S-phase Cell Population Research Articles

Total velvet-antler polypeptide extract from Cervus nippon Temminck induces cell proliferation and activation of the PI3K–Akt signalling pathway in human peripheral blood lymphocytes

Velvet antler (VA) has been used for centuries as a traditional medicine to treat a wide range of health problems. VA was first recorded in the Chinese medical classics Shen Nong Ben Cao Jing 2000 years ago. VA has been extensively used to strengthen bones and muscles, promote blood flow and increase immune function. In the present study, we used VA (Cervus nippon Temminck) protein extract (VAPE) to induce cell proliferation and differentiation in human peripheral blood lymphocyte cell line 1301 cells. The results showed that 100 µg/mL of VAPE could significantly induce 1301-cell proliferation (151.3%), increase the population of S-phase cells and increase the expression of CDK4. In addition, VAPE could effectively increase the expression levels of PI3K and Akt and activate PI3K–Akt signal pathway. These findings have provided novel insights into the further understanding of the molecular and signalling pathway mechanisms of VAPE in promoting proliferation of human peripheral blood lymphocyte cells.

Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, S-Phase Cycle Arrest and Extrinsic Apoptosis in Human Liver Hepatocellular Carcinoma HepG2 Cells.

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants. The toxic behavior and mechanism of PCBs individuals and congeners have been extensively investigated. However, there is only limited information on their metabolites. Our previous studies have shown that a synthetic PCB metabolite, PCB29-pQ, causes oxidative damage with the evidence of cytotoxicity, genotoxicity, and mitochondrial-derived intrinsic apoptosis. Here, we investigate the effects of PCB29-pQ on DNA damage checkpoint activation, cell cycle arrest, and death receptor-related extrinsic apoptosis in human liver hepatocellular carcinoma HepG2 cells. Our results illustrate that PCB29-pQ increases the S-phase cell population by down-regulating cyclins A/D1/E, cyclin-dependent kinases (CDK 2/4/6), and cell division cycle 25A (CDC25A) and up-regulating p21/p27 protein expressions. PCB29-pQ also induces apoptosis via the up-regulation of Fas/FasL and the activation of caspase 8/3. Moreover, p53 plays a pivotal role in PCB29-pQ-induced cell cycle arrest and apoptosis via the activation of ATM/Chk2 and ATR/Chk1 checkpoints. Cell cycle arrest and apoptotic cell death were attenuated by the pretreatment with antioxidant N-acetyl-cysteine (NAC). Taken together, these results demonstrate that PCB29-pQ induces oxidative stress and promotes p53-dependent DNA damage checkpoint activation, S-phase cycle arrest, and extrinsic apoptosis in HepG2 cells.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms.

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Abstract 3444: Identification of a miRNAs signature of radioresistance in a prostate cancer model

Abstract Introduction Radiotherapy (RT) has a prominent role when used as a single modality or in combination with androgen deprivation therapy (ADT) in the management of prostate cancer patients. However, even with currently available high-dose RT protocols, many patients develop recurrence or “biochemical failure” associated with a rise in PSA, and ultimately reduced overall survival. Key regulators of cancer cell behavior, dysregulation of miRNAs expression is associated with both prostate cancer pathogenesis and treatment resistance. This study aimed to identify a miRNAs signature of radioresistance in a prostate cancer model, as a prerequisite for the future development of a novel pre-treatment assay for the identification of radiotherapy prostate cancer patients at risk of biochemical failure. Methods Two models of radioresistance were used : (1) an isogenic radiation resistant model generated through weekly exposure to 2-Gy fractionated ionising radiation of 22Rv1 (primary) prostate cancer (CaP) cells to a cumulative dose of 60Gy. (2) chronically hypoxic 22Rv1 cells (48hrs). miRNA profiling of radioresistant and wild type cells was performed using the Exiqon miRCURY array and overlapping differentially expressed miRs were identified. Results The radiation survival curve of the resulting subline RR22Rv1 was associated with a significant increase in clonogenic survival (1.3 fold increase in survival after 2Gy and 2.2 fold increase after 10Gy), when compared to both parent 22Rv1 and aged-matched controls. The RR22Rv1 cell line is associated with decreased sensitivity to DNA damage (comet assay), enlargement of the S-phase cell population (PI staining) but no evidence for expansion of the senescent cell fraction (β-Gal assay). miRNA profiling identified 105 significantly differentially expressed miRNAs in RR22Rv1, when compared to the parent and age-matched control 22RV1 cells. A total of 12 miRNA were differentially expressed in chronically hypoxic compared to normoxic 22Rv1 cells. Three candidate miRNAs were associated with a radioresistant phenotype across the models: miR200a, miR210 and miR4284. Functional validation of these candidates is currently under way. Conclusion Available in tissues, serum and urine, miRNAs are ideal candidates for the development of novel biological tests. This study identifies a miRNAs signature of radioresistance in prostate cancer cells with potential for the development of a miRNA-based prognostic assay for radiotherapy prostate cancer patients. Citation Format: Niamh McDermott, Armelle Meunier, Christopher Hernandez, Karen J. Bowman, George D. Jones, Laure Marignol. Identification of a miRNAs signature of radioresistance in a prostate cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3444. doi:10.1158/1538-7445.AM2015-3444

