results in a difference between the thermal expansion of ice and glass as they undergo temperature changes. Rinzl plastic coverslips were selected as a possible substitute for glass because Rinzl has an αL (60 × 10−6/K) similar to that of ice (51 × 10−6/K) whereas glass has a much lower αL (5 × 10−6/K). V79-4 Chinese hamster fibroblasts were cultured on both glass and Rinzl coverslips until confluent and the area of coverage was measured before and after freezing at −9 °C. The glass coverslips showed significant loss of cells (coverage = 77.9 ± 8.0%) compared with Rinzl (coverage = 97.9 ± 1.4%). We concluded that Rinzl coverslips may improve cell attachment in future monolayer cryopreservation experiments.

Correlations of In Vitro Assays for Assessing Cytotoxicity and Biocompatibility of Contact Lens Multipurpose Solutions

To demonstrate correlations among in vitro assays used for assessing cytotoxicity of contact lens multipurpose solution (MPS) and propose the use of multiple assays as a part of preclinical evaluation for MPS biocompatibility assessment. The effect of four different MPS on cell cytotoxicity, metabolic activity, and membrane integrity was performed by evaluating toxicity, expression of tight junction protein zonula occludens-1, and transepithelial electrical resistance in human corneal epithelial cells and Chinese hamster fibroblast cells. Cytotoxicity of four MPS was assayed with five different experimental systems at various concentrations. In vitro MPS-induced cytotoxicity was dependent on assay choice, concentration of MPS used, and duration of treatment. Overall, MPS-1 and MPS-2 were comparable to MPS-4 and better than MPS-3 in maintaining corneal barrier integrity and cell viability. In vitro cytotoxicity testing with MPS exposure to monolayer of cells in culture could be used as a tool to understand the potential cytotoxicity profiles of MPS and possibly a predictor of clinical outcome. Furthermore, MPS effects on in vitro cytotoxicity are best demonstrated by performing multiple assays to evaluate cell viability, metabolic activity, and membrane integrity during development.

ChromaWizard: An open source image analysis software for multicolor fluorescence in situ hybridization analysis.

Multicolor image analysis finds its applications in a broad range of biological studies. Specifically, multiplex fluorescence in situ hybridization (M‐FISH) for chromosome painting facilitates the analysis of individual chromosomes in complex metaphase spreads and is widely used to detect both numerical and structural aberrations. While this is well established for human and mouse karyotypes, for which species sophisticated software and analysis tools are available, other organisms and species are less well served. Commercially available software is proprietary and not easily adaptable to other karyotypes. Therefore, a publically available open source software that combines flexibility and customizable functionalities is needed. Here we present such a tool called “ChromaWizard” which is based on popular scientific image analysis libraries (OpenCV, scikit‐image, and NumPy). We demonstrate its functionality on the example of primary Chinese hamster (Cricetulus griseus) fibroblasts metaphase spreads and on Chinese Hamster Ovary cell lines known for the large number of chromosomal rearrangements. The application can be easily adapted to any kind of available labeling kits and is https://dict.leo.org/englisch-deutsch/independent of the used organism and instrumentation. It allows direct inspection of the original hybridization signals and enables either manual or automatic assignment of colors, making it a functional and versatile tool that can be used also for other multicolor applications.

In situ formation of injectable chitosan-gelatin hydrogels through double crosslinking for sustained intraocular drug delivery

