Vol. 19 No. 1 March 2014

DOI: 10.2478/s11658-013-0111-2 Volume 19 (2014) pp 1-22
Authors Agata Piecuch and Ewa Obłąk*
Abstract Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cellsis the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiaeis often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found inhumans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects.
Keywords ABC proteins, PDR subfamily, Saccharomyces cerevisiae, Multidrug resistance, Regulation of ABC proteins, Transcription factors, P-glycoprotein, Modulators of ABC proteins, Flavonoids, Phenothiazines
Address and Contact Information Institute of Genetics and Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland
* Author for correspondence. e-mail: ewa.oblak@microb.uni.wroc.pl

DOI: 10.2478/s11658-013-0112-1 Volume 19 (2014) pp 23-36
Authors Raheleh Roudi1,2,4, Zahra Madjd1,3,4,*, Marzieh Ebrahimi2,*, Fazel Sahraneshin Samani2 and Ali Samadikuchaksaraei4,5
Abstract Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44+/24+ and CD44+/CD24-/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44+/CD24+ and CD44+/CD24-/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.
Keywords Cancer stem cells, Lung cancer, Cell line A549, Colony-formation assay, Sphere-formation assay, CD44, CD24, CD133, ALDH1, ABCG-2
Address and Contact Information 1Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran,
2Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran,
3Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran,
4Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran,
5Department of Medical Biotechnology, Iran University of Medical Sciences, Tehran, Iran
* Authors for correspondence: Marzieh Ebrahimi, Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. e-mail: Mebrahimi@royaninstitute.org, tel.: +982122306485, fax: +982122413720 and Zahra Madjd, Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran. E-mail: Zahra.madjd@yahoo.com, tel./fax: +982188622608

DOI: 10.2478/s11658-013-0113-0 Volume 19 (2014) pp 37-51
Authors Bregje Van Oorschot1, Arlene L. Oei1, Anna C. Nuijens1, Hans Rodermond1, Ron Hoeben2, Jan Stap2, Lukas J. Stalpers1 and Nicolaas A.P. Franken1,*
Abstract The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines:RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD2). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a 137Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.
Keywords p53, Radiation sensitivity, Potentially lethal damage repair (PLDR), Linear-quadratic model, Clonogenic assay, Colon cancer cells, RKO cells, RC10.1 cells, γ-H2AX foci, Flow cytometry
Address and Contact Information 1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Department of Radiation Oncology, and 2van Leeuwenhoek Center for AdvancedMicroscopy AMC, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands
* Author for correspondence. E-mail: n.a.franken@amc.uva.nl, tel.: +31 20 5663641

DOI: 10.2478/s11658-013-0115-y Volume 19 (2014) pp 52-64
Authors Qiu Chen1,§, Lei Li1,§, Yu Tu1, Lu Lin Zheng1, Wei Liu1, Xue Yong Zuo1, Yong Ming He2, Shu Yu Zhang1, Wei Zhu1, Jian Ping Cao1, Feng Mei Cui1,* and Jun Hou3,*
Abstract MicroRNAs (miRNAs) regulate gene expression by inhibiting translation or targeting messenger RNA (mRNA) for degradation in a posttranscriptional fashion. In this study, we show that ectopic expression of miR-34a-5p reduces the mRNA and protein levels of Krüppel-like factor 4 (KLF4). We also demonstrate that miR-34a targets the 3’-untranslated mRNA region of KLF4 and show that overexpression of miR-34a induces a significant level of apoptosis in BNL CL.2 cells exposed to doxorubicin or 10 Gy X-ray. Our data suggest that the effects of miR-34a on apoptosis occur due to the downregulation of KLF4.
Keywords MiR-34a, KLF4, Apoptosis, Liver, Irradiation, Doxorubicin
Address and Contact Information 11Department of Radiation Medicine, School of Radiation Medicine and Protection, Medical College of Soochow University, Suzhou City, China,
2Department of Cardiology, the First Affiliated Hospital of Soochow University, Suzhou City, China,
3Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai City, China
§ Qiu Chen and Lei Li contributed equally to this work
*Authors for correspondence. e-mail: cuifengmei@suda.edu.cn; hou.jun@zs-hospital.sh.cn

DOI: 10.2478/s11658-013-0114-z Volume 19 (2014) pp 65-76
Authors Maciej Grys, Zbigniew Madeja* and Włodzimierz Korohoda*
Abstract The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversibleand reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnosticsubstances into the cells and tissues.
Keywords Selective sensitization of cells, Local cationic anesthetics, Cationic dyes, Irreversible electroporation, Reversible electroporation, Loading with fluorescent dyes, Cell viability, Flow through an electric field, Direct current electric field, Focused electric field
Address and Contact Information Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, JagiellonianUniversity, Gronostajowa 7, 30-387, Kraków, Poland
* Authors for correspondence. e-mail: w.korohoda@uj.edu.pl, tel.: + 48 12 664 61 25, fax: +48 12 664 69 02; e-mail: z.madeja@uj.edu.pl, tel.: + 48 12 664 61 42

