Inactive Cells Research Articles

The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm.

Exposure to toxicants present in the environment, especially the so-called endocrine-disrupting chemicals (EDCs), has been associated with decreased sperm quality and increased anomalies in male reproductive organs over the past decades. Both human and animal populations are continuously exposed to ubiquitous synthetic and natural-occurring EDCs through diet, dermal contact and/or inhalation, therefore potentially compromising male reproductive health. Although the effects of EDC are likely induced via multiple genomic-based pathways, their non-genomic effects may also be relevant. Furthermore, spermatozoa are transcriptionally inactive cells that can come in direct contact with EDCs in reproductive fluids and secretions and are therefore a good model to address non-genomic effects. This review thus focuses on the non-genomic effects of several important EDCs relevant to mammalian exposure. Notably, EDCs were found to interfere with pre-existing pathways inducing a panoply of deleterious effects to sperm function that included altered intracellular Ca(2) (+) oscillations, induction of oxidative stress, mitochondrial dysfunction, increased DNA damage and decreased sperm motility and viability, among others, potentially jeopardizing male fertility. Although many studies have used non-environmentally relevant concentrations of only one compound for mechanistic studies, it is important to remember that mammals are not exposed to one, but rather to a multitude of environmental EDCs, and synergistic effects may occur. Furthermore, some effects have been detected with single compounds at environmentally relevant concentrations.

Biofilm Formation of <i>Sulfobacillus</i> <i>thermosulfidooxidans</i> on Pyrite in the Presence of <i>Leptospirillum ferriphilum</i>

In this study, initial attachment to and biofilm formation of Sulfobacillusthermosulfidooxidans DSM 9293T on pyrite in the presence of Leptospirillumferriphilum DSM 14647T were investigated. Interactions of S.thermosulfidooxidansT and L.ferriphilumT were studied by means of monitoring attachment behavior and biofilm formation on pyrite. Our preliminary results showed that 1): Pre-established biofilms of L.ferriphilumT had effects on attachment of S. thermosulfidooxidansT to pyrite; 2): physical contact between cells of L. ferriphilumT and S. thermosulfidooxidansT on pyrite were visible 3): Pyrite leaching by cells of S. thermosulfidooxidansT was inhibited by the presence of inactive cells of L.ferriphilumT.

Automatic tracking of neural stem cells in sequential digital images

Neural stem cells are the cells that give rise to the main cell types of the nervous system. Due to their varying size and shape, and random movement, the tracking of these cells in suspension in video sequences is challenging. This paper develops an automatic tracking system for neural stem cells. The system first detects and localizes cells in the image sequence, followed by a feature extraction step for the subsequent cell tracking. Then, the system tracks inactive cells using an improved mean shift algorithm, divisive cells through a context-based technique, and active cells by means of dynamic local prediction (DLP) and gray prediction (GP) algorithms. Experimental results show that the proposed system not only improves the accuracy of fast moving tracking, but also constructs accurately the trajectories of the cell movement and reduces the iterations during the center searching.

Intermittent PTH treatment can delay the transformation of mature osteoblasts into lining cells on the periosteal surfaces.

Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1-34, 80μg/kg) or vehicle daily for 10 (D36) or 20days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.

The influence of environmental factors on protistan microorganisms in grassland soils along a land-use gradient