Rapid clearance and low ocular bioavailability are drawbacks of conventional ophthalmic eye drops. To increase the ocular drug resistance time and improve efficacy, an in situ forming and thermosensitive chitosan-gelatin hydrogel was developed. The feasibility of using this hydrogel as a topical eye drop formulation for sustained release of timolol maleate was evaluated. The flexible hydrogel that was co-crosslinked with β‑glycerophosphate disodium salt hydrate (β-GD) and genipin showed a fast gel formation at 37 °C. The swelling properties and in vitro biodegradation characteristics showed a strong relationship with the initial genipin concentration. In vitro release profiles demonstrated that crosslinking with genipin reduced the release rate of entrapped model drugs and timolol maleate. In vitro cytotoxicity tests showed that the hydrogel was non-toxic to Chinese hamster fibroblast V79 cells. The hydrogel was further applied as eye drop formulations for sustained release of timolol maleate to reduce intraocular pressure (IOP). A fast gel formation was observed after instilling the chitosan-gelatin solution into the lower conjunctival sac of the rabbit eyes, and the in situ formed hydrogels protected the drugs from clearance by tears, and released the drugs in a sustained manner. Furthermore, administration of timolol maleate containing chitosan-gelatin hydrogels showed a long-lasting and effective IOP lowering efficacy for up to 24 h compared with the conventional eye drops. These results suggested that β-GD and genipin co-crosslinked chitosan-gelatin hydrogels could be a useful ocular drug delivery platform with enhanced therapeutic effects and reduced side effects.

Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.

Physicochemical and biological characterization of sustained isopropyl unoprostone-release device made of poly(ethyleneglycol) dimethacrylates.

Transscleral drug delivery is becoming increasingly popular to manage posterior eye diseases. To evaluate the clinical application of a transscleral, sustained, unoprostone (UNO)-release device (URD) constructed of photopolymerized tri(ethyleneglycol) dimethacrylate and poly(ethyleneglycol) dimethacrylate, we evaluated physicochemical and biological properties of this device. The URD consists of a drug-impermeable reservoir and a semi-permeable cover. The in vitro release rate of UNO from the URD increased with increasing temperatures from 20 to 45 °C. Scanning electron microscopy and atomic-force microscopy showed that the border between the reservoir and drug formulation was sharply defined but that between the cover and drug was poorly determined, indicating that UNO could permeate only through the cover. For stability tests, the URDs were sterilized with ethylene oxide gas and stored at 40 °C/75% for 3 and 6 months and at 25 °C/60% for 3, 6, 9, 12, 18, and 24 months; UNO content and release rate at 37 °C were then evaluated. There was no significant decrease in either UNO content or release rate after the storage conditions. Cytotoxicity was evaluated by examining the colony formation of Chinese hamster fibroblast V79 cells in a media extract of the URD without UNO. This extract did not affect colony formation of V79 cells, indicating the cytocompatibility of the URD. In conclusion, the URD was physically stable for 24 months and is potentially useful for clinical application.

Nuclear Trafficking of ABCB1‐Daunorubicin Vesicles Initiated by Sphingomyelinase Reverts Multidrug Resistance in Chinese Hamster Fibroblasts

Effective cancer chemotherapy treatment is hampered by multidrug resistance (MDR), defined by upregulation of ABCB1, which effluxes drugs. Intracellular ABCB1 also sequesters anthracycline drugs in acidic vesicles preventing nuclear targeting. Reports indicate sphingolipids affect vesicular trafficking. We hypothesized sphingomyelinase (SMase) activity might mobilize ABCB1/drug vesicles to revert MDR. Using drug‐resistant ADX fibroblasts expressing ABCB1‐EGFP, time‐lapse confocal microscopy revealed daunorubicin (DNR) sequestration in ABCB1‐EGFP‐containing vesicles within 5min. In ADX/ABCB1‐EGFP loaded with DNR (20μM,2h) followed by drug washout, exogenous bacterial (b)SMase, which hydrolyzes plasma membrane sphingomyelin and initiates sphingolipid signaling, induced rapid ABCB1‐EGFP/DNR vesicle movement to the nucleus, where DNR was deposited, while ABCB1‐EGFP remained in nuclear membranes. Immunoblots of isolated nuclei and nuclear envelope membranes purified by sucrose density gradient confirmed these observations. In DNR‐exposed ADX cells, bSMase‐induced ABCB1 translocation to the nucleus peaked at 1h, and was detectable at 4h but not at 8h, suggesting transient incorporation of ABCB1 into the nuclear membrane. No nuclear ABCB1 was observed in drug sensitive DC3F cells loaded with an isotoxic DNR dose (0.2μM, 2h), indicating ABCB1 nuclear translocation by bSMase is MDR specific. Cell viability studies by MTT assay demonstrate augmented DNR toxicity in the presence of bSMase and can be enhanced by co‐incubation with the ABCB1 inhibitor, PSC833/valspodar (1μM), to prevent cellular drug efflux, in an additive manner. Intriguingly, in the postnuclear supernatant of DNR‐treated ADX cells, ABCB1 primarily sediments at 8000g, but shifts to 18000g at 4h after bSMase suggesting altered vesicular density or ABCB1 transfer to a distinct vesicular pool. In conclusion, SMase activity mobilizes ABCB1/DNR vesicles to the nucleus where DNR is deposited and reverses MDR.Support or Funding InformationThe work was supported by the Max Kade Foundation and Intramural Research Program at Witten/Herdecke University (IFF2016‐20) (W.K.L).

