Plating Efficiency Of Cells Research Articles

Agar lot-specific inhibition in the plating efficiency of yeast spores and cells

Abstract The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single-celled, model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our three laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar were toxic. We report that this sporadic toxicity affects independently the agar stocks of multiple vendors, has occurred repeatedly over at least three decades, and extends to species in highly diverged taxa. Interestingly, the inhibitory effects displayed variable penetrance and were attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Abstract A006: Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells

Abstract Introduction: Nearly all the patients receiving hormone treatment eventually progress into castration resistance stage. In addition, approximately 30% to 40% of patients receiving radiation treatment will develop resistance. The aberrant alterations of androgen receptor (AR) and transforming growth factor-β (TGF-β) signaling play critical roles in such processes. Our studies identified five PMEPA1 (Prostate Transmembrane Protein Androgen Induced 1) gene isoforms in prostate cancer cells. PMEPA1-b isoform was androgen responsive and inhibited androgen signaling. In contrast, PMEPA1 isoforms a and d were TGF-β inducible and suppressed TGF-β signaling. Loss or reduced expressions of PMEPA1 was detected in over 60% of prostate cancer samples. In this study, we investigated the impacts of PMEPA1 isoforms (a, b and d) on the development of hormone and radiation therapies resistance in prostate cancer. Methods: RNA-seq data of unmatched 50 benign and 499 malignant prostate samples from The Cancer Genome Atlas (TCGA) dataset, as well as 101 metastatic castration resistant prostate cancer (mCRPC) biopsy samples from Quigley lab at UCSF (WCDT dataset) were used to study the transcript levels of PMEPA1 isoforms (a, b and d) and their correlations to disease progression. Hormone dependent LNCaP cells and androgen independent PC-3, DU-145 cells were utilized to investigate the impacts of PMEPA1 isoforms (a, b and d) on the responses to of androgen inhibitors (enzalutamide and bicalutamide, 0, 5.0 and 25.0 nM) and radiation (0, 1.0 and 5.0 Gy). The vectors of pcDNA3.1-PMEPA1-a, -b and -d were transfected into prostate cancer cells via Lipofectamine, and endogenous PMEPA1 isoforms were knocked down by isoforms specific siRNAs. The cell growth was assessed by cell counting, BrdU incorporation and cell plating efficiency assays. The protein levels of AR and PSA were evaluated by immunoblotting. The transcript levels of PSA, NKX3.1, NEDD9 and THBS1 were assessed by RT-PCR technology. Results: The reduced transcript levels of PMEPA1 isoforms (a, b and d) were detected in mCRPC group. Lower expression of PMEPA1-b was significantly associated with earlier biochemical recurrence. The growth inhibition and androgen signaling blockage mediated by androgen inhibitors were explicitly reversed by depletion of PMEPA1-b in LNCaP cells. Ectopic PMEPA1-b in isoform b depleted LNCaP cells reinstituted the growth inhibition induced by androgen inhibitors. Knockdown of isoforms a and d conspicuously rescued the decreased cell proliferation and suppressed TGF-β signaling resulted from radiation treatment in PC-3 and DU-145 cells. Similarly, over-expression of PMEPA1-a and -d in siRNA treated cells recapitulated the growth inhibitory effects of radiation treatments. Conclusion: Loss of PMEPA1 gene isoforms (a, b and d) induced the development of resistance to hormone ablation and radiation treatments in prostate cancer cells in vitro through activating androgen and TGF-β signaling. Our research provided a new insight into treatment resistance mechanism of prostate cancer cells. Citation Format: Shashwat Sharad, Hua Li. Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A006.

