Significant Difference In Cell Survival Research Articles

Subminute thermal damage to cell types present in the skin

Introduction Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. Materials and Methods Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2–20 sec) and temperature (45–70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. Results Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50–60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. Conclusion The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.

The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Effect of Proton Therapy on Tumor Cell Killing and Immune Microenvironment for Hepatocellular Carcinoma.

Radiotherapy with proton therapy (PT) has dosimetric advantages over photon therapy, which helps to enlarge the therapeutic window of radiotherapy for hepatocellular carcinoma (HCC). We evaluated the response of HCC to PT and examined the underlying mechanisms. The human liver cancer cell lines HepG2 and HuH7 and the murine liver cancer cell line Hepa1-6 were selected for cell and animal experiments to examine the response induced by PT irradiation. Biological changes and the immunological response following PT irradiation were examined. In vitro experiments showed no significant difference in cell survival following PT compared with photon radiotherapy. In a murine tumor model, the tumors were obviously smaller in size 12 days after PT irradiation. The underlying changes included increased DNA damage, upregulated IL-6 levels, and a regulated immune tumor microenvironment. Protein analysis in vitro and in vivo showed that PT increased the level of programmed cell death ligand 1 (PD-L1) expressed in tumor cells and recruited myeloid-derived suppressor cells (MDSCs). The increase in PD-L1 was positively correlated with the irradiation dose. In Hepa1-6 syngeneic mouse models, the combination of PT with anti-PD-L1 increased tumor growth delay compared with PT alone, which was associated with increased tumor-infiltrating T cells and attenuated MDSC recruitment in the microenvironment. Furthermore, when PT was applied to the primary HCC tumor, anti-PD-L1 antibody-treated mice showed smaller synchronous unirradiated tumors. In conclusion, the response of HCC to PT was determined by tumor cell killing and the immunological response in the tumor microenvironment. The combination with the anti-PD-L1 antibody to enhance antitumor immunity was responsible for the therapeutic synergism for HCC treated with PT. Based on our results, we suggest that PT combined with anti-PD-L1 may be a promising therapeutic policy for HCC.

Chronic nicotine impairs the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells in a murine model of peripheral arterial disease

ObjectiveLifestyle choices such as tobacco and e-cigarette use are a risk factor for peripheral arterial disease (PAD) and may influence therapeutic outcomes. The effect of chronic nicotine exposure on the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) was assessed in a murine model of PAD. MethodsMice were exposed to nicotine or phosphate-buffered saline (PBS) for 28 days, followed by induction of limb ischemia and iPSC-EC transplantation. Cells were injected into the ischemic limb immediately after induction of hindlimb ischemia and again 7 days later. Limb perfusion was assessed by laser Doppler spectroscopy, and transplant cell survival was monitored for 14 days afterward using bioluminescence imaging, followed by histological analysis of angiogenesis. ResultsTransplant cell retention progressively decreased over time after implantation based on bioluminescence imaging, and there were no significant differences in cell survival between mice with chronic exposure to nicotine or PBS. However, compared with mice without nicotine exposure, mice with prior nicotine exposure had had an impaired therapeutic response to iPSC-EC therapy based on decreased vascular perfusion recovery. Mice with nicotine exposure, followed by cell transplantation, had significantly lower mean perfusion ratio after 14 days (0.47 ± 0.07) compared with mice undergoing cell transplantation without prior nicotine exposure (0.79 ± 0.11). This finding was further supported by histological analysis of capillary density, in which animals with prior nicotine exposure had a lower capillary density (45.9 ± 4.7 per mm2) compared with mice without nicotine exposure (66.5 ± 8.1 per mm2). Importantly, the ischemic limbs mice exposed to nicotine without cell therapy also showed significant impairment in perfusion recovery after 14 days, compared with mice that received PBS + iPSC-EC treatment. This result suggested that mice without chronic nicotine exposure could respond to iPSC-EC implantation into the ischemic limb by inducing perfusion recovery, whereas mice with chronic nicotine exposure did not respond to iPSC-EC therapy. ConclusionsTogether, these findings show that chronic nicotine exposure adversely affects the ability of iPSC-EC therapy to promote vascular perfusion recovery and angiogenesis in a murine PAD model.