DOI: 10.2478/s11658-014-0181-9 Volume 19 (2014) pp 77-97
Authors Arthur Czarnowski, Sylvia Papp, Peter Szaraz and Michal Opas*
Abstract Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cellsubstratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, inducedextensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is thatY-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell’s underside by the thin lamellae and filopodia of a cell in close apposition.
Keywords Calreticulin, Insulin receptor substrate-1, Adhesion, Focal contacts, Close contacts, Motility
Address and Contact Information Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
* Author for correspondence. Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, room 6326, Toronto, Ontario, M5S 1A8 Canada, email: m.opas@utoronto.ca, phone: (416) 971-2140, fax: (416) 978-5959

DOI: 10.2478/s11658-014-0183-7 Volume 19 (2014) pp 98-115
Authors Elize Wolmarans1, Thandi V. Mqoco1, Andre Stander1, Sandra D. Nkandeu1, Katherine Sippel2, Robert Mckenna3 and Annie Joubert1,*
Abstract Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-Osulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16)was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 µM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells.This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.
Keywords Antiproliferative, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene, ESE-16, Esophageal carcinoma, In vitro, Apoptosis, Autophagy
Address and Contact Information 1Department of Physiology, University of Pretoria, South Africa,
2Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA,
3McKnight Institute, University of Florida, Gainesville, Florida, USA
* Author for correspondence. Email: annie.joubert@up.ac.za, phone: +27 12 319 2246, fax: +27 12 321 1679

DOI: 10.2478/s11658-014-0182-8 Volume 19 (2014) pp 116-125
Authors Ewa Górnicka-Michalska, Andrzej Kowalski* and Jan Pałyga
Abstract Two isoforms of the erythrocyte histone H1.a were identified in two conservative flocks of Rhode Island Red chickens and six conservative flocks of ducks. The H1.a1 and H1.a2 isoforms formed three phenotypes (a1, a2 and a1a2) and were electrophoretically similar in the two species. The frequency of phenotype and histone H1.a allele occurrence varied within the genetic groups of birds, but the relatively rare allele a2 was only detected in chicken and duck strains with colored feathers. Using mass spectrometry, we established that the difference between the measured masses of the duck H1.a isoforms was 156 Da. Since this value corresponds to the mass ofthe arginine residue alone or to the combined mass of the valine and glycine residues, we believe that the polymorphism of duck histone H1.a might have originated from sequence variation. A mass difference of 1 Da observed between chicken H1.a isoforms corresponded well to the previously detected Glu/Lys substitution (0.9414 Da) at position 117.
Keywords Chicken, Duck, Electrophoresis, Histone H1.a, Polymorphism, Mass spectrometry
Address and Contact Information Department of Biochemistry and Genetics, Institute of Biology, Jan Kochanowski University, ul. Świętokrzyska 15, 25-406 Kielce, Poland
* Author for correspondence. Email: a.kowalski@ujk.edu.pl

DOI: 10.2478/s11658-014-0186-4 Volume 19 (2014) pp 126-139
Authors Gloria Gutiérrez-Venegas*, Oscar Alonso Luna, Juan Antonio Arreguín-Cano and Cristina Hernández-Bermúdez
Abstract Periodontitis is an infectious disease caused by microorganisms present in dental bacterial plaque. Lipoteichoic acid (LTA) is a component of the external membrane of Gram-positive bacteria. It causes septic shock. Ingested flavonoids have been reported to directly affect the regulation of cyclooxygenase-2 (COX-2) expression induced by bacterial toxins. In this study, we examined the effects of four flavonoids (luteolin, fisetin, morin and myricetin) on the activation of ERK1/2, p38 and AKT, and on the synthesis of COX-2 in human gingival fibroblasts treated with LTA from Streptococcus sanguinis. We found that luteolin and myricetin blocked AKT and p38 activation and that myricetin blocked LTA-induced COX-2 expression. The results of our study are important for elucidating the mechanism of action of flavonoid regulation of inflammatory responses.
Keywords Periodontitis, Fisetin, Morin, Myricetin, Luteolin, Human gingival fibroblasts, Lipoteichoic acid, Cyclooxygenase
Address and Contact Information Laboratorio de Bioquímica, División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Ciudad de México, México
* Author for correspondence. Email: gloria@fo.odonto.unam.mx