Cover Feature: A Single‐Flow Battery with Multiphase Flow (ChemSusChem 4/2021)

Widespread adoption of redox flow batteries (RFBs) for renewable energy storage is inhibited by a relatively high cost of storage. This is due largely to typical RFBs requiring two flows, two external tanks, and expensive ion-exchange membranes. Here, we propose a potentially inexpensive Zn-Br2 RFB which is membraneless and requires only a single flow. The flow is an emulsion consisting of a continuous, Br2 -poor aqueous phase and a dispersed, Br2 -rich polybromide phase, pumped through the channel separating anode and cathode. With our prototype cell, we explore the effect of polybromide-phase volume fraction and Br2 concentration on cell performance and plating efficiencies. We demonstrate high discharge currents of up to 270 mA/cm2 , plating efficiencies up to 88 %, and dendriteless plating up to the highest Zn loading investigated of 250 mAh/cm2 . We provide mechanistic insights into cell behavior and elucidate paths towards unlocking ultra-low-cost single-flow RFBs with multiphase flow.

A Single-Flow Battery with Multiphase Flow.

Widespread adoption of redox flow batteries (RFBs) for renewable energy storage is inhibited by a relatively high cost of storage. This is due largely to typical RFBs requiring two flows, two external tanks, and expensive ion-exchange membranes. Here, we propose a potentially inexpensive Zn-Br2 RFB which is membraneless and requires only a single flow. The flow is an emulsion consisting of a continuous, Br2 -poor aqueous phase and a dispersed, Br2 -rich polybromide phase, pumped through the channel separating anode and cathode. With our prototype cell, we explore the effect of polybromide-phase volume fraction and Br2 concentration on cell performance and plating efficiencies. We demonstrate high discharge currents of up to 270 mA/cm2 , plating efficiencies up to 88 %, and dendriteless plating up to the highest Zn loading investigated of 250 mAh/cm2 . We provide mechanistic insights into cell behavior and elucidate paths towards unlocking ultra-low-cost single-flow RFBs with multiphase flow.

Performance degradation due to anodic failure mechanisms in lithium-ion batteries

We report a mechano-chemical model for anodic degradation during fast-charging of nickel-manganese-cobalt (NMC)/graphite (C) cell due to SEI growth, lithium plating/stripping, dead lithium storage, and film fracture of composite SEI and plated lithium film. Degradation of the battery is analyzed for a range of charging rates from 1 to 6 C-rates, and the influence of plating mechanisms – lithium plating and dead lithium deposition and recovery during stripping – on the film resistance of the anode are accounted for in the model. Dynamic evolution of the interfacial properties is modeled using rule-of-mixture approach. Model predictions of plating associated stress fields are used to compute critical energy release rate for film cracking. The results indicate an increased tendency of fracture for thinner SEI film with lithium plating at higher charging rates. The process of reforming the cracked film absorbs a significant portion of the electrode current thereby reducing the cell capacity and plating efficiency. The mechano-chemical model provides an extensive analytical framework for understanding the synergistic coupling of anodic degradation mechanisms, prognosticating conditions of SEI failure, and evaluating the capacity fade and efficiency of lithium-ion battery.

An experimental aerosol air–agar interface mouse lymphoma assay methodology

This work investigates a completely novel and experimental concept of exposing L5178Y cells at the air-agar-interface to mainstream cigarette smoke aerosol (Kentucky reference 3R4F). This study highlights the associated challenges of combining a suspension cell line alongside an in vitro aerosol exposure system. To achieve a monolayer, cells were ‘seeded’ in a concentrated cell super-mix suspension onto an RPMI/agar-matrix -base. The resulting cell suspension media was adsorbed into the agar base leaving the L5178Y cells lightly suspended on the agar surface, approximating a monolayer. Cells were deemed supportable on the agar-matrix, viable and recoverable. Using Vitrocell VC 10 exposure system and the Ames 4 exposure module, L5178Y cells were successfully exposed to a dynamic cigarette smoke aerosol, recovered and assessed for mutant frequencies, using standard assay procedures. Method development included assessment of flowing air conditions, plating efficiency and recovery of L5178Y cells from the agar-matrix surface. Positive controls MMS and B[a]P were successfully incorporated into the agar-matrix and metabolic activation was achieved by S-9 incorporation into the same agar-base-matrix. B[a]P demonstrated metabolic activation and positive response, suggesting a clear cellular interaction with the agar-matrix. Whole smoke exposed cells in the presence of metabolic activation showed a clear dose response and increasing mutant frequencies, well in excess of the controls (air and incubator) and the global evaluation factor following a 2 or 3 day expression period. This experimental concept demonstrates that L5178Y cells can be exposed to cigarette smoke aerosol, using a completely novel and a previously untested approach. Although this work successfully demonstrates the approach is viable and cells can be plated and maintained on an agar-matrix, more optimisation and robustness assessment is required before it can be considered fully adapted and used alongside other whole aerosol methodologies for the assessment of cigarette smoke and other inhaled aerosols.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Analysis of PMEPA1 Isoforms (a and b) as Selective Inhibitors of Androgen and TGF-β Signaling Reveals Distinct Biological and Prognostic Features in Prostate Cancer.

