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1.  Modulated Expression of Genes Encoding Estrogen Metabolizing Enzymes by G1-Phase Cyclin-Dependent Kinases 6 and 4 in Human Breast Cancer Cells 
PLoS ONE  2014;9(5):e97448.
G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers.
PMCID: PMC4029737  PMID: 24848372
2.  Step-Wise Epigenetic and Phenotypic Alterations Poise CD8+ T Cells to Mediate Airway Hyperresponsiveness and Inflammation 
The functional plasticity of CD8+ T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness (AHR) and inflammation and yet remains mechanistically undefined. We demonstrate that CD8+ T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8+ T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the step-wise molecular conversion of CD8+ T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA Pol II recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage following TCR stimulation, indicating that IL-4 induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro an atopic (IL-4) environment poises CD8+ T cells via step-wise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.
PMCID: PMC3622148  PMID: 23509358
CD8 T cells; IL-4; plasticity; IL-13; asthma
3.  Microbial Heat Shock Protein 65 Attenuates Airway Hyperresponsiveness and Inflammation by Modulating the Function of Dendritic Cells 
Heat shock proteins (HSPs), produced in response to stress are suppressive in disease models. We previously showed that Mycobacterium leprae HSP65 prevented development of airway hyperresponsiveness and inflammation in mice. Our goal here was to define the mechanism responsible for the suppressive effects of HSP. In one in vivo approach, BALB/c mice were sensitized to ovalbumin (OVA) followed by primary OVA challenges. Several weeks later, HSP65 was administered prior to a single, provocative secondary challenge. In a second in vivo approach, the secondary challenge was replaced by intratracheal instillation of allergen-pulsed bone marrow-derived dendritic cells (BMDCs). The in vitro effects of HSP65 on BMDCs were examined in co-culture experiments with CD4+ T cells. In vivo, HSP65 prevented development of airway hyperresponsiveness and inflammation. As well, Th1 cytokine levels in bronchoalveolar lavage (BAL) fluid were increased. In vitro, HSP65 induced notch receptor ligand Delta1 expression on BMDCs and HSP65-treated BMDCs skewed CD4+ T cells to Th1 cytokine production. Thus, HSP65-induced effects on allergen-induced airway hyperresponsiveness and inflammation were associated with increased Delta 1 expression on DCs, modulation of DC function, and CD4+ Th1 cytokine production.
PMCID: PMC3448847  PMID: 22933632
HSP65; asthma; dendritic cells; T cells
4.  Susceptibility to Vaccinia Virus Infection and Spread in Mice Is Determined by Age at Infection, Allergen Sensitization and Mast Cell Status 
Patients, especially young children, with atopic dermatitis are at an increased risk of developing eczema vaccinatum, a severe reaction to the smallpox vaccine, either through direct vaccination or indirect contact with a person recently vaccinated.
Using a mouse model of infection, the severity of vaccinia-induced lesions was assessed from their appearance and viral DNA content. The response to vaccinia inoculation was assessed in young and adult mice, allergen-sensitized mice, and in mast cell-deficient mice.
Young age, sensitization to an allergen prior to infection, and a mast cell deficit, accomplished by using mast cell-deficient mice, resulted in more severe viral lesions at the site of inoculation, according to lesion appearance and viral DNA content. All three factors combined demonstrated maximal susceptibility, characterized by the severity of primary lesions and the development of secondary (satellite) lesions, as occurs in eczema vaccinatum in humans. Resistance to the appearance of satellite lesions could be restored by adoptive transfer of bone marrow-derived mast cells from either wild-type or cathelicidin-related antimicrobial peptide-deficient mice. Primary lesions were more severe following the latter transfer, indicating that cathelicidin-related antimicrobial peptide does contribute to the protective activity of mast cells against infection.
The combination of young age, allergen sensitization and a mast cell deficit resulted in the most severe lesions, including satellite lesions. Understanding the factors determining the relative resistance/sensitivity to vaccinia virus will aid in the development of strategies for preventing and treating adverse reactions which can occur after smallpox vaccination.