Let-7b overexpression leads to increased radiosensitivity of uveal melanoma cells.

Uveal melanoma (UM) is an intraocular malignant tumor in adults that is characterized by rapid progression and recurrence. Irradiation has become the primary therapy for UM patients who are not candidates for surgery. However, after large-dose fraction irradiation treatment, some patients undergo subsequent enucleation because of radiotherapy-related complications. This situation has raised concerns on how to optimize the effectiveness of radiation treatment. Recent investigations of microRNAs are changing our understanding of UM tumor biology and are helping to identify novel targets for radiotherapy. The radioresistant UM cell lines OM431 and OCM1 were selected and exposed to irradiation, and let-7b was found to be downregulated after exposure. We then confirmed that let-7b mimics could inhibit UM growth both in vitro and in vivo. More specifically, transfection with let-7b mimics markedly resensitized OCM1 and OM431 cells to irradiation by reducing the population of S-phase cells. Cyclin D1 plays a vital role in cell cycle arrest, which is induced by let-7b overexpression. Cyclin D1 is also a target of let-7b and its expression is suppressed by upregulation of let-7b. Collectively, our results indicate that let-7b overexpression can in turn downregulate cyclin D1 expression and enhance the radiosensitivity of UM through cell cycle arrest. Let-7b could serve as a marker for radiosensitivity and could enhance the therapeutic benefit of UM cell irradiation.

The gadolinium-based contrast agent Omniscan® promotes in vitro fibroblast survival through in situ precipitation.

The current study aims to explore how the gadolinium (Gd)-based contrast agent (GBCA) Omniscan® enhanced cell viability of murine fibroblasts. The results of scanning electron microscopy showed that Omniscan® can precipitate in cell culture media and deposit on cell membranes. Energy-dispersive X-ray analysis and Fourier-transform infrared spectroscopy demonstrated the presence of Gd and phosphates in the agglomerated particles. By filtering the Omniscan®-containing medium through a 220 nm filter, it can be clearly found that the increased cell viability should be mainly attributed to the insoluble species of gadolinium rather than to chelated gadolinium. Moreover, the effects of other gadolinium-based contrast agents, Magnevist® and Dotarem®, were compared with that of Omniscan®. It is noted that the three contrast agents differed in their ability to induce cell viability, which is possibly ascribed to the different chemical stabilities of gadolinium chelates as demonstrated by the attenuation in cell growth upon the addition of excess ligands to the compounds. The results of flow cytometry analysis also showed that Omniscan® can promote cell growth via an increase in the S-phase cell population as evidenced by the elevated levels of cell cycle associated proteins cyclin D, cyclin A and the phosphorylated Rb protein. Furthermore, our results revealed that integrin-mediated signaling may play an important role in both Omniscan® and Magnevist®-enhanced focal adhesion formation since the blockade of integrins decreased the level of ERK phosphorylation induced by the two GBCAs. Taken together, these data suggested that in situ gadolinium phosphate precipitation formation mediated Omniscan®-promoted fibroblast survival, which is similar to that of gadolinium chloride. It was demonstrated that the application of GBCAs with more stable thermodynamic stability may cause less dissociation of the gadolinium ion and thus resulted in less precipitation, finally leading to lower occurrence of nephrogenic systemic fibrosis. The obtained results would also be helpful for the development of safe gadolinium-based contrast agents.