In this study, we investigated the effect of land use intensity, soil parameters and vegetation on protistan communities in grassland soils. We performed qualitative (T-RFLP) and quantitative (qPCR) analyses using primers specifically targeting the 18S rRNA gene for all Eukarya and for two common flagellate groups, i.e. the Chrysophyceae and the Kinetoplastea. Both approaches were applied to extracted soil DNA and RNA, in order to distinguish between the potentially active protists (i.e. RNA pool) and the total protistan communities, including potentially inactive and encysted cells (i.e. DNA pool). Several environmental determinants such as site, soil parameters and vegetation had an impact on the T-RFLP community profiles and the abundance of the quantified 18S rRNA genes. Correlating factors often differed between quantitative (qPCR) and qualitative (T-RFLP) approaches. For instance the Chrysophyceae/Eukarya 18S rDNA ratio as determined by qPCR correlated with the C/N ratio, whereas the community composition based on T-RLFP analysis was not affected indicating that both methods taken together provide a more complete picture of the parameters driving protist diversity. Moreover, distinct T-RFs were obtained, which could serve as potential indicators for either active organisms or environmental conditions like water content. While site was the main determinant across all investigated exploratories, land use seemed to be of minor importance for structuring protist communities. The impact of other parameters differed between the target groups, e.g. Kinetoplastea reacted on changes to water content on all sites, whereas Chrysophyceae were only affected in the Schorfheide. Finally, in most cases different responses were observed on RNA- and DNA-level, respectively. Vegetation for instance influenced the two flagellate groups only at the DNA-level across all sites. Future studies should thus include different protistan groups and also distinguish between active and inactive cells, in order to reveal causal shifts in community composition and abundance in agriculturally used systems.

Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers.

In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1.

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.

Irradiación subletal de fibroblastos murinos 3T3 con rayos X para su utilización como capa celular alimentadora

Keratinocytes, the main cells of the epidermis, require mitotically inactive but live feeder-layer cells for their survival and proliferation. We evaluated four X-ray irradiation doses on 3T3 murine fibroblasts for feeder-layer production. Each dose effect was evaluated by cell counting and cellular viability determination (MTT), cell adhesion (plating efficiency) and cell migration (scratch wound healing). Mitotic arrest was suggested by nuclear staining (GIEMSA). We determined that an 80 Gy X-ray dose generated enough cell damage to cause mitotic arrest yet keeping metabolically active cells up to 25 days.

Allogenic dendritic cell and tumor cell fused vaccine for targeted imaging and enhanced immunotherapeutic efficacy of gastric cancer

Dendritic cells (DCs) have displayed the promising potential in cancer immunity. How to enhance DCs immunotherapeutic effect in cancer targeted immunotherapy and prevention is still a great challenge. Herein, we report for the first time the allogenic DCs and tumor cell fused vaccine combined with cytokine induced killing cells (CIKs) for targeted imaging and enhanced immunotherapeutic efficacy of gastric cancer (GC). The fused vaccine was prepared by PEG mediated fusion between mature DCs and inactive gastric cancer MGC803 cells. The immunotherapeutic and prophylactic potential of the fused cells (FCs) were evaluated in tumor-bearing, post-surgery and tumor free mice models. The migration and homing process of near infrared region quantum dots (NIR-QDs) labeled FCs were investigated by real-time animal imaging system. Results showed that the FCs and FC + CIKs could trigger the tumor-specific CTLs against GC cells, target the tumor tissue initiatively and enhance the prophylactic effects, suppress the tumor growth remarkably in vivo. The potential mechanism is also investigated. In conclusion, allogenic DCs and tumor cell fused vaccine can be used for targeted imaging and enhanced immunotherapeutic efficacy of GC, and the FC + CIKs strategy own great potential in clinical applications such as early therapy and prevention of tumor-metastasis and relapse in near future.

Temporal-spatial changes in viabilities and electrochemical properties of anode biofilms.

Sustained current generation by anodic biofilms is a key element for the longevity and success of bioelectrochemical systems. Over time, however, inactive or dead cells can accumulate within the anode biofilm, which can be particularly detrimental to current generation. Mixed and pure culture (Geobacter anodireducens) biofilms were examined here relative to changes in electrochemical properties over time. An analysis of the three-dimensional metabolic structure of the biofilms over time showed that both types of biofilms developed a live outer-layer that covered a dead inner-core. This two-layer structure appeared to be mostly a result of relatively low anodic current densities compared to other studies. During biofilm development, the live layer reached a constant thickness, whereas dead cells continued to accumulate near the electrode surface. This result indicated that only the live outer-layer of biofilm was responsible for current generation and suggested that the dead inner-layer continued to function as an electrically conductive matrix. Analysis of the electrochemical properties and biofilm thickness revealed that the diffusion resistance measured using electrochemical impedance spectroscopy might not be due to acetate or proton diffusion limitations to the live layer, but rather electron-mediator diffusion.

