Cell Cycle Distribution Pattern Research Articles

Transcriptomic Analysis Reveals Candidate Ligand-Receptor Pairs and Signaling Networks Mediating Intercellular Communication between Hair Matrix Cells and Dermal Papilla Cells from Cashmere Goats.

Hair fiber growth is determined by the spatiotemporally controlled proliferation, differentiation, and apoptosis of hair matrix cells (HMCs) inside the hair follicle (HF); however, dermal papilla cells (DPCs), the cell population surrounded by HMCs, manipulate the above processes via intercellular crosstalk with HMCs. Therefore, exploring how the mutual commutations between the cells are molecularly achieved is vital to understanding the mechanisms underlying hair growth. Here, based on our previous successes in cultivating HMCs and DPCs from cashmere goats, we combined a series of techniques, including in vitro cell coculture, transcriptome sequencing, and bioinformatic analysis, to uncover ligand-receptor pairs and signaling networks mediating intercellular crosstalk. Firstly, we found that direct cellular interaction significantly alters cell cycle distribution patterns and changes the gene expression profiles of both cells at the global level. Next, we constructed the networks of ligand-receptor pairs mediating intercellular autocrine or paracrine crosstalk between the cells. A few pairs, such as LEP-LEPR, IL6-EGFR, RSPO1-LRP6, and ADM-CALCRL, are found to have known or potential roles in hair growth by acting as bridges linking cells. Further, we inferred the signaling axis connecting the cells from transcriptomic data with the advantage of CCCExplorer. Certain pathways, including INHBA-ACVR2A/ACVR2B-ACVR1/ACVR1B-SMAD3, were predicted as the axis mediating the promotive effect of INHBA on hair growth via paracrine crosstalk between DPCs and HMCs. Finally, we verified that LEP-LEPR and IL1A-IL1R1 are pivotal ligand-receptor pairs involved in autocrine and paracrine communication of DPCs and HMCs to DPCs, respectively. Our study provides a comprehensive landscape of intercellular crosstalk between key cell types inside HF at the molecular level, which is helpful for an in-depth understanding of the mechanisms related to hair growth.

Tension force causes cell cycle arrest at G2/M phase in osteocyte-like cell line MLO-Y4

Bone remodelling is the process of bone resorption and formation, necessary to maintain bone structure or for adaptation to new conditions. Mechanical loadings, such as exercise, weight bearing and orthodontic force, play important roles in bone remodelling. During the remodelling process, osteocytes play crucial roles as mechanosensors to regulate osteoblasts and osteoclasts. However, the precise molecular mechanisms by which the mechanical stimuli affect the function of osteocytes remain unclear. In the present study, we analysed viability, cell cycle distribution and gene expression pattern of murine osteocyte-like MLO-Y4 cells exposed to tension force (TF). Cells were subjected to TF with 18% elongation at 6 cycles/min for 24 h using Flexcer Strain Unit (FX-3000). We found that TF stimulation induced cell cycle arrest at G2/M phase but not cell death in MLO-Y4 cells. Differentially expressed genes (DEGs) between TF-stimulated and unstimulated cells were identified by microarray analysis, and a marked increase in glutathione-S-transferase α (GSTA) family gene expression was observed in TF-stimulated cells. Enrichment analysis for the DEGs revealed that Gene Ontology (GO) terms and Kyoto Encyclopedia Genes and Genomes (KEGG) pathways related to the stress response were significantly enriched among the upregulated genes following TF. Consistent with these results, the production of reactive oxygen species (ROS) was elevated in TF-stimulated cells. Activation of the tumour suppressor p53, and upregulation of its downstream target GADD45A, were also observed in the stimulated cells. As GADD45A has been implicated in the promotion of G2/M cell cycle arrest, these observations may suggest that TF stress leads to G2/M arrest at least in part in a p53-dependent manner.

Effects of β-glucan from S. cerevisiae on the expression of Casp9, Ccna2 and Sod1 genes in MCF-7 cells

β-Glucans (βG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of βG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. βG was not cytotoxic. The mRNA levels of CCNA2 of cells exposed to β-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9 suggest that βG could inhibit promotion and progression steps of carcinogenesis, eliminating neoplastic cells. The upregulation of CCNA2 gene in combined treatment could be occurred due to ability of βG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1 suggests that βG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of βG during chemotherapy

IGF2BP2 knockdown suppresses thyroid cancer progression by reducing the expression of long non-coding RNA HAGLR