PMCID: PMC3291886  PMID: 22286752
Allergen sensitization; Eczema vaccinatum; Mast cells; Neutrophils; Vaccinia virus
5.  Low-Dose Lipopolysaccharide Affects Lung Allergic Responses by Regulating Jagged1 Expression on Antigen-Pulsed Dendritic Cells 
Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.
Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR.
Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge.
These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.
PMCID: PMC3180653  PMID: 21912175
Asthma; Dendritic cells; Endotoxin; Notch ligands
6.  NF-κB-Dependent Induction of Cathelicidin-Related Antimicrobial Peptide in Murine Mast Cells by Lipopolysaccharide 
An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-κB and MAPK pathways were the focus of investigation.
Mouse bone marrow-derived mast cells were grown in culture and stimulated with LPS. MAPKs and NF-κB were monitored by immunoblot analysis. ERK, JNK and p38 MAPK were inhibited using siRNAs or a pharmacological inhibitor. Accumulation of the p65 component of NF-κB was inhibited by siRNA and NF-κB activation was inhibited by overexpression of IκBα. MEKK2 or MEKK3 were overexpressed by transfection. The effects of all of these treatments on CRAMP gene expression were monitored by RT-PCR.
Inhibition of ERK, JNK or p38 MAPK had little discernible effect on LPS-inducible CRAMP gene expression. Overexpression of MEKK2 or MEKK3 likewise had little impact. However, inhibition of the accumulation of p65 NF-κB prevented LPS-induced CRAMP mRNA. An important role for NF-κB in CRAMP gene expression was confirmed by overexpression of IκBα, which reduced both basal and induced levels of CRAMP mRNA.
NF-κB, but not MAPKs, plays an important role in LPS-mediated induction of CRAMP gene in mast cells. Defects which inhibit NF-κB activity may increase susceptibility to bacterial and viral pathogens which are sensitive to cathelicidins.
PMCID: PMC2814151  PMID: 19439978
Cathelicidin-related antimicrobial peptide; Inflammation; Mast cells; Transcription factors
7.  Inhibition of Spleen Tyrosine Kinase Prevents Mast Cell Activation and Airway Hyperresponsiveness 
Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor–mediated signaling.
Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo.
Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcɛRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow–derived mast cells (BMMCs) before cross-linking with allergen.
Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor α, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge.
Conclusion: This study delineates a functional role for Syk in the development of mast cell– and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.
PMCID: PMC2662982  PMID: 16192454
airway hyperresponsiveness; eosinophils; goblet cell metaplasia; mast cells; spleen tyrosine kinase
8.  Cell fusion and plasticity 
Cytotechnology  2003;41(2-3):103-109.
Cell plasticity is a central issue in stem cell biology. In many recent discussions, observation of cell fusion has been seen as a confounding factor which calls into question published results concerning cell plasticity of, particularly, adult stem cells. An examination of the voluminous literature of "somatic cell fusion" suggests the relatively frequent occurrence of "spontaneous" cell fusion and shows that the complicated cellular phenotypes which it can give rise to have long been recognized. Here, a brief overview of this field is presented, with emphasis on studies of special relevance to current work on cell plasticity.
PMCID: PMC3466689  PMID: 19002947
cell fusion; cloning; plasticity; stem cell; transdifferentiation
9.  Transcription and Transport of Virus-Specific Ribonucleic Acids in African Green Monkey Kidney Cells Abortively Infected with Type 2 Adenovirus 
Journal of Virology  1972;10(6):1109-1117.
The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of 14C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.
PMCID: PMC356590  PMID: 4345490
10.  Synthesis of Virus-Specific Ribonucleic Acid in KB Cells Infected with Type 2 Adenovirus 
Journal of Virology  1971;8(2):203-214.
By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 μm 5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of 3H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the 3H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition (“presaturation” hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4S. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication—it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.
PMCID: PMC356232  PMID: 4256015

Results 1-10 (10)