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2.
Elife ; 112022 05 04.
Article in English | MEDLINE | ID: mdl-35507394

ABSTRACT

Many age-associated changes in the human hematopoietic system have been reproduced in murine models; however, such changes have not been as robustly explored in rats despite the fact these larger rodents are more physiologically similar to humans. We examined peripheral blood of male F344 rats ranging from 3 to 27 months of age and found significant age-associated changes with distinct leukocyte population shifts. We report CD25+ CD4+ population frequency is a strong predictor of healthy aging, generate a model using blood parameters, and find rats with blood profiles that diverge from chronologic age indicate debility; thus, assessments of blood composition may be useful for non-lethal disease profiling or as a surrogate measure for efficacy of aging interventions. Importantly, blood parameters and DNA methylation alterations, defined distinct juncture points during aging, supporting a non-linear aging process. Our results suggest these inflection points are important considerations for aging interventions. Overall, we present rat blood aging metrics that can serve as a resource to evaluate health and the effects of interventions in a model system physiologically more reflective of humans.


Our blood contains many types of white blood cells, which play important roles in defending the body against infections and other threats to our health. The number of these cells changes with age, and this in turn contributes to many other alterations that happen in the body as we get older. For example, the immune system generally gets weaker at fighting infections and preventing other cells from developing into cancer. On top of that, the white blood cells themselves can become cancerous, resulting in several types of blood cancer that are more likely to happen in older people. Many previous studies have examined how the number of white blood cells changes with age in humans and mice. However, our understanding of this process in rats is still poor, despite the fact that the way the human body works has more in common with the rat body than the mouse body. Here, Yanai, Dunn et al. have studied samples of blood from rats between three to 27 months old. The experiments found that it is possible to accurately predict the age of healthy rats by measuring the frequency of populations of white blood cells, especially a certain type known as CD25+ CD4+ cells. If the animals had any form of illness, their predicted age deviated from their actual age. Furthermore, while some changes in the blood were gradual and continuous, others displayed distinct shifts when the rats reached specific ages. In the future, these findings may be used as a tool to help researchers diagnose illnesses in rats before the animals develop symptoms, or to more easily establish if a treatment is having a positive effect on the rats' health. The work of Yanai, Dunn et al. also provides new insights into aging that could potentially aid the design of new screening methods to predict cancer and intervene using a model system that is more similar to humans.


Subject(s)
Aging , Leukocytes , Aging/genetics , Animals , DNA Methylation , Male , Mice , Population Dynamics , Rats , Rats, Inbred F344
3.
Immunity ; 54(11): 2465-2480.e5, 2021 11 09.
Article in English | MEDLINE | ID: mdl-34706222

ABSTRACT

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Epigenomics , Gene Expression Regulation , Immunity , Transcription Factors/metabolism , Transcriptome , Epigenomics/methods , Humans , Immune System/cytology , Immune System/immunology , Immune System/metabolism , Transcription Factors/genetics
4.
Front Immunol ; 9: 549, 2018.
Article in English | MEDLINE | ID: mdl-29632530

ABSTRACT

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naïve CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naïve CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCd4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naïve CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naïve CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays a critical role in execution of activation-induced CD8+ T cell proliferation.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Cell Cycle/physiology , Cyclin-Dependent Kinase Inhibitor p16/physiology , Cyclin-Dependent Kinase Inhibitor p57/physiology , Enhancer of Zeste Homolog 2 Protein/physiology , Animals , Antigens/immunology , Apoptosis , Cell Proliferation , Listeria monocytogenes , Listeriosis/immunology , Mice, Knockout , Ovalbumin/immunology
5.
Cell Signal ; 36: 176-188, 2017 08.
Article in English | MEDLINE | ID: mdl-28495590

ABSTRACT

Activation of ß2-adrenergic receptor (ß2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a bivalent compound that agonizes ß2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent ß2AR activation and GPR55 blockade in C6 glioma cells using (R,R')-MNF as a marker ligand. Our data show that (R,R')-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R')-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of ß2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R')-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R')-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of ß-catenin occurred with (R,R')-MNF, and we provided new evidence of (R,R')-MNF-mediated inhibition of oncogenic ß-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of ß2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.


