Lymphocyte Surface Markers

[Archives Medical Review Journal 1992; 1(1.000): 5-24

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

Esendagli, Gunes/0000-0003-4865-2377;WOS: 000262088100011PubMed: 17922253Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.Eczacibasi Scientific Research and Award Fund; Hacettepe UniversityHacettepe University [05DO3104001]This study was supported by Eczacibasi Scientific Research and Award Fund, and Hacettepe University Scientific Research Unit (project no. 05DO3104001)

Comparison Of Phototoxic Effects Of Hypericin-Mediated Photodynamic Therapy In Ht-29 And Caco-2 Colon Cancer Cells

Hypericin (HYP) is a plant-derived photosensitizer. HYP is preferentially taken up by tumor cells. We designed this study to compare HYP-mediated photodynamic therapy (PDT) in HT-29 and Caco-2 colon cell lines. Cells were treated with 0.04, 0.08, and 0.15 mu M HYP concentrations and irradiated. The effect of HYP on metabolic profiles, alterations in lactate dehydrogenase (LDH) leakage, and cell cycle progression was investigated for the first time. Changes in glucose consumption, lactate production, and LDH leakage were analyzed. HYP-induced cell death was quantified by double staining (acridine orange/propidium iodide) and alterations in cell cycle regulation were analyzed with flow cytometry (using propidium iodide). LDH leakage and the number of dead cells were elevated, and glucose consumption and lactate production decreased in a dose-and time-dependent manner. PDT resulted in an induction of apoptosis, mostly at the 0.08 mu M HYP concentration. Apoptosis and/or necrosis were increased in the 0.15 mu M HYP group. The accumulation of cells in the G2/M phase might account for the growth inhibition in HT-29 and Caco-2 cells with 0.08 mu M HYP photoactivation. The observed G2/M arrest suggested that HYP may slow down the growth of colon cancer cells by regulating the cell cycle, leading either to growth inhibition or to initiation of apoptotic pathways.WoSScopu

A new nanosuspension prepared with wet milling method for oral delivery of highly variable drug Cyclosporine A: development, optimization and in vivo evaluation

Cyclosporine A (CsA) is a cyclic polypeptide, that has been widely used for immunosuppression. This study aims to develop nanosuspension for oral administration of CsA using the wet milling (WM) method one of the top-down technologies. The WM method was optimized by studying the effects of critical process parameters for WM on the particle size (PS), particle size distribution (PDI), and zeta potential (ZP) of nanosuspensions using the Design of Experiment (DoE) approach. Nanosuspension was developed using hydroxypropyl methylcellulose (HPMC) and sodium dodecyl sulfate (SDS) and in vitro characterization studies were performed. In vitro dissolution and in vivo pharmacokinetic studies were conducted with biorelevant media (fasted and fed state simulated fluids) and fasted and fed states in rats, respectively. In vivo immunological studies were also performed. PS, PDI, and ZP values for nanosuspension were approximately 600 nm, 0.4, -25 mV, respectively. The solubility of CsA was increased by 4.5-folds by nanosuspensions. Dissolution studies showed that nanosuspension had higher dissolution than the commercial product in the FeSSIF medium. The pharmacokinetic study indicated that AUC0–24 values of CsA nanosuspension were to be 2.09 and 5.51-fold higher than coarse powder in fasted and fed conditions, respectively. Immunological studies were carried out after oral administration of nanosuspension for 21 days, the ratio of CD4+/CD8+ was found to be more acceptable than the commercial product. These results demonstrated that nanosuspension is a promising approach for increasing the bioavailability and avoiding the food effect on absorption of CsA which one of the highly variable drugs

Does N-Acetyl Cysteine Protect Against Apoptosis in HL60 Cell Line? [N-Asetil Sistein HL-60 Hücrelerini Apoptoza Karşı Korur mu?]

ABSTRACT Objectives: The primary objective of this study is to determine the role of glutathione depletion and N-acetylcysteine on apoptotic signal formation in HL60 cell line. Methods: HL60 cells were exposed to t-BOOH which is a chemical used to induce oxidative stress. The effects of NAC on GSH in apoptotic cells were examined. Hence the GSH levels, caspase-3, 8, 9 activities and apoptosis percentages of the HL60 cells were determined in the presence or the absence of NAC. Results and Discussion: Our results showed that pretreatment with NAC, eventhough it increases intracellular GSH content, does not protect against t-BOOH induced apoptosis. Caspase activities were decreased as compared with control group in the absence of NAC. In the presence of NAC, enzyme activities were similar to the control group. Therefore, cells may have gone apoptosis through caspase independent mechanism. Key Words: HL 60 cell line, tertier-butylhydroperoxide, Glutathione, N-acetylcysteine, Apoptosis ÖZET Amaç: Bu çalışmada temel amaç HL-60 hücre dizisinde glutatyon tüketiminin ve N-asetilsisteinin apoptotik sinyal oluşumundaki rolünü belirlemektir. Yöntem: HL-60 hücreleri oksidatif stres oluşturmak için tersiyer-butilhidroperoksite maruz bırakıldı. Apoptotik hücrelerde redükte glutatyon düzeyi, kaspaz-3, 8, 9 aktiviteleri ve apoptoz yüzdesi NAC yokluğunda ve varlığında belirlendi. Sonuç ve Tartışma: Sonuçlarımız NAC ile önişleme maruz bırakılan hücrelerde, glutatyon içeriğindeki artışın t-BOOH ile indüklenen apoptoza karşı koruyucu olmadığını gösterdi. Kaspaz aktiviteleri NAC yokluğunda kontrol grubuna kıyasla düşük bulunurken, NAC varlığında, kontrol grubuna benzer aktivite sonuçları bulundu. Bu nedenle, hücrelerin kaspaz bağımsız bir mekanizma yoluyla apoptoza gitmiş olabileceği sonucuna varıldı. Anahtar Kelimeler: HL 60 hücre dizisi, tersiyer-butilhidroperoksit, Glutatyon, N-asetilsistein, Apoptoz Turk J Biochem, 2010; 35 (4) ; 333-339. Aksoy et al. 33