Cell membrane integrity and distinguishing between metabolically active and inactive cells as a means of improving viability PCR

Viability PCR uses cell membrane integrity to differentiate live cells from dead. Our new approach improves viability PCR by enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial homeostasis and cells that have an intact membrane but are metabolically inactive.

Nephrogenic Adenoma of the Urinary Bladder: A Review of the Literature.

Background. Nephrogenic adenoma of the urinary bladder (NAUB) is a rare lesion associated with nonspecific symptoms and could inadvertently be misdiagnosed. Aim. To review the literature. Methods. Various internet search engines were used. Results. NAUB is a benign tubular and papillary lesion of the bladder, is more common in men and adults, and has been associated with chronic inflammation/irritation, previous bladder surgery, diverticula, renal transplantation, and intravesical BCG; recurrences and malignant transformations have been reported. Differential diagnoses include clear cell adenocarcinoma, endocervicosis, papillary urothelial carcinoma, prostatic adenocarcinoma of bladder, and nested variant of urothelial carcinoma; most NAUBs have both surface papillary and submucosal tubular components; both the papillae and tubules tend to be lined by a single layer of mitotically inactive bland cells which have pale to clear cytoplasm. Diagnosis may be established by using immunohistochemistry (positive staining with racemase; PAX2; keratins stain positive with fibromyxoid variant), electron microscopy, DNA analysis, and cytological studies. Treatment. Endoscopic resection is the treatment but recurrences including sporadic malignant transformation have been reported. Conclusions. There is no consensus on best treatment. A multicentre study is required to identify the treatment that would reduce the recurrence rate, taking into consideration that intravesical BCG is associated with NAUB.

Ultrastructure of cells in an initiating lateral root primordium of Raphanus sativus

Lateral root primordia in &lt;i&gt;Raphanus sativus&lt;/i&gt; had developed 10 hours after main root decapitation. The primordia consisted of three cell layers — basal layer continuous with the pericycle. The primordia were initiated by activated groups of pericycle cells. Inactive pericycle cells with a thin layer of parietal cytoplasme large central vacuole and well developed leucoplasts with starch grains were trans-formed into meristematic cells. During transformation the amount of cytoplasm and number of cytoplasmic organelles greatly increased, the central vacuole disappeared, and an ER system continuous in many places with the nuclear envelope evolved. The lamellar structure of plastids underwent almost complete reduction; the dictyosomes became active. The newly formed meristem differed apparently from the apical root meristem only in the lack or scarcity of lipid bodies and starch.

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

Selective Photodepletion of Recipient-Alloreactive T-Cells Enables Safe and Efficacious Haploidentical HSCT: Initial Results from a Phase 2 Trial in Patients with AML, ALL, and MDS