Subject(s)
Brain Neoplasms/metabolism , Carcinogenesis/metabolism , Glioma/metabolism , Receptors, Adrenergic, beta-2/metabolism , Receptors, Cannabinoid/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Animals , Apoptosis/drug effects , Brain Neoplasms/pathology , Carcinogenesis/drug effects , Carcinogenesis/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Colforsin/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Fenoterol/analogs & derivatives , Fenoterol/pharmacology , Glioma/pathology , Humans , Isoproterenol/pharmacology , Mice, Inbred BALB C , Mice, Nude , Proto-Oncogene Proteins c-akt/metabolism , Rats , Serum , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
6.
Environ Mol Mutagen ; 58(2): 84-98, 2017 03.
Article in English | MEDLINE | ID: mdl-28181292

ABSTRACT

Base excision repair (BER) is the major pathway for coping with most forms of endogenous DNA damage, and defects in the process have been associated with carcinogenesis. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central participant in BER, functioning as a critical endonuclease in the processing of noncoding abasic sites in DNA. Evidence has suggested that APE1 missense mutants, as well as altered expression or localization of the protein, can contribute to disease manifestation. We report herein that the tumor-associated APE1 variant, R237C, shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1-deficient mouse embryonic fibroblasts. Overexpression of wild-type APE1 or the R237C variant in the nontransformed C127I mouse cell line had no effect on proliferation, cell cycle status, steady-state DNA damage levels, mitochondrial function, or cellular transformation. A human cell line heterozygous for an APE1 knockout allele had lower levels of endogenous APE1, increased cellular sensitivity to DNA-damaging agents, impaired proliferation with time, and a distinct global gene expression pattern consistent with a stress phenotype. Our results indicate that: (i) the tumor-associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells. Environ. Mol. Mutagen. 58:84-98, 2017. © 2017 Wiley Periodicals, Inc.


Subject(s)
Cell Transformation, Neoplastic/genetics , DNA Damage/genetics , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Animals , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Knockout Techniques , Genetic Complementation Test , HCT116 Cells , Humans , Mesylates/pharmacology , Mice, Transgenic , Tamoxifen/pharmacology
7.
J Immunol ; 196(8): 3385-97, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26983789

ABSTRACT

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells.


Subject(s)
Aging/immunology , B-Lymphocyte Subsets/cytology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Lymphocyte Activation/immunology , 4-1BB Ligand/metabolism , Adult , Aged , Animals , Autoimmunity/immunology , B-Lymphocyte Subsets/immunology , B7-2 Antigen/biosynthesis , B7-2 Antigen/metabolism , Cells, Cultured , Cellular Senescence , Enzyme Activation/immunology , Female , Granzymes/biosynthesis , Histocompatibility Antigens Class I/metabolism , Humans , Macrophages, Peritoneal/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Neoplasms/immunology , Neoplasms/pathology , Receptors, Interferon/biosynthesis , Receptors, Interferon/metabolism , Receptors, Tumor Necrosis Factor, Type II/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , Interferon gamma Receptor
8.
Cancer Res ; 75(17): 3456-65, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26183924

ABSTRACT

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfßR1/TgfßR2 signaling. MDSC fully educated in this manner exhibit an increased production of reactive oxygen species and NO and more efficiently suppress CD4(+) and CD8(+) T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfßR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immunosuppressive and prometastatic functions of MDSC.