Dysfunctions of androgen/TGF-β signaling play important roles in prostate tumorigenesis. Prostate Transmembrane Protein Androgen Induced 1 (PMEPA1) inhibits androgen and TGF-β signaling via a negative feedback loop. The loss of PMEPA1 confers resistance to androgen signaling inhibitors and promotes bone metastasis. Conflicting reports on the expression and biological functions of PMEPA1 in prostate and other cancers propelled us to investigate isoform specific functions in prostate cancer (PCa). One hundred and twenty laser capture micro-dissection matched normal prostate and prostate tumor tissues were analyzed for correlations between quantitative expression of PMEPA1 isoforms and clinical outcomes with Q-RT-PCR, and further validated with a The Cancer Genome Atlas (TCGA) RNA-Seq dataset of 499 PCa. Cell proliferation was assessed with cell counting, plating efficiency and soft agar assay in androgen responsive LNCaP and TGF-β responsive PC3 cells. TGF-β signaling was measured by SMAD dual-luciferase reporter assay. Higher PMEPA1-a mRNA levels indicated biochemical recurrence (p = 0.0183) and lower PMEPA1-b expression associated with metastasis (p = 0.0173). Further, lower PMEPA1-b and a higher ratio of PMEPA1-a vs. -b were correlated to higher Gleason scores and lower progression free survival rate (p < 0.01). TGF-β-responsive PMEPA1-a promoted PCa cell growth, and androgen-responsive PMEPA1-b inhibited cancer cell proliferation. PMEPA1 isoforms -a and -b were shown to be promising candidate biomarkers indicating PCa aggressiveness including earlier biochemical relapse and lower disease specific life expectancy via interrupting androgen/TGF-β signaling.

Changes in the Proliferation Rate, Clonogenicity, and Radiosensitivity of Cultured Cells During and After Continuous Low-Dose-Rate Irradiation.

We investigated the effects of continuous low-dose radiation on proliferation, clonogenicity, radiosensitivity, and repair of DNA double-strand breaks (DSBs) in human salivary gland (HSG) tumor cells. Human salivary gland cells were cultured on acrylic boards above very-low-dose (4.3 μSv/h) or low-dose (27 μSv/h) radiation-emitting sheets or without sheets. Total cell numbers and plating efficiencies were compared among the 3 groups every 1 or 2 weeks until 6 weeks after starting culture. At 2, 4, and 6 weeks, surviving fractions of HSG cells after irradiation at 2 to 8 Gy cultured on the very-low-dose or low-dose sheets were compared to those of the control. At 4 weeks, HSG cells irradiated at 2 Gy were assessed for phosphorylated histone (γH2AX) foci formation, and DSBs were evaluated. No significant differences were observed in total cell number or plating efficiencies with or without low-dose-emitting sheets. The surviving fractions after irradiation of the very-low-dose group at 2 to 6 weeks and those of the low-dose group at 2 to 4 weeks were higher than those of the control (P < .01). Thus, a radioadaptive response was clearly demonstrated. From the γH2AX foci quantification, the adaptive responses were considered to be associated with the efficient repair of DSB, especially slow repair, in this cell line.