Gastrokine 1 inhibits gastrin-induced cell proliferation.

Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth. We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGS(GKN1) and MKN1(GKN1) gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses. Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGS(mock) and MKN1(mock), but not in HFE-145, AGS(GKN1), and MKN1(GKN1), cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae. These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138+ multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

Abstract 856: Combination of crizotinib and radiation in the treatment of ALK-positive and cetuximab-resistant lung cancer

Abstract Approximately 4% of patients with non-small cell lung carcinoma (NSCLC) carry the EML4-ALK chromosomal rearrangement. Crizotinib, a c-MET and ALK inhibitor, has shown significant therapeutic benefit as a single agent with FDA approval for the treatment of patients with ALK-positive NSCLC. Since radiation commonly plays a key role in lung cancer treatment, we sought to evaluate the potential of crizotinib as a radiation sensitizer and the role of ALK in cellular radiation sensitivity. We examined the effect of crizotinib in combination with radiation on ALK signaling, cell proliferation, cell cycle distribution and radiosensitivity in ALK-positive NSCLC cell lines H3122 and H2228 in vitro. We also examined the in-vivo effects of crizotinib in combination with radiation in H3122 and H2228 xenograft models. Crizotinib blocked phosphorylation of ALK and its downstream effectors and inhibited cell proliferation in a dose-dependent manner. The combination of crizotinib and radiation resulted in increased inhibition of cell growth. Although cells were sensitized to radiation by crizotinib, molecular knockdown of ALK by siRNA had only modest impact on cell radiosensitivity. Cell cycle analysis by flow cytometry showed that ALK-siRNA induced G1 arrest. Interestingly, low dose crizotinib induced G1 arrest, while doses above 2µM induced G2 arrest. Combination treatment induced arrest in G1 and G2 phases and decreased the cell population in S-phase. Moreover, in xenograft tumor models, we observed combined treatment to produce additive tumor growth inhibition over that observed with either drug or radiation alone. We further examined the capacity of crizotinib overcome cetuximab resistance in our established in vitro resistance models. Compared with parental cells, cetuximab-resistant cells showed cross-resistance to radiation. Crizotinib inhibited the growth of cetuximab-resistant cells and resensitized cells to radiation. Compared with single-agent treatment, the combination of crizotinib and erlotinib resulted in increased growth inhibition and further re-sensitization to radiation. These data suggest that crizotinib in combination with radiation can enhance the inhibitory effect on ALK-positive tumor cell growth in vitro and in-vivo and also suggest the potential value of using crizotinib in combination with other EGFR-targeting therapeutics. Ultimately however, the potential value of crizotinib as a radiation sensitizer requires systematic clinical investigation. Citation Format: Chunrong Li, Shyhmin Huang, Noah Walters, Eric A. Armstrong, Paul M. Harari. Combination of crizotinib and radiation in the treatment of ALK-positive and cetuximab-resistant lung 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 856. doi:10.1158/1538-7445.AM2014-856

Flow cytometric determination of 5-bromo-2'-deoxyuridine pharmacokinetics in blood serum after intraperitoneal administration to rats and mice.