Subject(s)
B-Lymphocytes, Regulatory/immunology , Immunosuppression Therapy , Melanoma, Experimental/immunology , Myeloid Cells/immunology , Animals , B-Lymphocytes, Regulatory/pathology , Cell Lineage/immunology , Disease Models, Animal , Granulocytes/immunology , Granulocytes/pathology , Humans , Melanoma, Experimental/pathology , Mice , Monocytes/immunology , Monocytes/pathology , Myeloid Cells/pathology , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/immunology , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/immunology
9.
Aging Cell ; 14(2): 200-8, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25620312

ABSTRACT

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age-associated change is not fully understood. Here, we report the role of microRNA-125b (miR-125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T-cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age-related increase in CCL4. We then found the level of miR-125b was inversely correlated with the level of CCL4 in these cells, and the level of miR-125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock-down of miR-125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR-125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR-125b action requires the 'seed' sequence in 3'UTR of CCL4. Together these findings demonstrated that miR-125b is a negative regulator of CCL4 and its reduction is partially responsible for the age-related increase of CCL4.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cellular Senescence/immunology , Chemokine CCL4/biosynthesis , MicroRNAs/immunology , Monocytes/immunology , 3' Untranslated Regions , Age Factors , Aged , Chemokine CCL4/genetics , Chemokine CCL4/immunology , Humans , Lymphocyte Activation , MicroRNAs/biosynthesis , MicroRNAs/genetics
10.
Stem Cell Reports ; 3(1): 185-203, 2014 Jul 08.
Article in English | MEDLINE | ID: mdl-25068131

ABSTRACT

Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.


Subject(s)
Epitopes/analysis , Epitopes/immunology , Glycoproteins/immunology , Glycoproteins/metabolism , Pluripotent Stem Cells/immunology , Pluripotent Stem Cells/metabolism , Proteome/analysis , Cells, Cultured , Flow Cytometry , Humans , Immunohistochemistry , Real-Time Polymerase Chain Reaction
11.
PLoS One ; 8(4): e61836, 2013.
Article in English | MEDLINE | ID: mdl-23637916

ABSTRACT

Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , E2F1 Transcription Factor/metabolism , Ovarian Neoplasms/metabolism , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Diclofenac/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , E2F1 Transcription Factor/genetics , Female , Gene Expression , Gene Expression Regulation, Neoplastic/drug effects , Humans , Indomethacin/pharmacology , Mice , Mice, Nude , Ovarian Neoplasms/genetics , Xenograft Model Antitumor Assays
12.
Cancer Res ; 73(7): 2127-38, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23365136

ABSTRACT

The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivation of tumor-evoked regulatory B cells (tBreg). Here, we report the unexpected finding that B-cell depletion by CD20 antibody will greatly enhance cancer progression and metastasis. Both murine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo-targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) can block cancer metastasis by inhibiting CD20(Low) tBregs. Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B-expressing cytolytic CD8(+) T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo.


Subject(s)
4-1BB Ligand/metabolism , Antibodies, Monoclonal/pharmacology , Antigens, CD20/immunology , B-Lymphocytes, Regulatory/immunology , Breast Neoplasms/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lung Neoplasms/secondary , Melanoma, Experimental/pathology , Animals , Antigens, CD20/metabolism , B-Lymphocytes, Regulatory/metabolism , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , Disease Progression , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Oligodeoxyribonucleotides/pharmacology , Tumor Cells, Cultured
13.
PLoS One ; 7(8): e42350, 2012.
Article in English | MEDLINE | ID: mdl-22936984

ABSTRACT

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle/physiology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Trans-Activators/metabolism , Animals , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Line , Chromatin Immunoprecipitation , Mice , Models, Theoretical , Oligonucleotide Array Sequence Analysis , Trans-Activators/genetics
14.
J Pharmacol Exp Ther ; 343(1): 157-66, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22776956

ABSTRACT

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Apoptosis/physiology , Carcinoma, Hepatocellular/metabolism , Fenoterol/pharmacology , Liver Neoplasms/metabolism , Receptors, Cannabinoid/metabolism , Apoptosis/drug effects , Carcinoma, Hepatocellular/pathology , Fenoterol/chemistry , Hep G2 Cells , Humans , Liver Neoplasms/pathology
16.
Mol Cell Proteomics ; 11(8): 303-16, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22493178

ABSTRACT

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.