DOI: 10.2478/s11658-014-0187-3 Volume 19 (2014) pp 140-157
Authors Quansheng Li1, Ping Yu2, Wei Wang3, Peng Zhang4, Haiqing Yang5, Shengfu Li6 and Li Zhang6,*
Abstract Mesenchymal stem cells (MSCs) have both multi-lineage differentiation potential and immunosuppressive properties, making them ideal candidates for regenerative medicine. However, their immunosuppressive properties potentially increase the risk of cancer progression and opportunistic infections. In this study, MSCs isolated from human umbilical cord blood (UCMSCs) and adult bone marrow (BMMSCs) were infected with human cytomegalovirus (HCMV). Cytopathic changes were observed 10 days post infection. PCR products amplified from genomic DNA and cDNA were used to confirm the HCMV infection of the UCMSCs and BMMSCs. Real-time PCR was conducted to quantify the expression of immunomodulatory molecules, including cytokines, chemokines, growth factors, adhesion molecules and cancer-related genes. Our results indicate high upregulation of the majority of these molecules, including many growth factors, tumor necrosis factor alpha, interleukin-8, interleukin-6 and interferon gamma. Adhesion molecules (VCAM-1, TCAM-1 and selectin-E) were downregulated in the infected UCMSCs and BMMSCs. Antibody chip array evaluation of cell culture media indicated that the growth factor secretion by UCMSCs and BMMSCs was greatly influenced (p < 0.001) by HCMV. The stimulation of MSCs with HCMV led to the activation of downstream signaling pathways, including pSTAT3 and Wnt2. Our results show that HCMV can significantly alter the functions of both UCMSCs and BMMSCs, although not in the same way or to the same extent. In both cases, there was an increase in the expression of proangiogenic factors in the microenvironment following HMCV infection. The discrepancy between the two cell types may be explained by their different developmental origin, although further analysis is necessary. Futurestudies should decipher the underlying mechanism by which HCMV controls MSCs, which may lead to the development of new therapeutic treatments.
Keywords Mesenchymal stem cells, Bone marrow, Umbilical cord, Human cytomegalovirus, In vitro infection, Cytopathic change, Immunomodulatory molecules, Gene expression detection, Antibody chip, Kinase signal pathway
Address and Contact Information 1Department of Biliary Surgery, West China Hospital, Sichuan University, Chengdu, 610041 P.R. China,
2Laboratory of Cell and Gene Therapy, West China Second University Hospital, Sichuan University, Chengdu, 610041 P.R. China,
3Department of Pathology, West China Second Hospital, Sichuan University, Chengdu, 610041 P.R. China,
4Department of Urology, West China Hospital, Sichuan University, Chengdu, 610041 P.R. China,
5Department of Hematology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, 610041 P.R. China,
6Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041 P.R. China
* Author for correspondence. Email: zhangli7375@scu.edu.cn, phone: 86-28-85164030, fax: 86-28-85164034

DOI: 10.2478/s11658-014-0185-5 Volume 19 (2014) pp 158-179
Authors Dżamila M. Bogusławska1, Beata Machnicka1, Anita Hryniewicz-Jankowska2 and Aleksander Czogalla2,3,*
Abstract The spectrin-based membrane skeleton is crucial for the mechanical stability and resilience of erythrocytes. It mainly contributes to membrane integrity, protein organization and trafficking. Two transmembrane protein macro-complexes that are linked together by spectrin tetramers play a crucial role in attaching the membrane skeletonto the cell membrane, but they are not exclusive. Considerable experimental datahave shown that direct interactions between spectrin and membrane lipids are important for cell membrane cohesion. Spectrin is a multidomain, multifunctional protein with several distinctive structural regions, including lipid-binding sites within CH tandem domains, a PH domain, and triple helical segments, which are excellent examples of ligand specificity hidden in a regular repetitive structure, as recently shown for the ankyrin-sensitive lipid-bindingdomain of beta spectrin. In this review, we summarize the state of knowledge about interactions between spectrin and membrane lipids.
Keywords Spectrin–phospholipid interactions, Spectrin repeats, Spectrin tetramers, Ankyrin, Erythrocytes, Actin-binding domain, Pleckstrin homology domain, Dystrophin, Lipid bilayer, Membrane skeleton
Address and Contact Information 1University of Zielona Góra, Facultyof Biological Sciences, Poland,
2University of Wrocław, Biotechnology Faculty, Poland,
3Paul Langerhans Institute Dresden, Faculty of Medicine Carl Gustav Carus at the TU Dresden, Germany
* Author for correspondence. Aleksander Czogalla, University of Wrocław, Faculty of Biotechnology, Laboratory of Cytobiochemistry, F. Joliot-Curie 14A, 50-383 Wrocław, Poland. Email: alczog@ibmb.uni.wroc.pl, phone: + 48 71 3756 233, fax: + 48 71 3756 208

DOI: 10.2478/s11658-014-0184-6 Volume 19 (2014) pp 180
Title Erratum to the article
by: Abhijit Chakraborty, Atul Katarkar, Keya Chaudhuri, Ashis Mukhopadhyay and Jayasri Basak
published in: Cellular & Molecular Biology Letters, Vol. 18. No. 4, 2013, pp. 631-638, DOI: 10.2478/s11658-013-0110-3

The original version of the article title unfortunately contained mistake. The Acknowledgements should be:

We are grateful to the Indian Council ofMedical Research (ICMR), Government of India, New Delhi, India for their financial assistance (File No. 54/07/2011/ HUM-BMS; Project Number: 2011-03270).We owe our sincere thanks to the patients and their family members for their extensive cooperation with us. We would also like to thank all the staff at our hospital who helped to accomplish the study successfully. We wish to acknowledge West Bengal University of Health Sciences to which the Institute is affiliated.