5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

Metabolic analysis of the loss of Rb1 in vivo

Inactivation of the p16/pRB axis is a rate-limiting step in tumorigenesis. Loss of these tumor suppressors is well known to disrupt control of cell proliferation but, additionally, these mutations cause changes in cell metabolism. To investigate the metabolic changes resulting from pRB inactivation we have exploited a Drosophila model. Metabolomics and in vivo flux analysis revealed that RBF-deficient tissues have increased nucleotide production and struggle to maintain sufficient pools of reduced glutathione; ultimately leading to increased sensitivity to oxidative stress. We have now extended these studies to mammalian cell culture and mice. Depletion of pRB in non-tumorigenic hTERT-BJ cells leads to an increased cell population in S-phase and slight increases in the cell population doubling time. Acute loss of Rb1-/-in the mouse colon, but not the lung, leads to a robust increase in Ki67 staining within 96hrs. We therefore asked to what extent metabolic changes associated with proliferation increases are conserved between pRB-deficient cells in culture and acute loss of Rb1-/-in vivo. We performed isotopic tracer analyses with both glucose and glutamine and found that while loss of pRB in cell culture promotes increased isotopic enrichment from U13C-glutamine in TCA cycle intermediates, little to no change was seen in isotopic enrichment from U13C-glucose. In stark contrast, acute loss of Rb1 in the mouse colon leads to robust isotopic enrichment from U13C-glucose in TCA cycle intermediates and the pyrimidine precursor aspartate; while little to no change in isotopic enrichment from U13C-glutamine was found in all TCA cycle intermediates but alpha-ketoglutarate and no significant differences was seen in aspartate. Collectively these results reveal that loss of the tumor suppressor pRB has metabolic consequences in vivo.

Antitumor activity and induction of TP53-dependent apoptosis toward ovarian clear cell adenocarcinoma by the dual PI3K/mTOR inhibitor DS-7423.

DS-7423, a novel, small-molecule dual inhibitor of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), is currently in phase I clinical trials for solid tumors. Although DS-7423 potently inhibits PI3Kα (IC50 = 15.6 nM) and mTOR (IC50 = 34.9 nM), it also inhibits other isoforms of class I PI3K (IC50 values: PI3Kβ = 1,143 nM; PI3Kγ = 249 nM; PI3Kδ = 262 nM). The PI3K/mTOR pathway is frequently activated in ovarian clear cell adenocarcinomas (OCCA) through various mutations that activate PI3K-AKT signaling. Here, we describe the anti-tumor effect of DS-7423 on a panel of nine OCCA cell lines. IC50 values for DS-7423 were <75 nM in all the lines, regardless of the mutational status of PIK3CA. In mouse xenograft models, DS-7423 suppressed the tumor growth of OCCA in a dose-dependent manner. Flow cytometry analysis revealed a decrease in S-phase cell populations in all the cell lines and an increase in sub-G1 cell populations following treatment with DS-7423 in six of the nine OCCA cell lines tested. DS-7423-mediated apoptosis was induced more effectively in the six cell lines without TP53 mutations than in the three cell lines with TP53 mutations. Concomitantly with the decreased phosphorylation level of MDM2 (mouse double minute 2 homolog), the level of phosphorylation of TP53 at Ser46 was increased by DS-7423 in the six cell lines with wild-type TP53, with induction of genes that mediate TP53-dependent apoptosis, including p53AIP1 and PUMA at 39 nM or higher doses. Our data suggest that the dual PI3K/mTOR inhibitor DS-7423 may constitute a promising molecular targeted therapy for OCCA, and that its antitumor effect might be partly obtained by induction of TP53-dependent apoptosis in TP53 wild-type OCCAs.

Preclinical Evaluation of DO3A-Act-AQ: A Polyazamacrocyclic Monomeric Anthraquinone Derivative as a Theranostic Agent