Adhesion of beta1 integrin to fibronectin regulates CAM-DR phenotype via p21(WAF1/cip1) in HL60 acute myeloid leukemia (AML) cells

Esendagli, Gunes/0000-0003-4865-2377;WOS: 000254702300001Aims: Drug resistance is a major obstacle for a successful cancer therapy. Cell adhesion mediated drug resistance (CAM-DR) is a novel type of drug resistance and generated via interaction of cancer cells with the microenvironment. In this study, CAM-DR phenotype was analyzed in HL60 acute myeloid leukemia (AML) cells. Materials and Methods: Fibronectin (FN) adherence of HL60 cells was tested by a colorimetric adhesion assay. Flow cytometry analyses were performed to evaluate doxorubicin-incluced apoptosis and to determine cell cycle status. Proliferation rate was evaluated by [H-3]-thymidine incorporation assay. Western blot and RTPCR were used for analysis of the factors involved in cell cycle control. Results: Binding of HL60 to FN via alpha 4 beta 1 and alpha 5 beta 1 integrins exerted a CAM-DR phenotype, which shows resistance to apoptosis triggered by doxorubicin. FN-adherent HL60 cells accumulated in the G(0)/G(1) phase of cell cycle and stopped proliferation. However, after detachment from FN, cells entered S phase, proliferated, and became sensitive to apoptosis. The analysis of the factors involved in the G(0)/G(1) cell cycle checkpoint showed that CAM-DR phenotype might be regulated mainly by p21(waf/cip). Conclusions: Here we showed that CAM-DR may also represent a reversible drug resistance mechanism that decreases apoptosis and causes growth arrest in AML blasts

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia

Primary Tumor Cells Obtained from Mnu-Induced Mammary Carcinomas Show Immune Heterogeneity Which Can Be Modulated by Low-Efficiency Transfection of Cd40L Gene

The presence of CD40 on carcinoma cells is an important factor for the generation of tumor-specific responses induced by CD40 ligation. In an N-methyl-N-nitrosourea (MNU)-induced autochthonous mammary tumor model, we analyzed the immune features of primary tumor cells. Here, CD40 was frequently detected on the primary tumor cultures and selectively expressed on the malignant mammary tissue in vivo. On the other hand, every mammary tumor cell culture had a heterogeneous and reduced expression of proinflammatory TNF alpha, IL-1 beta, IL-6 and CXCL1 cytokines compared to normal mammary epithelial cells. Low-efficiency transfection of CD40 ligand (CD40L) gene enhanced the expression of proinflammatory cytokines in the tumor cells, and strengthened allogeneic immune reactions and costimulatory activity which may help overwhelming suppressive features of the tumor.WoSScopu

Dual actions of the antioxidant chlorophyllin, a glutathione transferase P1‐1 inhibitor, in tumorigenesis and tumor progression

Glutathione (GSH) and enzymes related to this antioxidant molecule are often overexpressed in tumor cells and may contribute to drug resistance. Blockade of glutathione transferases (GSTs) has been proposed to potentiate the efficacy of chemotherapeutic drugs in cancer. The aim of this study was to evaluate the effect of chlorophyllin that has antioxidant properties, and also interferes with the activity of GST P1-1, on breast cancers in vitro and in vivo. The in vivo studies were conducted using an N-methyl-N-nitrosourea (MNU)-induced chemical carcinogenesis model in laboratory rats. DNA damage, GST activity, and GSH levels were determined in liver and tumor tissues. Treatment with chlorophyllin increased the GSH levels in the liver and significantly decreased DNA damage in the blood, liver, and tumor tissues. Even though tumorigenesis was delayed in rats receiving chlorophyllin before MNU injections, once the tumors emerged, the progression of tumor appeared to be faster than in the animals that received the carcinogen only. Out of nine breast cell lines, GST P1-1 expression was detected in MCF-12A, MDA-MB-231, and HCC38. Concomitant incubation with chlorophyllin and docetaxel did not significantly affect cell proliferation and viability. Chlorophyllin displayed genoprotective effects that initially delayed tumorigenesis. However, once the tumors were established, it may act as a promoter that facilitates tumor growth, potentially by a mechanism independent of cell proliferation and viability. Our results underline the pros and cons of antioxidant treatment in cancer, even if it has a capacity to inhibit GST P1-1