P21 Cip1 Expression Research Articles

Stimulation of cellular senescent processes, including secretory phenotypes and anti-oxidant responses, after androgen deprivation therapy in human prostate cancer

Endocrine resistance is a major problem in prostate cancer. Recent studies suggest that cellular plasticity plays a key role in therapy resistance. Yet little is known about the cellular changes of human prostate cancer after androgen deprivation therapy (ADT). In this study, we investigated cellular senescence, senescence-associated secretory phenotypes (SASPs), and anti-oxidant responses. Hormone ablation upregulated senescence-associated (SA)-β-Gal activity in prostate glands, as well as the expressions of p27KIP1 and p53, in a mouse castration model. In line with this, the expressions of p21CIP1 and p27KIP1 were significantly more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. In a study of SASP markers, the expressions of IL6 and IL8 were also more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. IL6, IL8, and MMP2 were expressed more strongly in human prostate cancer specimens resected after ADT than in untreated tumors. Of note, treatment with the anti-oxidant reagent NAC significantly suppressed SA-β-Gal activity in androgen-sensitive human prostate cancer LNCaP cells. In immunohistochemical analyses on anti-oxidant response genes, NRF2 and NQO1 were more upregulated after hormone ablation in human prostate gland and carcinoma specimens after ADT than in untreated specimens or in murine prostate glands after castration. Taken together, these findings suggest that ADT induces cellular senescence processes accompanied by secretory phenotypes and anti-oxidant responses in prostate. These cellular changes may be attractive targets for preventing endocrine resistance in prostate cancer.

P27Kip1 represses the Pitx2-mediated expression of p21Cip1 and regulates DNA replication during cell cycle progression.

The tumor suppressor p21 regulates cell cycle progression and peaks at mid/late G1. However, the mechanisms regulating its expression during cell cycle are poorly understood. We found that embryonic fibroblasts from p27 null mice at early passages progress slowly through the cell cycle. These cells present an elevated basal expression of p21 suggesting that p27 participates to its repression. Mechanistically, we found that p27 represses the expression of Pitx2 (an activator of p21 expression) by associating with the ASE-regulatory region of this gene together with an E2F4 repressive complex. Furthermore, we found that Pitx2 binds to the p21 promoter and induces its transcription. Finally, silencing Pitx2 or p21 in proliferating cells accelerates DNA replication and cell cycle progression. Collectively, these results demonstrate an unprecedented connection between p27, Pitx2 and p21 relevant for the regulation of cell cycle progression and cancer and for understanding human pathologies associated with p27 germline mutations.

Potent anti-proliferative actions of a non-diuretic glucosamine derivative of ethacrynic acid

Ethacrynic acid (EA), a known inhibitor of the neoplastic marker glutathione S-transferase P1 and other GSTs, exerts a weak antiproliferative activity against human cancer cells. The clinical use of EA (Edecrin) as an anticancer drug is limited by its potent loop diuretic activity. In this study, we developed a non-diuretic 2-amino-2-deoxy-d-glucose conjugated EA (EAG) to target tumors cells via the highly expressed glucose transporter 1 (GLUT1). Cell survival assays revealed that EAG had little effect on normal cells, but was cytotoxic 3 to 4.5-fold greater than EA. Mechanistically, the EAG induced selective cell death in cancer cells by inhibiting GSTP1 and generating abundant reactive oxygen species. Furthermore, EAG induced p21cip1 expression and a G2/M cell cycle block irrespective of the p53 gene status in tumor cells. These data encourage the development of new EA analogs.

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Monitoring Ligand-Induced Protein Ordering in Drug Discovery

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: A platform for the preparation of single-enantiomer cis-imidazolines for protein–protein inhibition. J. Org. Chem. 79, 6913–6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity (Kd value of 150nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly (Kd values from 600nM to 12μM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21Cip1 expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure–activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation.

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21CIP1. Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34+ progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34+ progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34+ cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21CIP1 mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21CIP1 expression during APL differentiation.