An anthraquinone conjugated macrocyclic chelating agent, 2,2',2″-(10-(2-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid or DO3A-Act-AQ, was synthesized by reacting trisubstituted cyclen (DO3A) with 2-chloro-N-(9,10-dioxo-9,10-dihydro-anthracen-1-yl)-acetamide and radiolabeled with (68)GaCl3 in 84% efficiency and a specific activity of 4.62 MBq/nmol. The IC50 value for BMG-1 cells was 0.1 mM, while the same concentration of DO3A-Act-AQ rendered no significant toxicity in HEK cells. The exposure of BMG-1 cells with 0.1 mM DO3A-Act-AQ displayed a time-dependent increase in apoptosis (40.7% at 4 h and 53% at 24 h), and the effect was 2.8- and 3.6-fold % higher as seen in HEK cells. An increase in S-phase cell population suggested S-phase arrest concomitant with induction of apoptosis in BMG-1 cells reaching to 4.5 times after 24 h with respect to control cells. DNA binding studies on CT-DNA (calf thymus) revealed a quenching pattern in the presence of DO3A-Act-AQ (10-70 μM), and the Stern-Volmer quenching constant was 2.4157 × 10(6) L mol(-1), indicative of strong binding with ds-DNA. A decrease in the positive and negative bands of CT-DNA was seen at 278 nm and 240 nm, respectively, on addition of 0.05 mM of DO3A-Act-AQ in CD studies. (68)Ga-DO3A-Act-AQ was stable in vitro in both PBS and human serum for at least 2 h. The in vivo blood kinetics study performed on normal rabbits indicated fast clearance with t1/2(F) = 40 ± 0.3 min and t1/2(S) = 3 h 30 min ± 0.1 min. Ex vivo biodistribution analysis displayed a favorable tumor-to-muscle ratio of 8.4 after 2 h in athymic nude mice xenografted with BMG-1 cells, suggesting the specificity of (68)Ga-DO3A-Act-AQ toward tumors.

Bone Marrow Microenvironment Induces Stem Cell-Like Phenotype In Acute Myeloid Leukemia

Recent discoveries demonstrate that leukemia stem cells (LSCs) contribute towards leukemia progression and relapse. Several cell surface and intra-cellular markers including CD34+CD38-, CD123, aldehyde dehydrogenase (ALDH), side-population (SP) dye uptake, and TIM3 have been utilized to detect these cells, but the origin of LSCs has not been clearly understood. Here, we demonstrate for the first time that components of the bone marrow micro-environment (BME) such as mesenchymal stromal cells (MSCs) and hypoxia induce a stem cell phenotype in acute myeloid leukemia (AML) cells. Co-culture of AML cells with bone marrow (BM)-derived MSCs induced a time-dependent increase in ALDH activity (from 5.8 ± 3.3% to 36.9 ± 4.7%, p<0.01) in the leukemia cells. In addition, culture of AML cells in 1% oxygen increased the ALDH-active population from 0.5% ± 0.1% to 25.6% ± 3.2% (p<0.01), suggesting that stroma and hypoxia were sufficient by themselves for the induction of stem cell activity in leukemia cells. In addition, when AML cells were co-cultured with MSCs, SP cells were induced (from 0.2 ± 0.1% to 2.6 ± 0.8%; p<0.02). Similarly, SP cell phenotype was induced (from 0.2 ± 0.1% to 10.2 ± 2.4%; p<0.01) in AML cells cultured under hypoxia conditions, indicating that the micro-environment induces “stemness” in leukemia cells. Cell cycle analysis revealed that in OCI-AML3 cells cultured under hypoxic conditions, the S-phase cell population was reduced from 14.5 ± 0.1% to 5.02 ± 0.1% and cells G0/G1- increased from 68.02 ± 3.1% to 87.62 ± 2.7%, indicating that the leukemic cells become relatively quiescent in micro-environmental conditions. To examine the functional significance of stem-like changes, we analyzed chemo-resistance of leukemia cells. AML cells cultured under BME conditions were found to be >75% ± 8% resistant (based on AnnexinV+/PI+ staining) to cytrabine at 1, 2.5 and 5µM concentrations. Gene expression assessed by micro array and real-time RT-PCR of 19 ALDH isoforms identified ALDH1A3 and ALDH1A1 as upregulated by 10- and 3-fold, respectively in the leukemia cells cultured under BME conditions, suggesting that the increased ALDH activity was due to the upregulation of these ALDH isoforms. In addition, the embryonic stem cell markers oct4, nanog, and sox2, were distinctly upregulated by at least 2-fold when the leukemic cells were cultured in hypoxia. Recent findings from Look's group indicate an autocrine activation of cMet in AML. To investigate the expression of cMet, and its ligand hepatocyte growth factor (HGF), leukemia cells cultured under BME conditions were FACS-sorted and cMet expression was analyzed by qRT-PCR. We found that cMet expression increased by 8- to 10-fold in leukemia cells cultured under these conditions. In addition, cytokine antibody arrays revealed that HGF was upregulated by 6- to 8-fold in cell culture supernatants derived from the BME culture conditions. These data suggest a role for cMet signaling in the acquisition of stem cell features. Finally, to identify additional molecular factors that support our hypothesis, we performed a proteomics analysis using Kinexus® antibody arrays with 850 validated phospho and total proteins. These data revealed that proteins including β-catenin, Stat3, and Stat5B were upregulated, whereas GSK3a, p38a (T180+Y182), and IKKb were downregulated in the leukemic cells cultured under the BME conditions. Summary: the bone marrow microenvironment induces a stem cell-like phenotype in leukemia cells. We have identified several key signaling pathways including cMet, Wnt, NFκB and Stat3 that may lead to acquisition of stem cell phenotype under the influence of the BME. These factors may also facilitate leukemic cell survival during chemotherapy. Disclosures:No relevant conflicts of interest to declare.

