Regular Growth Medium Research Articles

Abstract 6512: Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines

Abstract Cancer cells are metabolically distinct from normal cells, and can adapt differently to adverse growth conditions. We sought to compare normal MCF10A and Ras-transformed MCF10AT cells under various conditions (regular growth media, doxorubicin-treatment, and glucose-deprived media) by imaging endogenous cellular FAD and NADH fluorescence and comparing it to proliferation rates (measured by MTS assay) and chromatin structure (measured by Hoescht staining). Under normal growth conditions, MCF10AT cells displayed similar levels of FAD and higher levels of mitochondrial NADH than MCF10A cells. Elevated NADH in MCF10AT cells seems to reflect the preference for anaerobic glycolysis rather than oxidative phosphorylation (the so-called Warburg effect). Our MTS data showed that doxorubicin treatment did not significantly affect MCF10AT cell proliferation but led to a modest reduction in cell proliferation in the normal cell line. Likewise, in MCF10AT cells, doxorubicin treatment led to an increase in FAD and a decrease in NADH. Conversely, in MCF10A cells, doxorubicin led to a decrease in FAD levels and an increase in mitochondrial NADH. These results suggest that the cancer line may be switching to oxidative phosphorylation to resist doxorubicin-induced toxicity, while the normal cells reduce oxidative phosphorylation with growth inhibition. In contrast to the doxorubicin response, glucose deprivation produced significant growth inhibition and cell death in MCF10AT cells while having a modest growth-inhibitory effect on the normal line. Interestingly, low glucose decreased FAD and mitochondrial NADH in MCF10AT cells, suggesting that both aerobic and anaerobic respiration was reduced and consistent with the marked reduction in observed cell viability. In contrast, MCF10A cells showed a slight decrease in FAD and an increase in NADH, suggesting a decrease in oxidative phosphorylation consistent with the modest growth inhibition observed. Finally, MCF10ATs displayed higher levels of Hoechst staining than MCF10A cells, consistent with a higher proportion of condensed chromatin observed in cancer cells. In addition, we found a larger increase in Hoechst staining in MCF10AT cells in response to both doxorubicin and low glucose, as compared to MCF10A cells. This data suggests that the chromatin state of the two lines is also differentially affected by these treatments. In conclusion, label-free measurement of autofluorescence of cellular FAD and mitochondrial NADH reveals the different metabolic and chromatin responses to adverse growth conditions between cancer and non-cancer cells. These differences are associated with the different proliferation rates and may have important clinical implications for cancer risk stratification. Citation Format: Katherine Junkins, Angel Perez Martinez, Margaret Rodgers, Shelley A. Phelan, Min Xu. Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6512.

Actinoalloteichus cyanogriseus: a broad spectrum bio-agent against mulberry root rot pathogens

BackgroundIn India, severe yield and economic losses in sericulture were reported due to mulberry root rot disease. Since the disease was caused by complex soil-borne pathogens with wide host range, an attempt was made to develop actinobacteria based biological management strategy to mitigate it.ResultsAs a result of continuous screening for anti-fungal actinobacteria, 2 novel and potent isolates (M11 and M12) belong to the rare genus, Actinoalloteichus were obtained from mulberry rhizosphere. Both isolates were gram positive, grayish, aerobic and produced good amount of melanin pigment during sporulation. They exhibited excellent anti-fungal activity against mulberry root rot pathogens viz., Macrophomina phaseolina, Lasiodiplodia theobromae, Fusarium solani, Sclerotium rolfsii and Rhizoctonia solani. In addition, these melanogenic isolates grew in wide temperature and pH ranges. Their multi-stress tolerance ability/polyextremophilic nature was exposed when amending regular growth medium with excess concentration of salt and alkali. In addition, the potent isolates also showed tolerance to carbendazim (up to 1000 ppm) and UVB irradiation (up to 180 min at 10 cm distance).ConclusionThe present study focused on the identification and characterization of melanogenic actinobacteria, further made it potential candidate that could be incorporated in eco-friendly, integrated disease management strategies against complex mulberry root rot pathogens.