Abstract B008: PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer

Abstract Introduction and Objectives: The dysfunctions of androgen receptor (AR) and TGF-β signaling contribute to prostate tumorigenesis and cancer progression. Our laboratory has identified PMEPA1 as an androgen responsive gene with prostate abundance, as well as PMEPA1/NEDD4 and AR negative feedback loop in the regulation of AR levels in prostate cancer cells. In addition, it has also been established by other groups that PMEPA1 inhibits TGF-β signaling via a similar negative feedback loop as a TGF-β responsive gene. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. This study focuses on understanding of the expression and biologic functions of PMEPA1 isoforms in CaP. Methods: The PMEPA1 isoforms were evaluated for expressions in multiple CaP cell lines, trensfectant derivatives, and prostate tumors. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145, and PC-3 cells individually. Cell proliferation was analyzed by cell counting, cell plating efficiency assay, and soft agar assay. The protein levels of PMEPA1 isoforms, AR, PSA, TGF-β receptor I, Smad2 were detected by immunoblotting, and the transcript levels of KLK3 (PSA), TGF-β responsive genes including THBS1, NEDD9 and COL1A1 were evaluated by QRT-PCR. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay. The transcript levels of PMEPA1-252, PMEPA3-287-STAG1, AR, PSA in 120 matched human benign and malignant frozen tissue were evaluated with Q-PCR. Results: The expressions of PMEPA1-252 and PMEPA2-344 isoforms were restricted to androgen-responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-287/STAG1, PMEPA4-259 and PMEPA5-237). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1-252 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Only PMEPA1-252 mediated AR protein degradation and inhibited AR signaling. In contrast, PMEPA3-287-STAG1 and PMEPA4-259 inhibited TGF-β signaling luciferase activity and transcript levels of THBS1, NEDD9 and COL1A1. All PMEPA1 isoforms were found to have no effects on protein level of TGF-β receptor I. Moreover, the deletion mutants of N-terminus and transmembrane domains interrupted the isoform specific inhibitory effects on AR/TGF-β signaling. The transcript level of PMEPA1-252 was higher than PMEPA3-287-STAG1 in benign prostate tissue. Different from PMEPA1-252, the expression of PMEPA3-287-STAG1 was not found to decrease in prostate tumor tissue. There was no significant correlation between the expression of PMEPA1-252 and PMEPA3-287-STAG1 in prostate tissue. The transcript of PMEPA1-252 was associated with PSA, but the expression of PMEPA3-287-STAG1 was found not to be associated with PSA or AR in CaP cells. Conclusions: The PMEPA isoforms appear to underscore distinct biologic functions in the context of androgen and TGF-β signaling. Intensively studied PMEPA1-252 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA1 isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Alagarsamy Srinivasan, Inger Rosner, Albert Dobi, Shiv Srivastava. PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B008.

MicroRNA-378 enhances radiation response in ectopic and orthotopic implantation models of glioblastoma.

Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor, which is virtually incurable due to its therapeutic resistance to radiation and chemotherapy. To develop novel therapeutic approaches for treatment of GBM, we examined the role of miR-378 on tumor growth, angiogenesis, and radiation response in ectopic and orthotopic U87 glioblastoma models. Cell and tumor growth rates, in vitro and in vivo radiation sensitivities, and tumor vascular density were evaluated in U87-GFP and U87-miR-378 tumor lines. Ectopic tumor response to radiation was evaluated under normal blood flow and clamp hypoxic conditions. Results show that in vitro, miR-378 expression moderately increased cell growth rate and plating efficiency, but did not alter radiation sensitivity. U87-miR-378 tumors exhibited a higher transplantation take rate than U87-GFP tumors. In vivo, under oxygenated condition, subcutaneous U87-miR-378 tumors receiving 25Gy showed a tendency for longer tumor growth delay (TGD) than control U87-GFP tumors. In contrast, under hypoxic condition, U87-miR-378 xenografts exhibited substantially shorter TGD than U87-GFP tumors, indicating that under normal blood flow conditions, U87-miR-378 tumors were substantially more oxygenated than U87-GFP tumors. Intracranial multi-photon laser-scanning microscopy demonstrated increased vascular density of U87-miR-378 versus control U87-GFP tumors. Finally, miR-378 increased TGD following 12Gy irradiation in U87 intracranial xenografts, and significantly prolonged survival of U87-miR-378 tumor-bearing mice (P = 0.04). In conclusion, higher miR-378 expression in U87-miR-378 cells promotes tumor growth, angiogenesis, radiation-induced TGD, and prolongs survival of orthotopic tumor-bearing hosts. Regulation of VEGFR2 by miR-378 significantly increased vascular density and oxygenation in U87 xenografts.