BackgroundN6-methyladenosine (m6A), a common internal modification on RNAs, has been found to be closely linked with RNA biosynthesis/metabolism and cancer development. In this text, the roles and molecular mechanisms of m6A-bind protein IGF2BP2 in the development of thyroid cancer (TC) were investigated in vitro. MethodsIGF2BP2 and lncRNA HAGLR were screened out through multiple public databases such as TCGA, Ualcan, POSTAR2, Starbase, and GEPIA. Cell proliferative, migratory and invasive abilities were assessed by CCK-8, Transwell migration and invasion assays, respectively. Cell cycle distribution and cell apoptotic patterns were measured by flow cytometry. The interaction between HAGLR and IGF2BP2 was examined by RIP, RNA pull-down and luciferase assays and bioinformatics analysis. The effect of IGF2BP2 knockdown on the m6A level of HAGLR was explored by meRIP assay. ResultsIGF2BP2 was highly expressed in TC tumor tissues. IGF2BP2 knockdown weakened cell proliferative, migratory, and invasive abilities, and induced cell cycle arrest and cell apoptosis in TC cells. LncRNA HAGLR expression was markedly upregulated and positively associated with IGF2BP2 expression in TC tissues. IGF2BP2 knockdown reduced HAGLR expression and transcript stability in TC cells. IGF2BP2 regulated HAGLR expression in an m6A-dependent manner. HAGLR overexpression weakened the effects of IGF2BP2 loss on cell proliferation, migration, invasion, apoptosis, and cell cycle progression in TC cells. ConclusionIGF2BP2 loss inhibited cell proliferation, migration and invasion, and induced cell apoptosis and cell cycle arrest by down-regulating HAGLR expression in an m6A-dependent manner in TC cells, providing some potential diagnostic and therapeutic targets for TC.

14-Deoxy-11,12-Didehydroandrographolide: A novel compound isolated from Andrographis paniculata Nees. induces robust apoptosis in leukemic cells

Objective: Andrographis paniculata is widely cultivated in South and Southeast Asian countries and popularly used in “Ayurveda” medicine. We attempted to investigate antileukemic activity of the biomolecules extracted from this plant and a probable mechanism of action. Materials and Methods: Biomolecules from methanolic extract were isolated using silica gel column chromatography and high-performance liquid chromatography. The structures were determined by liquid chromatography-mass spectrometry (LC-MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. In vitro antiproliferative activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic efficacy of the most potent molecule was investigated by annexin V and propidium iodide (V/PI) staining and flow cytometry. Caspase activation, cell cycle distribution pattern, and nuclear morphology of the treated leukemic cells were also investigated. Results: From the methanolic extract, four biomolecules were isolated, namely 14-deoxy-11,12-didehydroandrographolide (1), andrographolide (2), neoandrographolide (3), and 14-deoxy-11,12-didehydroandrographiside (4). Results of MTT assay exhibited that out of four compounds, compound (1) showed the most potent activity against all the cell lines tested with the lowest IC50 values of 13 μM on U937 cells. Annexin V/PI staining revealed that the compound was able to induce apoptosis in concentration-dependent manner with IC50 value being 17.66 μM. Apoptotic induction was mediated through elevated activation of caspase-3 and caspase-9. Cell cycle analysis revealed that the compound (1) effectively increased the sub-G0-G1 population in the treated U937 cells (73.25% at 50 μM) in comparison to control set (3.12%). DAPI nuclear staining indicated that compound (1) increased the number of deformed nuclei and an increased level of apoptotic body formation in the treated cells.

Meme kanseri kanser kök hücrelerinde PD-0332991 uygulanmasının hücre döngüsü düzenleyici genler üzerine etkisi

Aim: Palbociclib (PD-0332991) is an inhibitor for cyclin-dependent kinase 4/6 complex and exhibits more activity in luminal ER+ breast cancer. However, sensitivity of breast cancer stem cells (BCSCs) to PD-0332991 treatment and expression patterns of cell cycle regulatory genes after PD-0332991 treatment in BCSCs are still unclear. This study aims to determine response of BCSCs to PD-0332991 treatment.Materials and Methods: An experimental in vitro study was designed on breast cancer cell lines. MCF-7 and BCSCs cell lines were used in this study. Water soluble tetrazolium salt-1 (WST-1) test was used for the cytotoxicity assay. Cell cycle distribution pattern and apoptosis were examined with flow cytometry according to IC50 values at 48th h. Real-Time PCR was used to detect expression profiles of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes.Results: PD-0332991 decreased cell proliferation in both cell lines. G0/G1 arrest was detected for both cell lines. There was no apoptotic effect of PD-0332991 on MCF-7 cells and BCSCs. In MCF-7 cells, expression levels of CDKN1A, CDKN2A, and CCND1 genes were increased as 3.11, 3.21, and 1.05 folds, respectively. Expression levels of CHEK1 and CDC25A genes were decreased as 4.75 and 3.73 folds, respectively. In BCSCs, expression levels of CDKN1A, CHEK1, CDKN2A, and CCND1 were decreased as 1.15, 2.01, 1.32, and 1.68 folds, respectively. No expression of CDC25A gene was found in BCSCs group.Conclusion: In this study, it was observed that PD-0332991 leads to different expression profiles for cell cycle regulatory genes between BCSCs and breast cancer cells.