Abstract P5-09-04: Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy

Abstract Background: Breast cancer (BC) is one of the most diagnosed tumors in the United States. The mechanism of the disease development and progression is complex and still not fully understood. Dysregulation or change in the expression of transcription factors has been proposed as a possible cause of the disease severity and poor prognosis. The INhibitor of Growth family member 4 (ING4) is a tumor suppressor that has been connected to various cancer-related processes. Therefore, this study aims to assess the global gene regulation of BC cells by ING4 and identify the associated cellular processes and molecular pathways related to BC following ING4 loss. Methods: siRNA Knockdown and CRISPR Knockout technologies were used to deplete ING4 expression. Real-time PCR, RNA-Seq and Chip-Seq were used to identify gene expression alteration and ING4 enrichment on target promoters. Ingenuity Pathways Analysis (IPA®) was used to highlight candidates, networks and cellular processes. Results: Protein and RNA levels of ING4 were significantly reduced in MDA-231 knockdown and MCF7 knockout cells using siRNA and CRISPR technologies. RNA-Seq of BC cells revealed a large set of differentially expressed genes were regulated following ING4 loss. Key genes and biological networks related to cellular processes, including amino acid metabolism, cancer, cellular development, cellular growth and proliferation, were identified using IPA®. Chip-seq also revealed that ING4 was recruited to genes important in BC development and progression. Several genes were validated using the RT-qPCR method, and results were consistent with RNA-seq and Chip-seq data. Importantly, MDA-231 cells grown in stripped serum have shown an upregulation of the ribosomal protein S6 phosphorylation (PS6), an mTOR target protein, following ING4 knockdown. Treatment of MDA-231 cells with rapamycin blocked the phosphorylation of S6 induced by ING4 loss. An increase in S6 phosphorylation was also observed in MCF7 knockout cells grown either in regular or serum-free growth medium. The phosphorylation was reversed by amino acid transporter inhibitors JPH and BCH. Conclusion: The present study shows that ING4 regulates many genes involved in biological pathways and networks related to BC development and progression. Importantly, ING4 is a novel regulator of the mTOR pathway. Collectively, regulation of ING4 gene expression and its downstream molecules provides a novel approach in cancer treatment. Citation Format: Aymen Shatnawi, Travis Salisbury. Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-04.

The combination of TRAIL and the Smac mimetic LCL-161 induces an irreversible phenotypic change of MCF-7 breast cancer cells

IntroductionBreast cancer is the most common malignancy affecting women. Although the prognosis generally is good, a substantial number of patients still suffer from relapse, emphasizing the need for novel treatments. Smac mimetics were developed to facilitate cell death by blocking inhibitor of apoptosis proteins (IAPs). It has been suggested that TNF-related apoptosis inducing ligand (TRAIL) can be used together with Smac mimetics to induce cancer cell death. MethodsCell viability was studied with Trypan blue staining and Annexin V assay, siRNA was used to downregulate specific proteins, protein levels were estimated with Western blot, and mRNA levels were analyzed with qPCR, microarray and RNA-seq. For global expression, groups were compared with principal component analysis and the limma package in R. Gene enrichment was analyzed with Fisher's test. For other experiments, significance of difference was tested by one-way ANOVA, followed by Tukey's HSD test. ResultsThe combination of Smac mimetic LCL-161 and TRAIL induces an irreversible change in phenotype, but not cell death, of luminal MCF-7 breast cancer cells. The cells become small and circular and dissociate from each other and the effect could not be reversed by returning the cells to regular growth medium. The morphology change could be prevented by caspase inhibition using z-VAD-FMK and downregulation of caspase-8. Caspase-7 is also indicated to be of importance since downregulation of this caspase resulted in fewer morphologically changed cells. Enrichment analyses of changes in global gene expression demonstrated that genes associated with estrogen receptor (ER) signaling are downregulated, whereas nuclear factor kappa B- (NF-κB) and interferon- (IFN) driven genes are upregulated in altered cells. However, inhibition of these pathways did not influence the change in morphology. Induction of IFN-induced genes were potentiated but NF-ĸB-driven genes were slightly suppressed by caspase inhibition. ConclusionsThe results demonstrate that LCL-161 and TRAIL can irreversibly alter the MCF-7 breast cancer cell phenotype. However, the changes in morphology and global gene expression are mediated via separate pathways.