Mammalian cell encapsulation in monodisperse chitosan beads using microchannel emulsification

Microchannel emulsification can produce droplets of uniform size at high throughput for wide-ranging applications in the food and drug industry. Mammalian cell encapsulation using emulsion-based systems is of significant interest for immobilized culture or transplantation applications. Microchannel emulsification has recently been applied to mammalian cell encapsulation using alginate. Here, we extend this approach to thermoresponsive hydrogels using chitosan as a model. The performance of the microchannel emulsification process was compared to a previously established stirred emulsification method for mesenchymal stromal cell encapsulation. Chitosan microbeads with controlled sizes ranging from 600 µm to 1500 µm in diameter at a coefficient of variation less than 10 % were obtained after adapting the microchannel system design, channel geometries and operating parameters that affect the capillary number. In a single pass through a 21 G syringe needle, the fraction of ruptured beads was significantly reduced for monodisperse microchannel-generated vs polydisperse stirred emulsion-generated beads of matching De Brouckere mean diameter. The viability of the immobilized cells immediately after the process was 95 % ± 2 % and no significant difference in cell survival and growth factor secretion was observed between microchannel and stirred emulsion-generated beads over 3 days of culture. Microchannel emulsification is a promising and scalable approach to microencapsulate mammalian cells in hydrogels that undergo temperature-dependent gelation.

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p&amp;lt;0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p&amp;lt; 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 795.

Proliferation of osteoblast precursor cells on the surface of TiO2 nanowires anodically grown on a β-type biomedical titanium alloy

Studies have shown that anodically grown TiO2 nanotubes (TNTs) exhibit excellent biocompatibility. However, TiO2 nanowires (TNWs) have received less attention. The objective of this study was to investigate the proliferation of osteoblast precursor cells on the surfaces of TNWs grown by electrochemical anodization of a Ti-35Nb-7Zr-5Ta (TNZT) alloy. TNT and flat TNZT surfaces were used as control samples. MC3T3-E1 cells were cultured on the surfaces of the samples for up to 5 days, and cell viability and proliferation were investigated using fluorescence microscopy, colorimetric assay, and scanning electron microscopy. The results showed lower cell proliferation rates on the TNW surface compared to control samples without significant differences in cell survival among experimental conditions. Contact angles measurements showed a good level of hydrophilicity for the TNWs, however, their relatively thin diameter and their high density may have affected cell proliferation. Although more research is necessary to understand all the parameters affecting biocompatibility, these TiO2 nanostructures may represent promising tools for the treatment of bone defects and regeneration of bone tissue, among other applications.

IFNγ reduces viability in human conjunctival goblet cells in vitro

AbstractPurposeConjunctival goblets cells play an important role in the homeostasis of the ocular surface and are known to be affected by inflammatory cytokines in diseases of the ocular surface. An understanding of the role of interleukin (IL)‐4, IL‐13, IL‐17, and interferon (IFN)γ in conjunctival goblet cell survival and function is necessary to explore the pathogenesis of ocular surface diseases. Previous studies have shown that IFNγ reduces the survival of the goblet cells, whereas IL‐13, IL‐4 and IL‐17A induce proliferation in mouse and rat models (1). The purpose of this study was to investigate the effect of IL‐4, IL‐13, IL‐17A and IFNγ on human goblet cell survival assessed by LDH analysis.MethodsHuman goblet cell cultures were grown from cultured donor conjunctiva. The goblet cells were seeded into wells and stimulated with IL‐4, IL‐13, IL‐17A and IFNγ with 100 ng/mL for 48 hours, respectively. Cell viability was evaluated by LDH assays.ResultsStimulation with IFNγ showed significant decrease in goblet cell viability with LDH assay (p&lt;0.01). Stimulation with IL‐4, IL‐13 and IL‐17A showed no significant decrease in cell survival compared to control. Significant differences in cell survival were observed between stimulation with IFNγ and IL‐4 (p&lt;0.001) and IL‐13 (p&lt;0.05), respectively.ConclusionsOur study indicates that IFNγ reduces human conjunctival goblet cell survival in contrast to the Th2‐associated cytokines IL‐4 and IL‐13.ReferenceGarcía‐Posadas L, Contreras‐Ruiz L, Soriano‐Romaní L, Dartt DA, Diebold Y. Conjunctival Goblet Cell Function: Effect of Contact Lens Wear and Cytokines. Eye Contact Lens 2016;42(2): 83–90.Conflict of InterestThis work was supported by a research grant from LEO Pharma.

Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C.

Deinococcus radiodurans known for its extraordinary resistance to ionizing radiation contains bacterial phytochrome (BphP), a member of the family of red/far-red light-sensing proteins. In this study, we constructed a bphP mutant strain (ΔbphP) to investigate the role of D. radiodurans BphP (DrBphP) in the DNA damage response. When cells were incubated under light and dark conditions following exposure to DNA damaging agents, such as γ- and UV-radiation and mitomycin C (MMC), no significant difference in cell survival was observed between the wild-type D. radiodurans strain (WT) and ΔbphP. However, when continuously exposed to MMC under light conditions, the WT strain notably exhibited increased survival compared to cells grown in the dark. The increased survival was not observed in the ΔbphP strain. These results are indicative of the protective role of light-activated DrBphP in the presence of MMC. Site-directed mutagenesis revealed that the conserved amino acids Cys-24 and His-532 involved in chromophore binding and signal transduction, respectively, were essential for the protective function of DrBphP. Inactivation of the cognate response regulator (RR; DrBphR) of DrBphP increased MMC resistance in the dark. In trans complementation of the bphP bphR double mutant strain (ΔbphPR) with DrBphR decreased MMC resistance. Considering that DrBphP acts as a light-activated phosphatase that dephosphorylates DrBphR, it appears that phosphorylated DrBphR exerts a negative effect on cell survival in the presence of MMC. DrBphP overexpression resulted in an increase in MMC resistance of ΔbphPR, implying that other RRs might be involved in the DrBphP-mediated signaling pathway. A mutant lacking the dr_0781 gene (Δdr_0781) demonstrated the same MMC phenotype as ΔbphR. Survival was further increased in the bphR dr_0781 double mutant strain compared to each single mutant ΔbphR or Δdr_0781, suggesting that DR_0781 is also involved in the DrBphP-dependent MMC sensitivity. This study uncovered a previously unknown phenomenon of red/far-red light-dependent DNA damage survival mediated by BphP by identifying the conditions under which DrBphP exhibits a fitness advantage.

Temporal and Concentration Effects of Methanol on Cryopreservation of Zebrafish (Danio rerio) Sperm.

We evaluated the cryoprotective effects of methanol on zebrafish sperm at different concentrations, exposure times, and stages during cryopreservation. Samples were collected by crushing of dissected testes or abdominal stripping. After exposure to 0%, 2%, 5%, 8%, and 10% methanol for 0-11 min, fresh sperm (1 × 106 cells/mL) did not show changes in plasma membrane integrity (measured by flow cytometer), but cell size changes (light scatter) were observed after exposure to 8% or 10%. After exposure for 0-60 min, fresh sperm (1 × 108 cells/mL) did not show significant changes in survival or membrane integrity. Sperm cryopreserved in 5%, 8%, and 10% methanol showed high post-thaw survival, in 5% and 8% showed high post-thaw motility, and in 5% showed highest post-thaw membrane integrity compared to other concentrations between 0% and 10%. Within 0-60 min after thawing, no significant differences in cell survival and membrane integrity were found for any concentration (p ≥ 0.269). Comparison of 5% and 8% methanol for dissected testes (n = 20) revealed no difference in post-thaw motility, membrane integrity, cell survival, fertilization, or hatching, embryo viability; for stripped sperm (n = 10), no differences were observed in post-thaw membrane integrity, fertilization, and embryo viability, however, higher motility and survival were detected in 5% than in 8% methanol. Thus, a concentration of 5% methanol seems most suitable for cryopreserving zebrafish sperm based on post-thaw survival and motility.

Radiobiological Difference Between Flattening Filter Free (FFF) Vs. Flattening Filter (FF) X-Ray Beams in Lung Cancer Cells

Stereotactic Body Radiotherapy (SBRT) is a well-established therapeutic option for early lung cancer, and many clinical trials indicate that SBRT can provide excellent local control and survival with limited toxicity. Compared with conventional flattening filter (FF) beams, flattening filter free (FFF) beams are characterized by higher average dose rate with higher dose per pulse and inhomogeneous dose distribution with reduced peripheral dose. These benefits make FFF beams an attractive alternative for SBRT. Therefore, it is crucially to determine whether there are any differences in biological effects of FFF beams compared with FF beams. However, there are still conflicts on them between two beams. Therefore, we investigated the radiobiological effect between FF and FFF beam in human lung cancer cell lines. Three human lung cancer cell lines with different genetic characteristics (A549, NCI-H1299, NCI-H1650) were used. Irradiation was performed with a 10 MV linear accelerator (Versa HD, Elekta AB). The dose rate and dose per pulse for FF and FFF beams were 5 and 22 Gy/min, and 0.042 cGy and 0.171 cGy, respectively. To investigate the effect by different dose rate, but with same dose per pulse, FFF beam of 22 Gy/ min was modified to 5 Gy/min by increasing pulse interval. The radiobiologic effect was studied with three different conditions: FF (5 Gy/min, 0.042 cGy/pulse), FFF (5 Gy/min, and 0.171 cGy/pulse) and FFF (22 Gy/min, 0.171 cGy/pulse). Cell survival was measured using clonogenic assay and protein expression was measured using Western blot analysis. No significant differences in cell survival were observed upon three kinds of irradiation for all the three cell lines. No significant differences were observed in the expression of γ-H2AX and repair proteins (DNA-PKcs, Rad 51) after irradiation in all the three cell lines. There is no radiobiological difference (cell survival, DNA damage and repair) between FF and FFF beams in lung cancer cells.

Survival and immunomodulation of stem cells from human extracted deciduous teeth expanded in pooled human and foetal bovine sera

The immunomodulatory properties of mesenchymal stem cells (MSCs) from autologous and allogeneic sources are useful in stimulating tissue regeneration and repair. To obtain a high number of MSCs for transplantation requires extensive in vitro expansion with culture media supplements that can cause xeno-contamination of cells potentially compromising function and clinical outcomes. In this study stem cells from human extracted deciduous teeth (SHED) were cultured in Knockout™ DMEM supplemented with either pooled human serum (pHS) or foetal bovine serum (FBS) to compare their suitability in maintaining immunomodulatory properties of cells during in vitro expansion. No significant difference in cell survival of SHED grown in pHS (pHS-SHED) or FBS (FBS-SHED) was observed when co-cultured with complement, monocytes or lymphocytes. However, significant changes in the expression of sixteen paracrine factors involved in immunomodulation were observed in the supernatants of FBS-SHED co-cultures with monocytes or lymphocytes compared to that in pHS-SHEDs after both 24 and 120 h of incubation. Further analysis of changing protein levels of paracrine factors in co-cultures using biological pathway analysis software predicted upregulation of functions associated with immunogenicity in FBS-SHED and lymphocyte co-cultures compared to pHS-SHED co-cultures. Pathway analysis also predicted significant stimulation of HMGB1 and TREM1 signalling pathways in FBS-SHED co-cultures indicating activation of immune cells and inflammation. Though FBS supplementation does not impact survival of SHED, our combinatorial biological pathway analysis supports the idea that in vitro expansion of SHEDs in pHS provides optimal conditions to minimise xeno-contamination and inflammation and maintain their immunomodulatory properties.