▪IntroductionFor patients in need of a hematopoietic stem cell transplant (HSCT) but lacking an HLA matched donor, a haploidentical family donor is a particularly appealing alternative. However, to prevent graft-versus-host disease (GVHD), haploidentical HSCT necessitates intensive in vivo or ex vivo T-cell depletion that results in frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival. To overcome this limitation, we have developed a strategy that photodepletes host-reactive cells from the donor T cell graft, while preserving anti-infection and anti-leukemia reactivity.Patients and MethodsIn an open-label, multi-center phase 2 clinical trial (CR-AIR-007; NCT01794299), 12 of a planned 23 patients with high-risk hematologic malignancies were treated to date with this immunotherapy approach consisting of donor lymphocytes selectively allodepleted of host-reactive T-cells using photodynamic therapy (ATIR). ATIR was infused 28-32 days after haploidentical CD34-selected HSCT. No post-transplant GVHD prophylaxis was used. These patients were compared to a control group of 28 patients treated in a previous Phase 2 study with an investigational product manufactured using a process different from the Phase 1 trial and resulting mainly in dead and inactive cells instead of ATIR (CR-AIR-004).ResultsTwelve patients, mean age of 45 (range 21-64), 6 females/6 males with AML (n=9) and ALL (n=3) were treated with ATIR so far. ATIR consisted mainly of T-cells (>90%), with residual B and NK cells (≤10%). Selective depletion of recipient-reactivity in each ATIR cell graft was assessed using a CFSE-based proliferation assay. Cell division numbers upon stimulation were analyzed using Modfit LT software (Fig 1A), which generated a proliferation index representing viable/reactive T-cells in donor cells (blue) and final ATIR product (green)(Fig 1B). Selective depletion of recipient-reactive T-cells with preservation of reactivity towards 3rd party antigens and anti-CD3/CD28 was observed in all ATIR cell grafts and used as a release criteria in the 007 study. [Display omitted] Preparative regimen consisted of A) FTBI (1200 cGy; n=5) or B) melphalan (120 mg/m2; n=7), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x5 d) and ATG (2.5 mg/kg x4 d). Neutrophil and platelet engraftment was achieved in all patients at a median of 12 days (range: 9-35). No patient experienced graft rejection.Patients (n=28) in the 004 control group, mean age of 42 (range 18-61), 13 females/15 males had AML (n=19), ALL (n=6) or MDS (n=3). CFSE proliferation in T-cell grafts could not be assessed a posteriori due to low cell viability. These 004 patients received the same A) FTBI- (n=14), B) melphalan- (n= 10) based preparative regimen as 007 patients, except for 4 patients receiving single fraction (800 cGy) TBI. Neutrophil and platelet engraftment was achieved at a median of 16 days (range: 7-54). Three patients showed secondary graft rejection.Two patients in study CR-AIR-007 developed acute GVHD grade I (skin only) approximately 130 days post HSCT, which was of short duration, (18 and 41 days). Two patients died of infection and no patient relapsed at a mean follow-up of 8 months post HSCT (range 1-14 months). In the CR-AIR-004 control group, 2 patients developed grade I, 1 patient grade II and 3 patients grade III GvHD, none of these cases were lethal. Seventeen patients died of transplant related complications and 2 patients of relapse/disease progression. TRM is 20% in 007 group vs 63% in the 004 control group and OS is 80% in 007 group vs 35% in the 004 control group at 9 months post-transplant (Figures 2A and 2B). [Display omitted] [Display omitted] ConclusionsThese data confirm that a novel immunotherapy strategy consisting of donor lymphocytes selectively photodepleted of alloreactive cells (ATIR) can be manufactured consistently and reproducibly. Results to date show that ATIR is safe and does not cause any grade III/IV GvHD. Moreover, haploidentical HSCT patients treated with ATIR demonstrate very promising TRM and OS rates when compared to the control group. DisclosuresRoy:Kiadis Pharma: Consultancy, Research Funding. Foley:Hoffman-LaRoche: Advisory Board/Lectures Other, Honoraria; Lundbeck: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Sanofi: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Celgene: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Pfizer: Advisory Board/Lectures Other, Honoraria; Novartis: Advisory Board/Lectures Other, Honoraria; Jansen: Advisory Board/Lectures Other, Honoraria; Alexion: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Roche Canada: Honoraria, Research Funding, Unrestricted educational grant, Unrestricted educational grant Other. De Jong:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Wagena:Kiadis Pharma: Employment.

Fine-Grained Architecture in Dark Silicon Era for SRAM-Based Reconfigurable Devices

In this brief, we present a fine-grained dark silicon architecture to facilitate further integration of transistors in static random access memory-based reconfigurable devices. In the proposed architecture, we present a technique to power off inactive configuration cells in nonutilized or underutilized logic blocks. We also propose a routing circuitry capable of turning off the configuration cells of connection blocks (CBs) and switch boxes (SBs) in the routing fabric. Experimental results carried out on the Microelectronics Center of North Carolina benchmark show that power consumption in configuration cells of lookup tables, CBs, and SBs can, on average, be reduced by 27%, 75%, and 4%, respectively.