Long-term migratory velocity measurements of single glioma cells using microfluidics.

Microfluidic platforms enabling single-cell measurements notably contribute to the identification and observation of rare cancer cells that are involved in tumor invasion. Most aggressive, invasive, and heterogeneous glioblastoma cells cause incurable primary brain tumors. Infiltrating gliomas of a brain tumor microenvironment have been intensively studied using conventional assays. Still, quantitative, simple, and precise tools are required for long-term, steady-state migratory-velocity measurements of single glioma cells. To measure long-term velocity changes and investigate the heterogeneity of glioma cells under different growth conditions, we developed a microfluidic platform. We cultured U87 glioma cells in the microfluidic device using either regular growth medium or conditional medium composed of 50% basal medium and 50% macrophage-depleted medium. We microscopically monitored the behavior of 40 glioma cells for 5 days. Using acquired images, we calculated cellular circularity and determined the migratory velocities of glioma cells from 60 h to 120 h. The mean migratory velocity values of the glioma cells were 1.513 μm h-1 in the basal medium and 3.246 μm h-1 in the conditional medium. The circularity values of the glioma cells decreased from 0.20-0.25 to 0.15-0.20 when cultured in the conditional medium. Here, we clearly showed that the glioma cells lost their circularity and increased their steady-state velocities; in other words, they adopted an invasive glioma phenotype in the presence of macrophage-depleted medium. Besides, the heterogeneity of the circularity and the velocity of glioma cells were enhanced in the conditional medium.

Single-Cell Mechanophenotyping in Microfluidics to Evaluate Behavior of U87 Glioma Cells.

Integration of microfabricated, single-cell resolution and traditional, population-level biological assays will be the future of modern techniques in biology that will enroll in the evolution of biology into a precision scientific discipline. In this study, we developed a microfabricated cell culture platform to investigate the indirect influence of macrophages on glioma cell behavior. We quantified proliferation, morphology, motility, migration, and deformation properties of glioma cells at single-cell level and compared these results with population-level data. Our results showed that glioma cells obtained slightly slower proliferation, higher motility, and extremely significant deformation capability when cultured with 50% regular growth medium and 50% macrophage-depleted medium. When the expression levels of E-cadherin and Vimentin proteins were measured, it was verified that observed mechanophenotypic alterations in glioma cells were not due to epithelium to mesenchymal transition. Our results were consistent with previously reported enormous heterogeneity of U87 glioma cell line. Herein, for the first time, we quantified the change of deformation indexes of U87 glioma cells using microfluidic devices for single-cells analysis.

Osteogenic Potential of Periodontal Ligament Stem Cells Cultured in Osteogenic and Regular Growth Media: Confocal and Scanning Electron Microscope Study

To evaluate the ability of osteogenic culture media in comparison with regular growth culture media in enhancing the osteoblastic cell differentiation of human periodontal ligament stem cells (hPDLSCs). In vitro cultures of commercially obtained hPDLSCs were seeded onto xenograft bone blocks in both regular and osteogenic media. Confocal laser microscope images were obtained for cellular differentiation and adhesion, and scanning electron microscopy (SEM) images were obtained to validate the osteogenic differentiation by showing the morphological characteristics of the newly formed cells. Confocal laser microscope analysis showed positive staining for new bone cells with an increased signal intensity when samples were cultured in osteogenic culture media compared with regular culture media. These findings indicate the effect of the active ingredients of the osteogenic culture media in enhancing the osteogenic differentiation hPDLSC. Scanning electron microscopy images validated the osteogenic differentiation showing a flattened, polygonal morphology with multiple extending cytoplasmic processes of new cells. Xenograft bone blocks are biocompatible scaffold for the osteogenic differentiation of seeded hPDLSCs. Osteogenic culture media enhances and increases the osteogenic differentiation of hPDLSCs into new bone cells more than regular growth culture media. Periodontal ligament stem cells are a predictable biological input as a cell-based tissue-engineered construct and biologically acceptable when it is cultured in a suitable growth media that mimics the intended environment. Consideration of the clinical use of equine bone blocks and periodontal ligament stem cells in a suitable biological environment as a potential new option for bone regeneration techniques.