Combined internal-external radiotherapy (CIERT) in a cell model

Combined internal-external radiotherapy (CIERT) requires a unified assessment of biologic radiation effects in addition to the total dose. The concept of biological effective dose (BED) was evaluated in a cell model. The thyroid NIS-positive cell line FRTL-5 was irradiated with X-ray and the radiotracer Tc-99m pertechnetate either alone or in combination. The cellular uptake of the radionuclide during the incubation time of 24 h was controlled by the presence or absence of perchlorate. Dose calculation was performed based on measured uptake values. Cell specific radiobiologic parameters were derived from dose effect curves using the colony forming assay as biological endpoint. For the combination of the radiation qualities the sequence and time difference were varied. Cell survival was compared with the prediction of the BED model. The radiobiologic parameters from X-ray dose response were α = (0.22 ± 0.02) Gy-1 and β = (0.021 ± 0.001) Gy-2. The half life for repair was (1.51 ± 0.21) h. These values could also explain the dose response curves for Tc-99m-irradiation with exponential decreasing dose rate. CIERT experiments showed no significant differences in cell survival regarding sequence and irradiation break. When the radionuclide uptake was not prevented the cell survival for the combination of X-ray and Tc-99m was lower than the prediction by BED calculations. The validity of the BED formalism for different dose rates and radiation qualities was verified. Supraaddive effects measured in the combination of X-ray and intracellular Tc-99m might be caused by Auger and conversion electrons, however further experiments are necessary.

Increased production of omega-3 fatty acids protects retinal ganglion cells after optic nerve injury in mice

Injury to the central nervous system causes progressive degeneration of injured axons, leading to loss of the neuronal bodies. Neuronal survival after injury is a prerequisite for successful regeneration of injured axons. In this study, we investigated the effects of increased production of omega-3 fatty acids and elevation of cAMP on retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) crush injury in adult mice. We found that increased production of omega-3 fatty acids in mice enhanced RGC survival, but not axonal regeneration, over a period of 3 weeks after ON injury. cAMP elevation promoted RGC survival in wild type mice, but no significant difference in cell survival was seen in mice over-producing omega-3 fatty acids and receiving intravitreal injections of CPT-cAMP, suggesting that cAMP elevation protects RGCs after injury but does not potentiate the actions of the omega-3 fatty acids. The observed omega-3 fatty acid-mediated neuroprotection is likely achieved partially through ERK1/2 signaling as inhibition of this pathway by PD98059 hindered, but did not completely block, RGC protection. Our study thus enhances our current understanding of neural repair after CNS injury, including the visual system.

Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field

Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells is not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant increase in γ-H2AX foci formation when irradiated to 20 cGy in-field in comparison to out-of-field. However, no significant difference in cell survival or colony volume was observed whether the PNT1A cells were irradiated in-field or out-of-field. Out-of-field radiation dose alone can have a damaging effect on the proliferation of PNT1A cells when a clinically relevant dose of 2 Gy is delivered in in-field. Out-of-field radiation with the transfer of bystander factors induces an adaptive response in the PNT1A cells.

Clinical evaluation of inter-fractional organ motion using 3D ultrasound image-guided radiotherapy for positioning prostate cancer patients

Clinical evaluation of inter-fractional organ motion using 3D ultrasound image-guided radiotherapy for positioning prostate cancer patients

Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing

We have investigated whether inkjet printing technology can be extended to print cells of the adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia, and the effects on survival and growth of these cells in culture, which is an important step in the development of tissue grafts for regenerative medicine, and may aid in the cure of blindness. We observed that RGC and glia can be successfully printed using a piezoelectric printer. Whilst inkjet printing reduced the cell population due to sedimentation within the printing system, imaging of the printhead nozzle, which is the area where the cells experience the greatest shear stress and rate, confirmed that there was no evidence of destruction or even significant distortion of the cells during jet ejection and drop formation. Importantly, the viability of the cells was not affected by the printing process. When we cultured the same number of printed and non-printed RGC/glial cells, there was no significant difference in cell survival and RGC neurite outgrowth. In addition, use of a glial substrate significantly increased RGC neurite outgrowth, and this effect was retained when the cells had been printed. In conclusion, printing of RGC and glia using a piezoelectric printhead does not adversely affect viability and survival/growth of the cells in culture. Importantly, printed glial cells retain their growth-promoting properties when used as a substrate, opening new avenues for printed CNS grafts in regenerative medicine.