Repression of Cyclin D1 Expression Is Necessary for the Maintenance of Cell Cycle Exit in Adult Mammalian Cardiomyocytes

The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

CDK inhibitors, p21Cip1 and p27Kip1, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21Cip1 and p27Kip1 but not p57Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21Cip1 and p27Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21Cip1 and p27Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21Cip1 and p27Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.

Further studies and biological activities of macromolecular protein R-Phycoerythrin from Portieria hornemannii

In the present study, the purified R-Phycoerythrin (R-PE) from a red alga Portieria hornemannii was subjected to the analysis of stability under the influence of different agents. Among the various inhibitors tested on R-PE EDTA at lower concentrations (<1mM) supported the activity of R-PE. When R-PE was exposed to different organic solvents, ethanol supported the activity at the maximum followed by acetone, ethyl acetate, chloroform and methanol. Citric acid, as a preservative maintained the stability of R-PE both under 0±5°C and 30±5°C with 59.34% and 56.23% respectively, on 30th day. Thermal decomposition of the R-PE began near 60°C. Maximum weight loss was occurred between 150°C and 500°C. Complete weight loss was recorded around 875°C. Thermal denaturation was observed between 19°C and 40°C. Moderate to low antioxidant activities were observed in R-PE in relation to total antioxidant activities. After characterization, R-PE was taken for in vitro anticancer studies against selected cancer cell lines. Further studies involving AO/EB fluorescence staining and phase contrast microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, and nuclear DNA fragmentation, etc. Likewise, FACS analysis revealed the cell cycle distribution pattern of A549 and HepG2 cells.

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin-dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono- and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono- to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.

Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

Anomalous retinal pigment epithelial (RPE) cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL) supplement (20ug or 90ug/ml) for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml), Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

Ex VivoObservation of Human Nucleus Pulposus Chondrocytes Isolated From Degenerated Intervertebral Discs

Study DesignWe performed an ex vivo study to observe cell morphology and viability of human nucleus pulposus (NP) chondrocytes isolated from degenerated intervertebral discs (IVD).PurposeTo better understand the biological behavior of NP chondrocytes in monolayer cultures.Overview of LiteratureBiological repair of IVDs by cell-based therapy has been shown to be feasible in clinical trials. As one of the most promising transplanting seeds, how the isolated NP chondrocytes behavior ex vivo has not been fully understood.MethodsHuman NP chondrocytes were harvested from 20 degenerated IVDs and cultured in monolayers. Histological and immunochemistry staining was used to detect cell morphology change. Cell viability was studied by analyzing cell cycle distribution and apoptotic rate in the primary and subculuted cells.ResultsThe round or polygonal primary NP chondrocytes had an average adherence time of 7 days and took nearly 31 days to reach 95% confluence. The spindle-shaped P1 NP chondrocytes increased growth kinetics and took about 12 hours to adhere and 6.6 days to get 95% confluent. Immunochemistry staining of collagen II was positive in the cell cytoplasm. Nearly 90% of the confluent NP chondrocytes stayed in G1 phase while 16% underwent apoptosis. No significant difference of the collagen II expression, cell cycle distribution or the apoptosis indices were detected between the primary and subcultured NP chondrocytes.ConclusionsHuman NP chondrocytes undergo significant morphological change in monolayer cultures. Cell cycle distribution pattern and apoptosis index of the cutured NP chondrocytes potentially influence their clinical transplantation or laboratory use.

Neoplastic transformation of breast epithelial cells by genotoxic stress

BackgroundExposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation.MethodsTo study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray) or cigarette smoke condensate (Csc - 10 microgram/ml of medium) or a combination of Rad + Csc. Following treatments, cells were analyzed for cell cycle distribution patterns and the ability to extrude the Hoechst 33342 dye. In addition, in vitro invasion and migration as well as mammosphere formation assays were performed. Finally, differential gene expression profiles were generated from the individual and combination treatment.ResultsExposure of MCF 10A cells to the combination of radiation plus cigarette smoke condensate generated a neoplastic phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases with a doubling of the population in S phase, and increased invasion and motility. Also, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. In addition, changes in gene expression include the up regulation of genes encoding proteins involved in metabolic pathways and inflammation.ConclusionsThe results indicate that when normal breast cells are exposed to low dose radiation in combination with cigarette smoke condensate a phenotype is generated that exhibits traits indicative of neoplastic transformation. More importantly, this is the first study to provide a new insight into a possible etiology for breast cancer formation in individuals exposed to low dose radiation and tobacco smoke.