Targeting the Hedgehog Signaling Pathway By PF-04449913 Limits the Self-Renewal of MDS-Derived Induced Potent Stem Cells (iPSC): Molecular Mechanisms

Background:Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. In xenograft models of human coloirectal and pancreatic cancer, treatment with PF-04449913 in combination with other anticancer agents reduced the tumor growth. Furthermore, PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. In the present study, we investigated the molecular mechanisms by which PF-04449913 regulate the self-renewal of MDS-derived iPS cells (iPSCs) in vivo.Methods:We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO clssification) with chromosome 5 deletion and complex karyotypic abnormalities, respectivly. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected sucutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line establised from MDS patient with del(5q) and complex karyotypic abnormalities for in vitro studies.Results:Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38-positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38-positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. We further examined the effects of Nanog pathway modulation on in vitro clonogenic growth. CD34+CD38- cells from MDS-derived iPSCs transferred NOD/SCID mice and MDS-L cells were treated with 2 mM of PF-04449913 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. PF-04449913 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by PF-04449913 (p<0.001). To identify the mechanisms that limit the self-renewal of MDS-initiating ells by PF-04449913, NOD/SCID mice engrafted with CD34+CD38- fractions from MDS-derived iPSCs were treated with PF-04449913 (100 mg/kg; p.o.) for 14 days. PF-04449913 induced the expressions of p21Cip1, cleaved PARP and reduced the expression of BMI-1, c-Myc, Nanog, and Bcl-XL.Conclusion:Our preclinical results indicate that PF-04449913 have potential as an important option for controlling the drug-resistant MDS-initiating cells. It is expected that the PF-04449913 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including MDS, where the persistence of cancer stem cells. DisclosuresOhyashiki:Sumitomo Dainippon: Membership on an entity's Board of Directors or advisory committees; Chugai Pharna KK: Research Funding; Bristol Meyer Squib KK: Research Funding; Jansen Pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis Pharma KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Kirin KK: Honoraria; MSD KK: Honoraria; Nippo Shinyaku KK: Speakers Bureau; Toyama Kagaku KK: Speakers Bureau; Shinbaio Pharma KK: Honoraria; Asteras: Research Funding; Alexion Pharma KK: Research Funding; Teijin Pharma KK: Research Funding; Asahikasei: Research Funding; Taiho Yakuhin KK: Research Funding.

Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization

IntroductionPrimary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. MethodsPrimary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. ResultsThese cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21cip1, a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. DiscussionTaken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.

P27Kip1 and p21Cip1-independent proliferative inhibition of vascular smooth muscle cells cultured in type-I collagen matrix honeycombs

The proliferation of vascular smooth muscle cells (SMCs) contributes to atherosclerotic plaque formation and restenosis. Cyclin-dependent kinase inhibitors, such as p27Kip1 and p21Cip1, are known to play significant roles in the control of the aberrant proliferation of SMCs. Primary cultured SMCs stop proliferating immediately when cultured in three-dimensional matrices of type-I collagen “honeycombs” structures. To clarify whether p27Kip1 and p21Cip1 are involved in the proliferative inhibition of SMCs cultured in honeycombs, the characteristics of SMCs derived from the aorta of both wild-type mice (p27[+/+] SMCs) and p27Kip1 knockout mice (p27[−/−] SMCs) were investigated. Although the growth of p27(−/−) SMCs cultured on plates was faster than that of p27(+/+) SMCs, the number of both p27(+/+) and p27(−/−) SMCs did not change when they were cultured in honeycombs. p21Cip1 expression was decreased but maintained in p27(−/−) SMCs cultured on plates and in honeycombs. Knockdown of p21Cip1 in p27(−/−) SMCs promoted proliferation on plates. On the contrary, p21Cip1 knockdown had no effect on the proliferation of p27(−/−) SMCs cultured in honeycombs. In conclusion, p27Kip1 and p21Cip1 are insufficient for the proliferative inhibition of SMCs cultured in honeycombs.

Transcription factor LSF (TFCP2) inhibits melanoma growth.

Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus.

The RNA-binding protein Sam68 regulates tumor cell viability and hepatic carcinogenesis by inhibiting the transcriptional activity of FOXOs.