Strategic growth of limnic green microalgae with phycoremediation potential for enhanced production of biomass and biomolecules for sustainable environment.

The approach of combining the microalgae cultivation with wastewater provides a cost-effective and eco-friendly perspective in the production of microalgae-based bio-products. In the present investigation, microalgae Scenedesmus rubescens KACC 2 isolated from catchment region of River Noyyal was found to be efficient in removing nitrogen, phosphorus, and heavy metals from industrial and domestic effluents, which was optimized through central composite design matrix for higher biomass generation. Nutrient requirements for the growth were optimized and evaluated using Plackett-Burman design to check the effect of variables. Three variables, viz., nitrate, phosphate, and inoculums, were found to be significant among the 11 variables tested, and the interaction between these variables and its optimum concentrations were statistically studied using central composite design matrix. The optimized growth conditions of this strain were found to be as nitrate (0.2%), phosphate (0.018%), and inoculums (7.5%). These conditions yielded a higher biomass of 0.73g/L from the optimized media which was 5.4 times higher than the regular growth media. FT-IR analysis showed the variations in the spectra and also in biomolecular composition with 2-fold increase in the lipid and protein region when grown in optimized culture conditions. Lipid profile showed the presence of saturated and monounsaturated fatty acids in the biomass accepting it as a source of energy feedstock. This study concludes that nitrate, phosphate, and inoculums play a significant role in biomass production of S. rubescens with phycoremediation potential that can be exploited for simultaneous wastewater treatment-coupled biomass production.

Influence of media supplements on inhibition of oxidative browning and bacterial endophytes of Camellia sinensis var. sinensis.

Explant oxidative browning and necrosis of Camellia sinensis var. sinensis is a severe problem in tissue culture, often associated with the exuded phenolic compounds and microbial contamination from the explants. In this study, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase (PAL), and different antibiotics were tested to control tea explant necrosis and browning. These compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron at different concentrations. Our data indicated that application of 2µM of AIP was able to effectively inhibit callus browning, significantly reduce EGC abundance, and greatly improve callus induction and growth. Moreover, the use of 150mg/L of timentin and 30mg/L gentamycin resulted in an effective elimination of the surface and endophytic microbes associated with explants of C. sinensis var. sinensis. Our study revealed that the inhibition of PAL using AIP combined with the two tested antibiotics could open up new doors to control oxidative tissue browning and endophyte contamination in tissue culture for tea genetic manipulation.

Hierarchically aligned fibrillar fibrin hydrogel designed for spinal cord injury repair

Event Abstract Back to Event Hierarchically aligned fibrillar fibrin hydrogel designed for spinal cord injury repair Xiumei Wang1* and Shenglian Yao1* 1 Tsinghua University, School of Materials Science and Engineering, China Biomaterial-based strategies for nerve regeneration have attracted extensive attentions in recent years. In this study, a hierarchical aligned fibrillar fibrin hydrogel was prepared through electrospinning technique and the concurrent molecular self-assembly process at the moment of collection. The aligned fibrin hydrogel that simulated the elasticity and oriented structures of nerve tissue simultaneously provided biomimetically biophysical cues to direct in vivo or in vitro cells thus constructing nerve-like microtissues rapidly. In vitro studies showed that human umbilical cord mesenchymal stem cells could “feel” the cues afforded by aligned fibrin gels and extend along the long axis of the fibers soon after cell attachment and undergo strong neural differentiation after two weeks cell culture in regular growth medium without any soluble growth factors. More interestingly, the rat dorsal root ganglion neurons also could follow the fiber structure in a highly oriented longitudinal manner with three-dimensionally neurite elongation to total 3.92 mm just in 3 days basic culture. In rat T9 dorsal hemisection spinal cord injury. the host neural cells with β-tubulin III-positive migrated into the aligned fibrin hydrogels very quickly. And the axon reconstruction and locomotor performance was improved starting from 2 weeks after the surgery in the aligned fibrin hydrogel group. Our study suggested that the hierarchically aligned fibrillar fibrin hydrogels will not only have great promising for directing stem cells towards nerve lineage, but also may have great potential applications in spinal cord regeneration. Tsinghua University Initiative Scientific Research Program (20121087982, 20131089199); 973 Program (No. 2011CB606205)