MiR-150-5p inhibits the proliferation and promoted apoptosis of pancreatic cancer cells

To investigate the role of miR-150-5p in cell proliferation and apoptosis in human pancreatic cancer cell lines. The expression of miR-150-5p in pancreatic cancer was detected by real time qPCR analysis in 11 pairs of pancreatic cancer tissue and matched adjacent normal tissue samples and in 4 pancreatic cancer cell lines. PANC-1, MIA PaCa-2,BxPC-3 and AsPC-1 cells were transfected with chemically synthesized MiR-150-5p mimics, and CCK-8 assays was then performed to assess cellular functions. To fully understand the mechanisms by which miR-150-5p exerted its function, cell cycle analysis was performed on MIA PaCa-2 and PANC-1 cells 48 hours after transfection, by incubating with propidium iodide (PI)and subsequently analyzed by fluorescence-activated cell sorting (FACS) . Apoptosis assay was performed on MIA PaCa-2 and PANC-1 cell lines 24 hours after transfection using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) and analyzed by FACS. The expression of miR-150-5p was consistently lower in the pancreatic cancer tissues than in normal tissues, and the miR-150-5p was also down-regulated in pancreatic cancer cell lines (P < 0.05) . MiR-150-5p mimics transfection significantly raised the expression level of miR-150-5p mRNA in PANC-1 and MIA PaCa-2 (P < 0.01) . The CCK-8 proliferation assay showed that cell growth was reduced in 4 pancreatic cancer cell lines (AsPC-1, BxPC-3,MIA PaCa-2, PANC-1) of miR-150-5p transfected cells compared with NC-transfected cells. The inhibition rates were 50.7%, 48.6%, 30.8% and 42.3%, respectively (P < 0.01). The apoptotic rate was increased in cells transfected with miR-150-5p mimics (P < 0.01) . The cell cycle analysis in MIA PaCa-2 indicated that miR-150-5p treatment induced cell cycle arrest in G1 phase with a significant increase in the percentage of cells in G1 phase (P < 0.01), and a reduction of the S-phase cell population in MIA PaCa-2 and PANC-1 (P < 0.01). MiR-150-5p is down-regulated in pancreatic cancer. Over-expression of miR-150-5p inhibits cell proliferation, blocked the cell cycle, but promotes cell apoptosis in pancreatic cancer cells.

Abstract 4121: Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia.

Abstract The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. The type III receptor tyrosine kinase, FLT3 is widely expressed in AML and approximately 30% of AML patients carry an oncogenic FLT3-internal tandem duplication (ITD) mutation. We observed that Grb10 physically associates with normal and oncogenic FLT3 through FLT3 phospho-tyrosine 572 and 793 residues. Although Grb10 expression did not affect FLT3 activation or stability, its expression enhanced FLT3-mediated Akt and STAT5 phosphorylation without affecting Erk or p38 phosphorylation. Selective Grb10 depletion reduced Akt and STAT5 phosphorylation in Ba/F3 and OCI-AML-5 cells. Grb10 regulates cell cycle by accelerating G2-M transition followed by an increased cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation, survival and colony formation of Ba/F3-FLT3-ITD cells. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression correlated to relapse as well as to poor prognosis. In addition, Grb10 expression led to resistance to chemotherapy. These results suggest that Grb10 binds to FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells that furthermore initiates drug-resistance and poor prognosis in AML. Citation Format: Kazi J. Uddin, Lars Rönnstrand. Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2013-4121