Optimization of Culture Conditions to Support Undifferentiated Growth of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5 approximately 10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies.

The cytotoxic effect of Bowman–Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

Selective FLT3 Inhibitor FI-700 Neutralizes Mcl-1 and Enhances p53-Mediated Apoptosis in Acute Myeloid Leukemia (AML) Cells with Activating Mutations of FLT3 Via Mcl-1/Noxa Axis.

Abstract 2597Poster Board II-573 Introduction:Activating mutations of the Fms-like tyrosine kinase-3 gene (FLT3) occur in approximately 30–40% of acute myeloid leukemia (AML) patients. FLT3 mutations confer numerous oncogenic properties, including dysregulated proliferation, resistance to apoptosis and a block in differentiation. FLT3 mutations result in abnormal activation of the downstream pathways, including signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase kinase (Mek)/extracellular signal–regulated kinase (Erk) and phosphatidylinositol-3 kinase (PI3K)/Akt. Activation of these downstream effectors has been thought to allow leukemia cells to evade apoptosis. Targeting of FLT3 mutations is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML.Results:FI-700 induced G1-phase cell cycle arrest and apoptosis as evidenced by increased sub-G1 DNA content and phosphatidylserine externalization in FLT3/ITD MOLM-13 (FLT3-ITD, wild-type (wt)-p53) and MV4-11NR (FLT3-ITD, mutated-p53) AML cells. FI-700 did not affect cell cycle distribution patterns nor did it induce apoptosis in FLT3/WT OCI-AML-3 (FLT3/WT, wt-p53) and HL-60 (FLT3/WT, del (del)-p53). Wt-p53 MOLM-13 and OCI-AML-3 cells were susceptible to Nutlin-induced apoptosis. FI-700 augmented Nutlin-induced Bax activation, mitochondrial membrane potential (MMP) loss, caspase-3 activation and phosphatidylserine externalization in MOLM-13 cells. FI-700 rapidly reduced Mcl-1 levels in FLT3/ITD cells, mainly by enhancing proteasomal Mcl-1 degradation. Levels of other Bcl-2 family proteins examined did not change significantly. Mcl-1 levels were only modestly reduced upon Nutlin treatment. The FI-700/Nutlin-3 combination profoundly reduced Mcl-1 levels. Immunoprecipitation/ immunoblotting results suggested that the drug combination results in a profound decrease in Mcl-1-bound Bim. FI-700 enhanced doxorubicin-induced apoptosis in FLT3/ITD MOLM-13 and MV4-11NR cells, suggesting that FI-700 can enhance both the p53-dependent and the p53-independent apoptotic effects of doxorubicin. Finally, cooperative apoptotic effects of FI-700/Nutlin-3 were seen in primary AML cells with FLT3/ITD. Conclusion:FLT3 inhibition by FI-700 immediately reduces anti-apoptotic Mcl-1 levels and enhances Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/ITD-expressing AML cells via the Mcl-1/Noxa axis. FLT3 inhibition, in combination with p53-inducing agents, might represent a potential therapeutic approach in AML with FLT3/ITD. Disclosures:No relevant conflicts of interest to declare.

Use of Human Amnion Epithelial Cells as a Feeder Layer to Support Undifferentiated Growth of Mouse Embryonic Stem Cells

Mouse and human embryonic stem (ES) cells are usually maintained in an undifferentiated state by coculture with mouse embryonic fibroblasts (MEFs) as feeder cells. In the case of mouse ES cells, addition of leukemia inhibitory factor (LIF) to culture media is also necessary to prevent cell differentiation. Here, we report the use of primary human amnion epithelial cells (hAECs) as feeder cells to culture mouse ES cells using our newly developed protocols. We found that mouse ES cells grown on hAECs express ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT. Interestingly, the expression levels of these genes are three- to five fold higher on hAECs than on MEFs by quantitative real-time PCR. The quicker growing ES cells on hAECs showed a normal 19XY karyotype on passages 25, and ruled out the transformation of ES cells. Using flow cytometry analysis, we show that ES cells grown on hAECs have the same cell cycle distribution pattern as those on MEFs. Further, mouse ES cells cultured on hAECs for at least 20 passages retain the capability of teratoma formation in mice. Finally, we reveal that hAECs express highly LIF that allows for ES growth without the need of addition of commercially obtained LIF. Taken together, our data suggest that hAECs are suitable for mouse ES cell culture and may prove to be a useful alternative to MEFs for human ES cell culture.