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells.

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

The Extracellular Calcium-Sensing Receptor as a Contributor in Human Cartilaginous Endplate Degeneration

Introduction Intervertebral discs (IVDs) are virtually avascular tissue relying on the diffusion of nutrients and metabolites from blood vessels in the outer annulus fibrosus, and in the vertebral bodies that partially penetrate the subchondral plate, and terminate at the cartilaginous endplate (CEP). Degeneration of CEP is viewed as a catalyst to IVD degeneration, as it impedes nutrient diffusion to the disc. Similar to articular cartilage, degeneration of CEP involves decreased proteoglycan and type II collagen (Col II) content, thinning, increased type X collagen (Col X), and advanced calcification. Although biological repair strategies of the degenerative disc involving supplementation of growth factors are being pursued, disc nutrition is an important factor in considering their success in disc repair. The extracellular calcium-sensing receptor (CaSR) is a G protein-coupled receptor, and currently, is the only known mechanism in sensing both local and systemic calcium fluctuations. CaSR is the principle regulator of parathyroid hormone synthesis and secretion from the parathyroid gland, and in addition to its expression in other calcium-handling tissues (intestine, kidney), evidence suggests a direct involvement in bone and cartilage development. Recently, CaSR has been shown to modulate vascular calcification; however, its function in the calcification of other tissues remains unknown. Objectives Calcification of CEP is suggested to be a catalyst in the degeneration of IVDs. However, the mechanism(s) of CEP degeneration are currently unknown. We propose that CaSR activation modulates the degeneration of CEP, by increasing the expression of catabolic factors in CEP chondrocytes, resulting from the accumulation of ionic calcium in the milieu. Materials and Methods Human CEPs were obtained from donor IVDs of Thompson grades 2, 3, and 4 through organ donations within 24 hours of death. CEP chondrocytes were isolated from tissue by sequential digestion with pronase followed by collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on CEP tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting, and MAPK signaling were performed on cultured CEP chondrocytes of various grades to demonstrate expression of CaSR and degenerative markers, including the function of the receptor. CEP cells of various grades were cultured in medium containing either high calcium (5 mM Ca2+), CaSR allosteric agonist (cinacalcet, 1 µM), or regular growth medium (control, 1 mM Ca2+) for 7 days, and monitored for changes in markers of degeneration and CaSR expression. Results Ionic calcium content in CEP tissue increased significantly with IVD degeneration (Fig. A). Interestingly, immunohistochemistry on CEP tissue also revealed an increase in the expression of CaSR that paralleled the degree of IVD degeneration. Immunocytochemistry and Western blotting on isolated CEP cells demonstrated an upregulation of CaSR (Fig. B) and type X collagen that varied with Thompson grades. The expression of matrix metalloproteinase (MMP)-13 was also increased with degeneration; however, the expression of Col II and the proteoglycan aggrecan (Agg) were inversely related to Thompson grade. The magnitude of CaSR signaling in CEP cells when stimulated with 5 mM Ca2+, as determined by ERK phosphorylation, was significantly enhanced in grade 4 versus grade 2 CEP chondrocytes. To determine a role of CaSR in the degeneration of CEP, we cultured Thompson grade 2 CEP chondrocytes in 1 mM Ca2+ (control medium), 5 mM Ca2+ or cinacalcet (allosteric CaSR agonist, 1 µM) for 7 days. Western blot analysis on lysate from G2 CEP cells treated with 5 mM Ca2+ or cinacalcet demonstrated significant increases in the expression of Col X and MMP-13 (Figs. D and E), however, decreased expression of Col II and Agg was observed (Fig. C). Conclusion CaSR has been described as a mediator in ectopic calcification, and its expression is upregulated in CEP of degenerative discs. We provide evidence that CaSR upregulation may be a hallmark of CEP calcification. Activation of CaSR either by its agonist, calcium or cinacalcet, lead to increases in the synthesis of degenerative factors (Col X and MMP-13), and decreases in the production of matrix proteins (Col II and Agg). An understanding on the mechanism(s) of CEP degeneration is key when considering growth factor substitution in IVD repair, as disc nutrition may be an important factor in determining their success. Disclosure of Interest None declared References Lotz JC, Fields AJ, Liebenberg EC. The role of the vertebral end plate in low back pain. Global Spine J 2013;3(3):153–164 Brown EM. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract Res Clin Endocrinol Metab 2013;27(3):333–343