Subject(s)
Cell Separation/methods , Glycoproteins/analysis , Immunophenotyping/methods , Membrane Proteins/analysis , Pluripotent Stem Cells/metabolism , Proteomics/methods , Animals , Cells, Cultured , Cytokine Receptor gp130/analysis , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/transplantation , Fibroblasts/cytology , Fibroblasts/metabolism , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Mass Spectrometry , Mice , Mice, 129 Strain , Mice, Transgenic , Microscopy, Confocal , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Teratoma/metabolism , Teratoma/pathology
17.
Aging Cell ; 11(3): 500-8, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22385834

ABSTRACT

An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it affects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30 months. In fresh and early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, an increase in the acceleration of cell cycle S and G2 phases, decrease in the G1/G0 phase, and an increase in PDGF and its receptors confer elevated proliferative capacity, compared to young VSMC. Increased coexpression and physical interaction of MFG-E8 and integrin αvß5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or overexpressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation, and promotes proliferation, via αvß5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.


Subject(s)
Antigens, Surface/metabolism , Integrins/metabolism , Milk Proteins/metabolism , Muscle, Smooth, Vascular/cytology , Age Factors , Animals , Antigens, Surface/biosynthesis , Antigens, Surface/genetics , Cell Growth Processes/physiology , Immunohistochemistry , MAP Kinase Signaling System , Male , Milk Proteins/biosynthesis , Milk Proteins/genetics , Muscle, Smooth, Vascular/metabolism , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Rats , Rats, Inbred BN , Rats, Inbred F344 , Rats, Sprague-Dawley
18.
Nucleic Acids Res ; 40(4): 1621-35, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22013166

ABSTRACT

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.


Subject(s)
Antigens, Neoplasm/metabolism , Cell Cycle , DNA Topoisomerases, Type II/metabolism , DNA, Catenated/metabolism , DNA-Binding Proteins/metabolism , RecQ Helicases/metabolism , Apoptosis , Cell Cycle Checkpoints , Cell Line , Cell Proliferation , Chromosome Aberrations , Exodeoxyribonucleases/metabolism , Humans , Metaphase/genetics , RecQ Helicases/antagonists & inhibitors , Werner Syndrome Helicase
19.
Stem Cells Int ; 2011: 214203, 2011.
Article in English | MEDLINE | ID: mdl-21912557

ABSTRACT

Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs) are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

20.
Mol Cell Biol ; 31(18): 3790-801, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21768308

ABSTRACT

The cellular abundance of topoisomerase IIα (TOP2A) critically maintains DNA topology after replication and determines the efficacy of TOP2 inhibitors in chemotherapy. Here, we report that the RNA-binding protein HuR, commonly overexpressed in cancers, binds to the TOP2A 3'-untranslated region (3'UTR) and increases TOP2A translation. Reducing HuR levels triggered the recruitment of TOP2A transcripts to RNA-induced silencing complex (RISC) components and to cytoplasmic processing bodies. Using a novel MS2-tagged RNA precipitation method, we identified microRNA miR-548c-3p as a mediator of these effects and further uncovered that the interaction of miR-548c-3p with the TOP2A 3'UTR repressed TOP2A translation by antagonizing the action of HuR. Lowering TOP2A by silencing HuR or by overexpressing miR-548c-3p selectively decreased DNA damage after treatment with the chemotherapeutic agent doxorubicin. In sum, HuR enhances TOP2A translation by competing with miR-548c-3p; their combined actions control TOP2A expression levels and determine the effectiveness of doxorubicin.


Subject(s)
Antigens, Neoplasm/genetics , Antigens, Surface/metabolism , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Doxorubicin/pharmacology , RNA-Binding Proteins/metabolism , 3' Untranslated Regions , Antibiotics, Antineoplastic/pharmacology , Antigens, Neoplasm/biosynthesis , Antigens, Surface/genetics , DNA/metabolism , DNA Damage/drug effects , DNA Topoisomerases, Type II/biosynthesis , DNA-Binding Proteins/biosynthesis , ELAV Proteins , ELAV-Like Protein 1 , Gene Expression Regulation , HeLa Cells , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Poly-ADP-Ribose Binding Proteins , Protein Biosynthesis , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , RNA-Induced Silencing Complex/metabolism
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