Synthesis, structure, interaction with DNA and cytotoxicity of a luminescent copper(II) complex with a hydrazone ligand

A luminescent copper(II) complex [CuII(L)H2O]2SO4,1a was synthesized with the acyclic tridentate 2-hydroxy naphthaldehyde-2-pyridylhydrazone ligand, HL, 1. The molecular structure of 1a clearly shows that it is a monomeric copper(II) complex with CuN2OOW coordination with slightly distorted square planar geometry. The asymmetric unit of 1a contains two independent complex molecules and a sulfate anion. DNA binding interactions of 1a have been investigated by absorption, emission, viscosity and thermal denaturation studies. The cytotoxic activity of 1a was measured in vitro against the HeLa cells. The LD50 value was calculated as 4.97μM. There was no nuclear fragmentation observed after treatment with 1a, which revealed that the copper(II) complex 1a was not capable of inducing apoptosis. The cell cycle analysis of 1a indicated a dose dependent decrease of G0/G1 and S-phase cell population and dose-dependent increase of G2/M population compared with untreated control cell.

FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia

The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. We observed that Grb10 physically associates with FLT3 in response to FLT3-ligand (FL) stimulation through FLT3 phospho-tyrosine 572 and 793 residues and constitutively associates with oncogenic FLT3-ITD. Furthermore endogenous Grb10–FLT3 association was observed in OCI-AML-5 cells. Grb10 expression did not alter FLT3 receptor activation or stability in Ba/F3-FLT3 cells. However, expression of Grb10 enhanced FL-induced Akt phosphorylation without affecting Erk or p38 phosphorylation in Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD. Selective Grb10 depletion reduced Akt phosphorylation in Ba/F3-FLT3-WT and OCI-AML-5 cells. Grb10 transduces signal from FLT3 by direct interaction with p85 and Ba/F3-FLT3-ITD cells expressing Grb10 exhibits higher STAT5 activation. Grb10 regulates the cell cycle by increasing cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation and survival of Ba/F3-FLT3-ITD cells as well as increased colony formation in semisolid culture. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression partially correlated to relapse as well as to poor prognosis. These results suggest that Grb10 binds to both normal and oncogenic FLT3 and induces PI3K–Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells.

Egr-1 Promotes Cell Proliferation and Invasion by Increasing β-Catenin Expression in Gastric Cancer

Abnormal expression of early growth response gene 1 (Egr-1) and β-catenin may play a crucial role in the development and progression of human cancer. However, little is known about the expression and underlying molecular mechanisms in which Egr-1 and β-catenin are involved in the development and progression of gastric cancer. The purpose of this study was to elucidate the potential relationship between Egr-1 and β-catenin expression in gastric cancer, which contributes to finding new molecular carcinogenesis as a potential therapeutic target for gastric cancer. In a sample of 102 cases of human gastric cancer, the expression of Egr-1 and β-catenin was detected using immunohistochemistry. Egr-1 gene was transfected into gastric cancer SGC7901 cells and its role in proliferation and cell invasion was detected by MTT assay, flow cytometry, wound-healing and transwell invasion assay. Western blot analysis was used to study the expression of β-catenin and cyclin D1 proteins. Upregulated Egr-1 and β-catenin protein expression were strongly correlated with cancer progression and depth of invasion in gastric cancer. β-catenin, present mainly in cytoplasmic and nucleus of gastric cancer cells, was also positively correlated with Egr-1 expression in gastric cancer. Furthermore, the overexpression of Egr-1 upregulated β-catenin expression level, promoted cell proliferation, increased cell population in S-phase and enhanced gastric cancer cell migration and invasion in vitro. Egr-1 might contribute to gastric cancer proliferation and invasion through activation of the β-catenin signaling pathway.