Proteolytic Processing of the Extracellular Scaffolding Protein LEV-9 Is Required for Clustering Acetylcholine Receptors

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.

Serum resistance, gallium nitrate tolerance and extrapulmonary dissemination are linked to heme consumption in a bacteremic strain of Acinetobacter baumannii

Bacteremia caused by Acinetobacter baumannii is a highly lethal complication of hospital-acquired pneumonia. In the present study, we investigated the serum resistance, gallium nitrate tolerance and heme consumption of A. baumannii strain LAC-4 which was recently reported to display high virulence in a mouse pneumonia model with extrapulmonary dissemination leading to fatal bacteremia. This strain showed enhanced growth in mouse and fetal bovine serum that was independent of complement and was not observed with regular growth media. The LAC-4 strain was found to possess a high tolerance to gallium nitrate (GaN), whereas serum synergized with GaN in inhibiting A. baumannii strain ATCC 17978. We found that LAC-4 contains a heme oxygenase gene and expresses a highly efficient heme consumption system. This system can be fully blocked in vitro and in vivo by gallium protoporphyrin IX (GaPPIX). Inhibition of heme consumption by GaPPIX completely abrogated the growth advantage of LAC-4 in serum as well as its tolerance to GaN. More importantly, GaPPIX treatment of mice intranasally infected with LAC-4 prevented extrapulmonary dissemination and death. Thus, we propose that heme provides an additional source of iron for LAC-4 to bypass iron restriction caused by serum transferrin, lactoferrin or free gallium salts. Heme consumption systems in A. baumannii may constitute major virulence factors for lethal bacteremic isolates.

Characterization of small spheres derived from various solid tumor cell lines: are they suitable targets for T cells?

T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion.

Oxygen Relieves the CO2 and Acetate Dependency of Lactobacillus johnsonii NCC 533

Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO2 during growth. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas phase. Switching off the CO2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO2 dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO2 requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO2 and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO2 dependency.