Cytotoxicity evaluation of three light-cured dentin adhesive materials on human gingival fibroblasts, ex vivo

To evaluate the cytotoxic effects of three current light-cured dentin adhesives, in both uncured and post-cured conditions, on human gingival fibroblasts. The materials tested were Heliobond, Adper Single Bond 2 and Xeno V, which are characterized by various compositions and application procedures. Each agent, in volumes of 5 and 10 μL, was tested after polymerization, and those unpolymerized were diluted in DMEM to 10-3 and 10-5. The cytotoxicity of the adhesives was assessed on the basis of a test of cell viability in a culture of human gingival fibroblasts, with the use of tetrazolic salt (MTT assay). The results showed that, among the adhesive/bonding systems tested, Xeno V was the least cytotoxic. There were statistically significant differences in cell survival between polymerized Xeno V, Adper Single Bond 2 and Heliobond in the amount of 5 μL as well as between the Xeno V and Adper Single Bond 2 in 10-5 dilutions. The tested adhesives were more toxic in the polymerized form than in the dilutions. Samples of 10 μL resulted in a lower survival percentage of fibroblasts compared to 5 μL. All the tested adhesives demonstrated cytopathic effects towards human gingival fibroblasts, but varied in their cytotoxicity. This has clinical implications. Dentists should follow the rules of adhesive application, precisely dose them and not allow direct contact with the gums as, even after polymerization, adhesive agents exhibit potential cytotoxic activity.

Abstract B68: Differential gene expression associated with temozolomide resistance development in glioblastoma xenografts.

Abstract Purpose/Background: The first-line chemotherapeutic temozolomide (TMZ) does not offer any therapeutic benefit in some cases of glioblastoma (GBM) and emergence of resistance leads to tumor progression in almost all patients. While the expression of O-6-methylguanine-DNA-methyltransferase (MGMT) correlates well with TMZ resistance, its expression varies among GBM tumors. The goal of this study is to identify additional genes with potential roles in TMZ resistance. Materials and Methods: Five primary GBM xenografts were generated from patient samples, and subsequent secondary models for resistance were developed by subjecting tumor-bearing mice to escalating doses of TMZ. Triplicate samples from parental and TMZ-resistant xenografts were analyzed for gene expression using the Illumina BeadChip microarray platform. The top 100 differentially expressed genes for each line were considered candidates. Genes were selected for qRT-PCR validation after ontological and pathway analysis. A lentivirus-mediated shRNA knockdown was performed to validate the role of several candidate genes in TMZ resistance in U87 and primary GBM12 cell lines. Knockdown was confirmed with qRT-PCR and western blots. TMZ responsiveness was assessed using CyQUANT and Neurosphere assays. Results: Upregulated MGMT expression was found in 3 of the 5 xenografts (GBM12TMZ, GBM14TMZ and GBM28TMZ) and was mechanistically linked with TMZ resistance in these lines using O6-benzylguanine to reverse the resistance phenotype. Differential expression of other DNA damage response genes included CCND2, CDKN1A, GADD45B, UBB as well as apoptosis regulators NFKB2 and BIRC3. In lines with unchanged MGMT expression (GBM22TMZ and GBM39TMZ), changes in anti-apoptosis genes NFKB2 and MAP3K1, transcription factor SRF and signal transduction gene PRKCDBP were validated by qRT-PCR. In GBM12TMZ, in addition to MGMT upregulation, expression of Ubiquitin B (UBB) was found to be the most significantly down-regulated gene both on microarray (Log2FC = −4.55, Fold change = 0.043) and qRT-PCR (Log2RQ =-4.04, RQ = 0.061). To test a possible role of UBB in TMZ resistance, knockdown was achieved in U87 (knockdown efficiency=91.2%) and in primary xenograft line GBM12 (knockdown efficiency=84.4%). The CyQUANT assay for U87UBB-KD and U87NT treated with escalating doses of TMZ revealed no significant difference in cell survival (10 M TMZ relative survival= 89.7% for U87UBB-KD and 79.2% for U87NT; p-value=0.53). Similarly, a neurosphere assay for GBM12UBB-KD and GBM12NT also yielded no significant difference in cell survival (10 M TMZ relative survival= 25.13% for GBM12UBB-KD and 23.35% for GBM12NT; p-value=0.83). Conclusions: We have used a TMZ resistance xenograft model to identify candidate TMZ resistance genes. The current data provides evidence on the role of MGMT in the development of acquired TMZ resistance while ruling out UBB as potential TMZ resistance gene in GBM. Additional studies are underway to assess the functional significance of other candidate genes, particularly those with differential expression in the setting of constant MGMT expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B68.