Radiation Protection by a New Chemical Entity, Ex-Rad™: Efficacy and Mechanisms

Ex-Rad is among a series of small molecule kinase inhibitors developed for modifying cell cycle distribution patterns in cancer cells subjected to radiation therapy, and it has been identified as a potential candidate for radiation protection studies. We have investigated its radioprotective efficacy using mouse and in vitro models. Thirty-day survival studies with C3H/HeN male mice revealed 88% survival when 500 mg/kg of Ex-Rad was injected subcutaneously 24 h and 15 min before gamma irradiation with 8.0 Gy. To understand Ex-Rad's mechanism of action, we also studied its radioprotective efficacy in lung fibroblast (HFL-1), skin fibroblast (AG1522) and human umbilical vein endothelial cells (HUVECs). Colony-forming assays indicated that Ex-Rad protected cells from radiation damage after exposure to (60)Co gamma radiation. A study using single-cell gel electrophoresis (SCGE; also known as the alkaline comet assay) showed that Ex-Rad protected cells from radiation-induced DNA damage. Western blot analyses indicated that the radiation protection provided by Ex-Rad resulted in reduced levels of pro-apoptosis proteins such as p53 as well as its downstream regulators p21, Bax, c-Abl and p73, indicating that Ex-Rad could rescue cells from ionizing radiation-induced p53-dependent apoptosis. In conclusion, it appears that Ex-Rad's radioprotective mechanisms involve prevention of p53-dependent and independent radiation-induced apoptosis.

In vitro sequence-dependent interaction between nedaplatin and paclitaxel in human cancer cell lines

To define the most effective combination schedule of paclitaxel and nedaplatin, a new platinum derivative, we investigated the in vitro interaction between these drugs in AZ-521 and NUGC-4 gastric adenocarcinoma and KSE-1 esophageal squamous carcinoma cell lines. Cytotoxic activity was determined by the WST-1 assay. Different treatment schedules of the two drugs were compared and evaluated for synergism, additivity, or antagonism using a quantitative method based on the median-effect principle of Chou and Talalay. Cell-cycle perturbation and apoptosis were evaluated by means of flow cytometry. Upon 24-h sequential exposure, the sequence paclitaxel followed by nedaplatin induced greater than additive effects in all of the cell lines, with synergistic interactions in NUGC-4 and KSE-1 cells. By contrast, antagonistic effects were observed with the reverse sequence. Simultaneous treatment resulted in either a synergistic or antagonistic effect, depending on the cell line. Therefore, the sequence paclitaxel followed by nedaplatin appears most active, at least in these three cell lines. Flow cytometric analyses at IC50 indicated that paclitaxel induced G2/M arrest with subsequent induction of apoptosis (56%) in the sub-G1 phase. When paclitaxel preceded nedaplatin, apoptosis was most prominent (70%) with pronounced G2/M arrest. By contrast, the reverse sequence yielded only 28% induction of apoptotic cells, with almost identical cell-cycle distribution patterns to those observed with nedaplatin alone, indicating that the activity of paclitaxel is abolished by pretreatment with nedaplatin. Our findings suggest that the interaction of nedaplatin and paclitaxel is highly schedule dependent and that the sequential administration of paclitaxel followed by nedaplatin should be thus incorporated into the design of a clinical trial.

Water-soluble polymeric polyphenols from cinnamon inhibit proliferation and alter cell cycle distribution patterns of hematologic tumor cell lines

To explore possible anti-cancer properties of water-soluble, polymeric polyphenols from cinnamon, three myeloid cell lines (Jurkat, Wurzburg, and U937) were exposed to increasing concentrations of an aqueous extract prepared from cinnamon (CE) for 24 h. Cell growth and cell cycle distribution patterns responded in a dose-dependent manner to CE. That is, an increase in the percentage of cells distributed in G2/M was observed in all three cell lines as the amount of CE increased. At the highest dose of CE, the percentage of Wurzburg cells in G2/M was 1.5- and 2.0-fold higher than those observed for Jurkat and U937 cells, respectively. Wurzburg cells lack the CD45 phosphatase and may be more sensitive to imbalances in signaling through kinase/phosphatase networks that promote growth. The results suggest the potential of CE to interact with phosphorylation/dephosphorylation signaling activities to reduce cellular proliferation in tandem with a block at the G2/M phase of the cell cycle.