Cytotoxicity of Local Anesthetics on Human Mesenchymal Stem Cells

Local anesthetics are frequently delivered intra-articularly to provide perioperative pain control. Previous studies have shown that the commonly used drugs lidocaine, ropivacaine, and bupivacaine can be toxic to human chondrocytes. The present study was conducted to determine whether the toxic effects of local anesthetics on human chondrocytes also extend to human mesenchymal stem cells. Human mesenchymal stem cells from three healthy donors were grown in tissue culture and exposed to the following anesthetic treatments for sixty minutes: (1) 1% lidocaine, (2) 2% lidocaine, (3) 0.25% bupivacaine, (4) 0.5% bupivacaine, (5) 0.2% ropivacaine, and (6) 0.5% ropivacaine. The cells were then allowed to recover for twenty-four hours in regular growth media, and viability was measured with use of fluorescent staining for live cells or a luminescence assay for ATP content. The live cell counts and ATP content were correlated (r2 = 0.79), and 2% lidocaine was found to be significantly more toxic than all doses of bupivacaine and ropivacaine. Treatment with 1% lidocaine resulted in significantly fewer live cells (49%) compared with the control, and the live cell count was also significantly less than that for the other anesthetics. However, the ATP level in the 1% lidocaine group was not significantly lower than those in the other groups. Bupivacaine and ropivacaine did not exhibit significant differences in toxicity compared with the control or with each other. Ropivacaine and bupivacaine had limited toxicity in human mesenchymal stem cells. However, lidocaine could significantly decrease mesenchymal stem cell viability. Since other studies have shown ropivacaine to be less toxic to chondrocytes than bupivacaine, ropivacaine may be a safer intra-articular anesthetic. Mesenchymal stem cells likely play a key role in healing following surgical procedures such as microfracture and ligament reconstruction. If local anesthetics are used following joint surgery, selection of an agent with low toxicity toward mesenchymal stem cells, such as ropivacaine, may maximize tissue healing potential.

Abstract 497: H2O2 in Fumarase Insufficiencies in Dahl Salt-Sensitive Rats.

We have shown previously that renal insufficiencies of fumarase, an enzyme in the tricarboxylic acid cycle, appear to contribute to salt-induced hypertension in Dahl salt-sensitive (SS) rats. The present study examined the molecular mechanism underlying fumarase insufficiencies in SS rats. Protein abundance and enzymatic activity of fumarase was significantly lower in the renal medulla of SS rats compared to salt-insensitive Sprague-Dawley rats. We screened several factors including TGFβ1, TNFα, and H 2 O 2 for their effects on fumarase expression. These factors were selected for screening because they had been reported to be elevated in the kidneys of the SS rat and they represent a pro-fibrotic, pro-inflammatory, and oxidative milieu present in the kidneys of the SS rat. Cultured human renal epithelial cells were treated with TGFβ1 (3 ng/ml), TNFα (25 ng/ml), or H 2 O 2 (500 μM) for 24 hours. H 2 O 2 substantially reduced fumarase mRNA levels (normalized by 18S rRNA) to less than 10% of control. TNFα tended to suppress fumarase expression, but the effect did not reach statistical significance. TGFβ1 did not have significant effects on fumarase mRNA levels. The greater than 90% loss of fumarase following the H 2 O 2 treatment was accompanied by significant cell death. To determine if the down-regulation of fumarase was simply a consequence of the cytotoxicity of H 2 O 2 , we treated HRE cells with 100 μM of H 2 O 2 for 48 hours. The lower dose of H 2 O 2 did not cause significant cell death. Yet, fumarase mRNA was still down-regulated to 79.6% ± 4.7% of control (n=4, P<0.05 vs. control). Moreover, we repeated the treatment in cells cultured in the Dulbecco’s Modified Eagle Medium (DMEM) instead of the regular epithelial growth medium. HRE cells cultured in DMEM were highly resistant to the cytotoxicity of H 2 O 2 . H 2 O 2 (500 μM) did not cause any significant cell death in HRE cultured in DMEM, yet it again significantly suppressed fumarase expression to 77.1% ± 6.6% of control (n=7-8, P<0.05 vs. control). The result suggests that elevated levels of H 2 O 2 in the renal medulla might contribute to fumarase insufficiencies in SS rats.