Plasma Membrane Organization and Dynamics is Probe and Cell Line Dependent: An Imaging FCS Study

The plasma membrane is a complex assembly comprising hundreds of different lipids and proteins. Its properties are temperature dependent and change from cell line to cell line. This raises the question whether measurements at different temperatures and within different cell lines are comparable even when using the same probe. Here, we investigate the dynamics and organization of five different cell lines - Chinese hamster ovary (CHO-K1), Hela, neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) - using up to four different lipid probes. For the outer membrane leaflet we use DiI-C18, a common liquid disordered marker and GFP-GPI, a GFP tagged glycosylphosphatidylinositol anchored protein as a marker for the liquid ordered phase. For the inner membrane leaflet we use PH-PLCδ-RFP, a PIP2 binding domain, and PMT-GFP, a GFP tagged plasma membrane targeting domain that had been shown to be sensitive to the cytoskeleton. We applied Imaging Fluorescence Correlation Spectroscopy (imaging FCS) on a Total Internal Reflection Microscope (TIRFM) which provided diffusion coefficients (D), the Arrhenius activation energy for diffusion (EArr), and the FCS diffusion law intercept (τ0), which report on membrane fluidity, molecular packing, and diffusion mode of the probes (free, domain, or hop diffusion), respectively. The combination of these parameters is unique for each of the probes. Our results showed, that each of the labels is characterized by a unique set of parameters (D, EArr, τ0). Furthermore, the parameters do not stay constant for a particular probe or change in a concerted manner, but can change individually from cell to cell line, indicating that the organization and mode of diffusion of these molecules depends strongly on the environment. This variability has important implications for membrane measurements in general and the comparability of measurements is only given in a particular cell line and at a given temperature.

A Nucleoside Anticancer Drug, 1-(3-C-Ethynyl-β-D-Ribo-Pentofuranosyl)Cytosine, Induces Depth-Dependent Enhancement of Tumor Cell Death in Spread-Out Bragg Peak (SOBP) of Proton Beam.

The effect of 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd) on proton-induced cell death was evaluated in human lung carcinoma cell line A549 and Chinese hamster fibroblast cell line V79 to enhance relative biological effectiveness (RBE) within the spread-out Bragg peak (SOBP) of proton beams. Treatment with ECyd significantly enhanced the proton-induced loss of clonogenicity and increased senescence at the center, but not at the distal edge of SOBP. The p53-binding protein 1 foci formation assay showed that ECyd decelerated the rate of DNA double-strand break (DSB) repair at the center, but not the distal region of SOBP, suggesting that the ECyd-induced enhancement of proton-induced cell death is partially associated with the inhibition of DSB repair. This study demonstrated that ECyd enhances proton-induced cell killing at all positions of SOBP, except for the distal region and minimizes the site-dependent differences in RBE within SOBP. Thus, ECyd is a unique radiosensitizer for proton therapy that may be useful because it levels the biological dose within SOBP, which improves tumor control and reduces the risk of adverse effects at the distal edge of SOBP.