Src associated in mitosis (Sam68; 68 kDa) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family, and has been implicated in the oncogenesis and progression of several human cancers. Our study aimed to investigated the clinicopathologic significance of Sam68 expression and its role in cell proliferation and the underlying molecular mechanism in hepatocellular carcinoma (HCC). We demonstrated that Sam68 expression was significantly increased in HCC and high expression of Sam68 was significantly associated with Edmondson grade, tumor size, tumor nodule number, HBsAg status and Ki-67 expression. The Kaplan-Meier survival curves showed that increased expression of Sam68 was correlated with poor prognosis in HCC patients and served as an independent prognostic marker of overall survival in a multivariable analysis. In addition, through serum starvation and refeeding assay, we demonstrated that Sam68 was lowly expressed in serum-starved HCC cells, and was progressively increased after serum-additioning. Furthermore, siRNA knockdown of endogenous Sam68 inhibited cell proliferation and tumourigenicity of HCC cells in vitro, through blocking the G1 to S phase transition. Moreover, we reported that the anti-proliferative effect of silencing Sam68 was accompanied with up-regulated expression of cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), enhanced transactivation of FOXO factors (FOXO4), and dysreuglation of Akt/GSK-3β signaling. Taken together, these findings provide a rational framework for the progression of HCC and thereby indicated that Sam68 might be a novel and useful prognostic marker and a potential target for human HCC treatment.

Wogonin inhibits the proliferation of myelodysplastic syndrome cells through the induction of cell cycle arrest and apoptosis.

The present study aimed to assess the effects of the flavonoid, wogonin, and its underlying mechanism on myelodysplastic syndrome (MDS) in SKM-1 cells. In the present study, wogonin inhibited the cell proliferation of SKM-1 cells in a dose- and time-dependent manner, with the concentration required to yield a half maximal inhibitory concentration (IC50) of 212.1 µmol/l at 24 h, and 43.4 µmol/l at 72 h. Furthermore, wogonin induced cell cycle arrest at the G0/G1 phase and induced the apoptosis of the SKM-1 cells, which possibly accounted for the antiproliferative effects of wogonin. Notably, the data in the present study revealed that wogonin upregulated the expression of p21Cip1 and p27Kip1, and downregulated the expression of cyclin D1 and cyclin-dependent kinase 4, causing a G0/G1 phase arrest, halting cell cycle progression, and inducing apoptosis in the MDS cells, which was mediated by the mitochondrial pathway through a modulation of the ratio of Bcl-2 to Bax. Therefore, the present study suggests that wogonin may be a logical therapeutic target in the treatment of MDS.

Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p&lt;0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p&lt;0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p&lt;0.05) and activation of Nox1 (~1.7 fold; p&lt;0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.

Elevated expression of FoxM1 promotes the tumor cell proliferation in hepatocellular carcinoma.

The Forkhead box M1 (FoxM1) transcription factor plays crucial roles in multiple biological processes, including cell proliferation, differentiation, migration, and transformation. Recent studies have reported that aberrant expression of FoxM1 was found in a variety of human cancers. However, the expression pattern of FoxM1 and its clinical significance in human hepatocellular carcinoma (HCC) have not been well characterized to date. In this study, the expression of FoxM1 was evaluated in 46 pairs of human HCC, the adjacent non-tumorous liver tissues, and 12 pairs of normal liver tissues by immumohistochemistry. FoxM1 expression was upregulated in the HCC (76.09%) compared with non-tumorous liver tissues (39.13%) and normal liver tissues (8.33%) (P < 0.05). FoxM1 expression was significantly associated with tumor stage, tumor size, tumor number, integrality of tumor encapsulation, tumor thrombus, and AFP level (P < 0.05). Functionally, enforced expression of FoxM1 in HCC cell line (HHCC) remarkably enhanced cell proliferation in vitro and in vivo. Further analysis of cell cycle-related molecules showed that FoxM1 overexpression increased expressions of cyclin B1 and cyclin D1 but reduced expressions of p27(Kip1) and p21(Cip1). Our findings suggest that FoxM1 overexpression promotes HCC cell proliferation by cell cycle regulation, which is a potential target for hepatocellular carcinoma therapy.

Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue.