Abstract A25: Genome-wide methylation analyses in advanced-stage prostate cancer models

Abstract Introduction: Aberrant DNA methylation is a frequent event in prostate cancer (PCa). Investigation of genome-wide methylation changes in PCa promises to provide novel biomarkers that discriminate between indolent and aggressive diseases and to facilitate the development of novel treatment options. Most methylation studies to date have focused on CpG islands (CGI) in the promoter region of known genes. The current study investigated genome-wide methylation changes with the aim to identify recurrent regions differentially methylated in advanced PCa, including metastases and castration-resistance diseases. Methods: Using comprehensive high-throughput array-based relative methylation analysis (CHARM), we assessed genome-wide methylation status in metastatic PCa represented by two PCa cell lines (LNCap and VCap) as well as castration-resistant cancer represented by PCa xenografts cultivated in castrated mice (LuCaP23.1v, 35v, and 96v) and APIPC cells derived from AR-null LNCap maintained in hormone depleted media. To identify differentially methylated regions (DMR), each metastatic PCa cell line was compared with benign prostate epithelial RWPE1 cells. The castration-resistant xenografts were compared with the corresponding castration-sensitive xenografts, LuCaP23.1, 35, and 96, respectively. APIPC was compared with the parent line maintained in regular growth media. Unlike most methylation analyses that only target promoter CGI, CHARM analyses target CpG enriched regions of the entire genome, including genomic sequences up to 2kb outside of promoter CGIs, “CpG island shores”. The shores have been recently demonstrated to harbor more methylation alterations than the islands in tissue differentiation and cancer development. Results: Methylation changes were frequent in PCa metastases and castration-resistant diseases. Using RWPE1 as the reference, 306 DMRs were identified in LNCap (114 hyper- and 192 hypo-methylated), while 128 DMRs (32 hyper- and 96 hypo-methylated) were discovered in VCap cells. Compared with the parent LNCap line, APIPC showed 284 DMRs (80 hyper- and 204 hypomethylated). Using each corresponding castration-sensitive xenograft as the reference, castration-resistant LuCaP23.1v, LuCaP35v, and LuCaP96v demonstrated 587 (378 hyper- and 209 hypo-methylated), 150 (17 hyper- and 133 hypo-methylated), and 761 (353 hyper- and 408 hypo-methylated) DMRs, respectively. Distribution analyses showed that DMRs associated with these advanced PCa are enriched in regions 500bp outside CpG islands, i.e., CpG island shores, rather than in the islands. As expected, recurrent DMRs were evident. LNCap and VCap, representing PCa metastases, demonstrated 20 DMRs in common. Two of these common DMRs were of special interest: one is located at the 3′ UTR of STAT6, the expression of which was previously shown to correlate with high histological grades of prostate cancer and with tumor size; another DMR is located at intron 1 of HOXA9 within the CGI shore. Aberrant methylation of HOXA9 promoter CGI was reported in multiple cancers such as bladder and non-small cell lung cancer. Among those representing castration-resistant PCa, 11 DMRs were shared between APIPC and LuCaP23.1v, while 2 DMRs were in common between APIPC and LuCaP96v. Conclusion: CHARM studies in advanced prostate cancer enabled us to better understand methylation changes at the whole genome level. Our data confirmed the importance of CpG enriched regions outside the promoter CGI during the development of cancer, specifically, PCa metastases and castration-resistant diseases. Multiple novel recurrent DMRs were identified in the study, which call for further investigation of their role in primary PCa as well as the related changes in gene expression and histone modification, and the prognostic significance of those changes. Citation Format: Xiaoyu Qu, Jerry Davison, Eric Bluemn, Peter Nelson, Robert Vessella, Min Fang. Genome-wide methylation analyses in advanced-stage prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A25.

A putative in vitro organotypic model of molting with human skin explants

We report finding a simple method to partially reproduce the characteristic process of molting that takes place in invertebrates using human skin explants in vitro. In this method, human skin explants discarded from regular plastic surgery procedures were kept, submersed, in regular growth medium for 10 days at 4°C. After that period, the skin explants were cultured at the air-liquid interface for another 10 days. Histological analysis of the skin revealed the formation of one full epidermal structure and an additional intermediate epidermal structure containing a putative stratum corneum, superimposed one of top of the other, in which we consider an equivalent model of "molting" or "ecdysis". Basic analysis of cell proliferation and differentiation of the explants at different stages of the process are briefly presented. We believe this model can be used in the study of certain human skin diseases as well as in comparative animal physiology.