Ionizing radiation response of primary normal human lens epithelial cells.

Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

HOPX functions as a tumour suppressor in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is generalized term that encompasses a diverse group of cancers that includes tumours of the oral cavity (OSCC), oropharynx (OPSCC) and nasopharynx (NPC). Genetic alterations that are common to all HNSCC types are likely to be important for squamous carcinogenesis. In this study, we have investigated the role of the homeodomain-only homeobox gene, HOPX, in the pathogenesis of HNSCC. We show that HOPX mRNA levels are reduced in OSCC and NPC cell lines and tissues and there is a general reduction of HOPX protein expression in these tumours and OPSCCs. HOPX promoter methylation was observed in a subset of HNSCCs and was associated with a worse overall survival in HPV negative tumours. RNAseq analysis of OSCC cells transfected with HOPX revealed a widespread deregulation of the transcription of genes related to epithelial homeostasis and ectopic over-expression of HOPX in OSCC and NPC cells inhibited cell proliferation, plating efficiency and migration, and enhanced sensitivity to UVA-induced apoptosis. Our results demonstrate that HOPX functions as a tumour suppressor in HNSCC and suggest a central role for HOPX in suppressing epithelial carcinogenesis.

Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk

The poor clinical outcome of pancreatic cancer (PC) is largely attributed to its aggressive nature and refractoriness to currently available therapeutic modalities. We previously reported antitumor efficacy of honokiol (HNK), a phytochemical isolated from various parts of Magnolia plant, against PC cells in short-term in vitro growth assays. Here, we report that HNK reduces plating efficiency and anchorage-independent growth of PC cells and suppresses their migration and invasiveness. Furthermore, significant inhibition of pancreatic tumor growth by HNK is observed in orthotopic mouse model along with complete-blockage of distant metastases. Histological examination suggests reduced desmoplasia in tumors from HNK-treated mice, later confirmed by immunohistochemical analyses of myofibroblast and extracellular matrix marker proteins (α-SMA and collagen I, respectively). At the molecular level, HNK treatment leads to decreased expression of sonic hedgehog (SHH) and CXCR4, two established mediators of bidirectional tumor-stromal cross-talk, both in vitro and in vivo . We also show that the conditioned media (CM) from HNK-treated PC cells have little growth-inducing effect on pancreatic stellate cells (PSCs) that could be regained by the addition of exogenous recombinant SHH. Moreover, pretreatment of CM of vehicle-treated PC cells with SHH-neutralizing antibody abolishes their growth-inducing potential on PSCs. Likewise, HNK-treated PC cells respond poorly to CM from PSCs due to decreased CXCR4 expression. Lastly, we show that the transfection of PC cells with constitutively active IKKβ mutant reverses the suppressive effect of HNK on nuclear factor-kappaB activation and partially restores CXCR4 and SHH expression. Taken together, these findings suggest that HNK interferes with tumor-stromal cross-talk via downregulation of CXCR4 and SHH and decreases pancreatic tumor growth and metastasis.

Biological responses of human solid tumor cells to X-ray irradiation within a 1.5-Tesla magnetic field generated by a magnetic resonance imaging-linear accelerator.