ObjectiveMacrophages are important producers of obesity-induced MCP-1; however, initial obesity-induced increases in MCP-1 production precede M1 macrophage accumulation in visceral adipose tissue (VAT). The initial cellular source of obesity-induced MCP-1 in vivo is currently unknown. Preliminary reports based on in vitro studies of preadipocyte cell lines and adherent stroma-vascular fraction cells suggest that resident stromal cells express MCP-1. In the past several years, elegant methods of identifying adipocyte progenitor cells (AdPCs) have become available, making it possible to study these cells in vivo. We have previously published that global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet-induced obesity, but it is unclear if Id3 plays a role in diet-induced MCP-1 production. We sought to determine the initial cellular source of MCP-1 and identify molecular regulators mediating MCP-1 production.MethodsId3+/+ and Id3−/− mice were fed either a standard chow or HFD for varying lengths of time. Flow cytometry, semi-quantitative real-time PCR, ELISAs and adoptive transfers were used to assess the importance of AdPCs during diet-induced obesity. Flow cytometry was also performed on a cohort of 14 patients undergoing bariatric surgery.ResultsFlow cytometry identified committed CD45−CD31−Ter119−CD29+CD34+Sca-1+CD24− adipocyte progenitor cells as producers of high levels of MCP-1 in VAT. High-fat diet increased AdPC numbers, an effect dependent on Id3. Loss of Id3 increased p21Cip1 levels and attenuated AdPC proliferation, resulting in reduced MCP-1 and M1 macrophage accumulation in VAT, compared to Id3+/+ littermate controls. AdPC rescue by adoptive transfer of 50,000 Id3+/+ AdPCs into Id3−/− recipient mice increased MCP-1 levels and M1 macrophage number in VAT. Additionally, flow cytometry identified MCP-1-producing CD45−CD31−CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue, with a higher percentage in omental adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in visceral adipose tissue.ConclusionsThis study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.

BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).

Abstract 2222: Possible involvement of senescence bypass in mesenchymal stem cells for sarcomagenesis identified through a comparative gene expression profiling in rat sarcoma model

Abstract INTRODUCTION. Mesenchymal stem cells (MSCs) exist in a variety of tissues such as bone marrow and adipose tissue contributing tissue repair and maintenance, and have been used in clinical cell transplantation therapy. On the other hand, MSCs are also believed to be one of the cell of origins for certain sarcomas including osteosarcoma and malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (MFH/UPS). To identify the signaling pathways that are involved in sarcoma pathogenesis, we compared gene expression profiles in rat osteosarcoma and MFH cells with those in syngeneic rat MSCs. MATERIALS &amp; METHODS. Rat osteosarcoma cell line COS1NR and MFH cell line MFH1NR, that were established in our laboratory from chemically induced tumors by 4-hydroxy(amino)-quinolin 1-oxide, and syngeneic rat MSCs isolated from rat femur bone marrow were used in this study. To investigate the difference of gene expression between sarcoma cells and MSCs, gene expression profiling was performed by Agilent array analysis. Data analysis was carried out using Agilent Feature Extraction software, analyzing pathways that were differentially expressed in sarcoma cells compared to MSCs. RESULTS &amp; DISCUSSION. Analysis of genes that characterize MSCs such as CD44, CD105, CD73 and CD90 showed higher expression in MSCs compared to sarcomas. Pathways involved in focal adhesion, cytokines, extracellular matrix-receptors and Wnt signaling were down-regulated in both sarcomas. In those factors, the genes involved in senescence acceleration in MSCs including Wnt4 and 5a, Lef1/tcf, β-catenin, p21Cip1, lysophosphatidic acid receptor 1 (LPAR1) are significantly down-regulated in both sarcomas. Meanwhile, DNA replication, cell cycle, mismatch repair, hedgehog signaling and metabolic pathways were up-regulated in both sarcomas and those include the genes involved in inhibiting senescence of MSCs such as DKK1, SOD1, FOXO4 and SIRT1. These results indicated that senescence factors for MSCs, which several previous reports suggested, such as Wnt pathways, p21Cip1 and CDKs-cyclins expression are mal-functionally regulated in sarcomas, and those have been suggested the involvement of cellular senescence in MSCs as the responses to oxidative stress and DNA damages. Taken together, acquiring the ability to bypassing senescence in MSCs is possibly involved in sarcomagenesis. CONCLUSIONS. The current study suggests that the loss of senescence mechanism in MSCs might be involved in sarcomagenesis. Further study will clarify the critical pathways containing the driver genes in sarcoma development upon the data that current results indicated and targeting those genes and pathways will be beneficial to develop novel treatment for sarcomas. Citation Format: Kanya Honoki, Hiromasa Fujii, Akira Kido, Shinji Tsukamoto, Toshio Mori, Yasuhito Tanaka, Toshifumi Tsujiuchi. Possible involvement of senescence bypass in mesenchymal stem cells for sarcomagenesis identified through a comparative gene expression profiling in rat sarcoma 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 2222. doi:10.1158/1538-7445.AM2015-2222