The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

Bacteriophages and Biofilms

Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce) enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

Genetic inhibition of the atypical kinase Wee1 selectively drives apoptosis of p53 inactive tumor cells.

BackgroundTumorigenesis is the result of genomic or epigenomic insults and subsequent loss of the proper mechanisms to respond to these alterations leading to unscheduled growth. Tumors arising from these mutations often have altered cell cycles that offer proliferative advantages and lead to the accumulation of additional mutations that can lead to more aggressive phenotypes. Nevertheless, tumor cells must still adhere to the basic tenets of the cell cycle program to ensure their survival by DNA duplication, chromosomal segregation and cytokinesis. The atypical tyrosine kinase Wee1 plays a key role in regulating the cell cycle at the DNA synthesis and mitotic checkpoints via phosphorylation and subsequent inactivation of cyclin-dependent kinases (CDKs) in both healthy and tumorigenic cells.MethodsTo assess the role of Wee1 in tumor cell proliferation we performed small interfering RNA (siRNA) experiments in a panel of diverse cell lines derived from various tissue origins. We also tested the hypothesis that any potential effects would be as a result of the kinase activity of Wee1 by siRNA rescue studies with wild-type or kinase-dead versions of Wee1.ResultsWe find that, in general, cells with wild-type p53 activity are not susceptible to loss of Wee1 protein via siRNA. However, Wee1 siRNA treatment in tumor cells with an inherent loss of p53 activity results in a deregulated cell cycle that causes simultaneous DNA synthesis and premature mitosis and that these effects are kinase dependent. These cumulative effects lead to potent inhibition of cellular proliferation and ultimately caspase-dependent apoptosis in the absence of co-treatment with cytotoxic agents.ConclusionsThese results suggest that, while Wee1 acts as a tumor suppressor in the context of normal cell growth and its functional loss can be compensated by p53-dependent DNA damage repairing mechanisms, specific inhibition of Wee1 has deleterious effects on the proliferation and survival of p53 inactive tumors. In total, targeting the atypical kinase Wee1 with an siRNA-based therapeutic or a selective ATP competitive small molecule inhibitor would be a feasible approach to targeting p53 inactive tumors in the clinic.

Interplay of phosphorylation kinetics and methylation dynamics leads to complex behavior of Foxp3 and PTEN in computational model of T cell differentiation (IRM6P.709)

Abstract We have recently published a mathematical model that demonstrates that differential activation of signaling pathways downstream of TCR by high and low concentrations of antigen can be modeled by varying the duration of antigen stimulation, which results in a mixed population of regulatory (Treg), helper (Th) and inactive cells, a prediction we confirmed experimentally. Analysis of the dynamics of this scenario demonstrated an important role for a feedback loop involving Foxp3, PTEN, and Akt-mTOR signaling, which affects the timing of transient Foxp3 expression and PTEN activity, and the ratio of different phenotypes in the mixed population. We hypothesized that the correlation in the dynamics of Foxp3 and PTEN, which we previously modeled as a direct link between Foxp3 and PTEN, stems from the regulation of both Foxp3 and PTEN expression by the transcription factor Foxo1. We updated the model to test this hypothesis, and preliminary results suggest that the interplay of rapid modulation of Foxo1 activity by Akt with the slower kinetics of methylation/demethylation at the Foxp3 promoter mediated by mTOR can produce dramatic changes in the resulting distribution of phenotypes when the timing of these events is varied. To calibrate this timing in the model, we are currently conducting experiments that will help us determine the time scale for Foxo1 inactivation by Akt and for the effect of Foxo1 on Foxp3 and PTEN expression.