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc.

679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line

Discovery of cancer stem cells (CSC) as driving force behind cancer cells tumorigenesis and heterogeneity in solid tumors led to a paradigm shift. The recognition of non-CSC to CSC transient introduces the cancer cell plasticity concept. This behavior appeared to be brought into existence majorly by the hostility of tumor microenvironment and stress imposed by hypoxia, pH, and nutrient depletion in the tumor niche. This recognition draw attention to the correlation between cancer hallmarks such as metastasis, chemotherapy, radiotherapy resistant and the transition of non-CSC to CSC by determination of CSC markers in cells subpopulations. Different types of cancer cell lines were shown to have a subset of cells expressing CSC markers such as CD44, ALDH, C133, Oct 3/4 with tumorigenicity in vivo. Recently, metabolic stress in a long-term nutrient deprivation showed to induces conversion of non-CSC to CSC like state. Additionally the hypoxic microenvironment was shown to upgraded stem-like properties of gastric cancer cells. Since it was recognized that metabolic alterations in tumors often combined by hyperthermia, we hypothesized that hyperthermia could be one of the stress components imposed by microenvironment that may manipulate non-CSC to CSC status and we asked if this elevation in temperature would affect phenotypic characteristics or up-regulate the genes responsible for stress resistant and the genes of cancer stem cell markers. To answer this, three different types of cancer cell lines were investigated by incubating in elevated temperature without CO2 supplement. They were rhebdomyosarcoma cell line (RD), cervical carcinoma cell line (HeLa), and mice mammary adenocarcenoma (AMN3). In all experiments cell lines were seeded in 12 well tissue culture plates and incubated at 37°C with 5% CO2 until they reach absolute confluent monolayer, after that subsets of plates incubated either at 40°C or 37°C without CO2 for 24 or 48 hr. Cultivation of cell lines in RPMI1640 at 37°C for 24hr without CO2 served as control untreated experiments. We followed changes in cells viability, phenotypic alterations, cologencity, and genetic markers of heat resistant (HSP90-beta and HIF), genetic markers of cancer stem cells (CD44, ALDH, Oct4, and CD133) by RT- qPCR. Results showed that upon incubation at 40°C for 24 hr or 48 hr without CO2 supplement cells were detached floated and acquired spherical shapes and aggregated. Noticeably the formed aggregation resembled to some extent the cell spheres that induced in cancer stem cells at sphere forming assay (fig.1,A,Bfig.1,A,B). The count of viable floated cells for RD, HeLa, and AMN3 cells were 3.93E–6, 2.1E–6, and 5.7E–6 cell/ml at 40°C for 48 hr respectively, and generally it was temperature and time dependant (Fig 1,CFig 1,C). All viable floated cells from the cell lines investigated were capable to form colonies in colony forming assay, the plating efficiency for RD, HeLa, and AMN3 cells were 48%, 64%, and 57% after incubation at 37°C for 10 days with CO2 supplement respectively (Fig.1,DFig.1,D). Up regulation of heat stress gene HSP90-beta and HIF was detected in floated cells derived from all experiment and time of incubation. This was combined by up regulation of CSC markers CD44, ALDH, Oct4 and CD133 in floated cells derived from all experiment. These results may indicate a shift of non-CSC to CSC during heat stress and the increment of cancer stem cell population in the cell line under investigation which need more careful investigation.View Large Image | Download PowerPoint Slide

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BackgroundThe proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications.MethodsSingle cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients.ResultsSingle cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFβ super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn’t support the prognostic value of the ASCs in AML.ConclusionsASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1233-x) contains supplementary material, which is available to authorized users.

Abstract 5566: Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer

Abstract Introduction: Androgen receptor (AR) stability has been shown to be regulated by MDM2 E3 ubiquitin ligase. Our data suggest a role for PMEPA1 and NEDD4-1 in the regulation of AR protein in prostate cancer cells. The AR inducible PMEPA1 recruits NEDD4-1, an E3 ubiquitin ligase, to degrade AR. Decreased or absent PMPEA1 was observed in more than 65% of prostate tumors. PMEPA1 depleted transfectant is established in LNCaP cells with a shRNA lentivirus system and shown elevated AR protein levels, AR signaling and increased cell growth. In the current study, we investigated the growth and AR signaling of PMEPA1 depleted prostate cancer cells in the presence of AR inhibitors. Also we investigated the role of PMEPA1 silencing by DNA methylation in the regulation of AR. Methods: PMEPA1 depleted LNCaP cells were treated with AR inhibitors (MDV3100 or bicalutamide) in the presence or absence of the synthetic androgen R1881. Cell growth was monitored by cell counting, BrdU incorporation, colony plating and soft agar assays. Cell cycle was analyzed with propidium iodide staining based flowcytometry. AR, PMEPA1, PSA proteins were assessed by immunoblot assay. Methylation of the PMEPA1 gene was evaluated in LCM-enriched tumor samples by combing precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). The transcript levels of PMEPA1 gene was assessed by qRT-PCR. Results: PMEPA1 depleted LNCaP cells are more resistance to growth inhibition by MDV3100 or bicalutamide in both hormone and hormone-depleted conditions, which demonstrated stronger BrdU incorporation abilities, cell plating efficiency and colony formation capacities in soft agar under MDV3100 or bicalutamid treatment compared to controls. The half life of AR was increased, and more cells in S-phase and less apoptotic cells in PMEPA1 depleted LNCaP cells. Methylation of PMEPA1 gene and mRNA levels showed significant correlation in prostate tumors (n=77, P=0.001). The higher frequency of PMEPA1 gene methylation was noticed in Caucasian American patients. Conclusions: PMEPA1 depleted prostate cancer cells develop increased resistance to AR inhibitors through enhanced AR signaling. Silencing of PMEPA1 may contribute to AR activation in prostate cancer. These data highlight that PMEPA1 deficiency may confer resistance to AR inhibitors during prostate cancer progression. This study is supported by the NIH grant (R01CA106653) to SS. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Michael Haffner, Daniel Heidenberg, Srinivasan Yegnasubramanian, Yongmei Chen, Ahmed Mohamed, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava. Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2014-5566

Loss of Malignancy in Mouse Melanoma Cells by Long-Term Impact of Interferon-Beta In Vitro is Associated with N- and VE-Cadherins Suppression without Inhibition of Expression of EMT-Associated Twist and Slug Proteins

Aim: To study the effect of long-term impact of mouse interferon-beta (IFN-b) on the behavior of mouse melanoma cells in vitro and in vivo and the expression of epithelial-mesenchymal transition (EMT) associated proteins. Materials and Methods: Studies were performed on mouse B16 melanoma cells as a tumor model (MM-4 cell line). Immunocytochemical and tumor cell biology approaches have been used in this study. Results: Long-time treatmentof MM-4 melanoma cellswith low-dose IFN-b (1/2 of IC50) leads to change their morphology, significant inhibition of cell growth and plating efficiency, suppression of cell migration and anchorage-independent growth in semisolid agar. Moreover, IFN-modification of melanoma cells is accompanied by the significant suppression of their malignancy in vivo: growth of tumor induced by IFN-treated cells has inhibited on 50% and growth of metastases - on 90%. Also, IFN-modification of MM-4 cells affects on the expression of proteins involved in cell cycle regulation and inhibits expression of some molecules of adhesion (N-, VE-cadherins), but not influence on the expression of EMT-associated Twist and Slug proteins and E-cadherin. Conclusions: Long-term impact of mouse IFN-beta in low dose on melanoma cells in vitro changes their phenotype and inhibits their proliferative potential, signs of malignancy in vitro, tumorigenicity and metastatic ability in vivo. Loss of malignancy is associated with inhibition of N- and VE-cadherins expression, but not associated with the change of expression and subcellular localization of E-cadherin and EMT transcription factors Twist and Slug.