Abstract 3493: Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including pancreatic cancer. MGMT has been the focus of significant research for its role in the repair of DNA damage caused by chemotherapeutic agents. Aldehyde Dehydrogenase as a progenitor/stem cell marker has been linked to chemotherapy and radiation resistance. Methods: We tested the administration of Antabuse (disulfiram, DSF) at nontoxic doses in combination with gemcitabine (GEM) and Abraxane (ABX) on pancreatic cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases pancreatic cancer cell growth in a dose-dependent manner. DSF further sensitizes pancreatic cancer cells to GEM and ABX and significantly inhibits pancreatic cancer growth without causing unwanted side effects to the normal pancreatic cells. Dose effect and isobologram studies confirm synergistic activity of DSF and ABX combination and moderate synergism for DSF and GEM combination. DSF, either alone or in combination with GEM (DSF ± GEM) and/or ABX (DSF ± ABX), significantly inhibits expression of MGMT, aldehyde dehydrogenase, antigen Ki-67 and BIRC5 gene (survivin) - all involved in pancreatic cancer tumorigenesis. DSF+/-GEM+/-ABX induces the expression of p21Cip1, a cell cycle inhibitor. We show an inverse correlation between aldehyde dehydrogenase activity and MGMT expression, where MGMT expression is high, aldehyde dehydrogenase activity is low and where aldehyde dehydrogenase activity is high, MGMT levels were low. The ALDH3A1 expression is significantly higher in highly metastatic cancer cells (L3.6pl) compared to other metastatic cancer cells (PANC1, Mia PaCa2, ASPC1). We further report that in our model DSF at very low doses (achievable in human serum with standard DSF clinical dosing) effectively inhibits both aldehyde dehydrogenase and MGMT in pancreatic cancer cells. DSF also induces caspase activation indicative of mitochondrial induced apoptosis in these cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and aldehyde dehydrogenase potentially provides a novel therapeutic approach to inhibiting pancreatic cancer growth/metastasis and synergistically enhances GEM and ABX activity. Citation Format: George C. Bobustuc, Alisher Holmuhamedov, Kalkunte S. Srivenugopal, Jacob C. Frick, JAMES L. WEESE, Santhi D. Konduri. Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane. [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 3493. doi:10.1158/1538-7445.AM2015-3493

Abstract 2620: Plumbagin induces growth inhibition of human glioma cells by downregulating the expression and activity of FOXM1

Abstract Plumbagin, a natural quinonoid constituent isolated from the root of medicinal plant Plumbago zeylanica L, has exhibited anti-tumor and anti-proliferative activities in various tumor cell lines as well as in animal tumor models. However, its anticancer effects and the mechanisms underlying its suppression of glioma cell growth have not been elucidated. Oncogenic transcription factor Forkhead Box M1 (FOXM1) has garnered particular interest in recent years as a potential target for the prevention and/or therapeutic intervention in glioma, nevertheless, less information is currently available regarding FOXM1 inhibitor. Here, we reported that plumbagin could effectively inhibit cell proliferation, migration and invasion and induce apoptosis of glioma cells. Cell cycle assay showed that plumbagin induced G2/M arrest. Interestingly, we found that plumbagin decreased the expression of FOXM1 both at mRNA level and protein level. Plumbagin also inhibited the transactivation ability of FOXM1, resulting in down-regulating the expression of FOXM1 downstream target genes, such as cyclin D1, Cdc25B, survivin, and increasing the expression of p21CIP1 and p27KIP1. Most importantly, down-regulation of FOXM1 by siFOXM1 transfection enhanced plumbagin-induced change in viability. On the contrary, over-expression of FOXM1 by cDNA transfection reduced plumbagin-induced glioma cell growth inhibition. Further investigation revealed that plumbagin inhibition of glioma cell growth was also evident in a nude mouse model. Taken together, these results suggest that plumbagin exhibits its anticancer activity partially by inactivation of FOXM1 signaling pathway in glioma cells. Our findings indicate that plumbagin may be considered as a potential natural FOXM1 inhibitor, which could contribute to the development of new anticancer agent for therapy of gliomas. Citation Format: Xuejiao Liu, Wei Cai, Mingshan Niu, Rutong Yu. Plumbagin induces growth inhibition of human glioma cells by downregulating the expression and activity of FOXM1. [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 2620. doi:10.1158/1538-7445.AM2015-2620

Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21(Cip1) expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21(Cip1) expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21(Cip1) promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21(Cip1) gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21(Cip1) promoter, and that this is associated with enhanced p21(Cip1) expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation.