Vol. 17 No. 4 December 2012

DOI: 10.2478/s11658-012-0024-5 Volume 17 (2012) pp 501-525
Title ANTILEUKEMIC ACTIVITY OF COMBINED EPIGENETIC AGENTS,DNMT INHIBITORS ZEBULARINE AND RG108 WITH HDAC INHIBITORS, AGAINST PROMYELOCYTIC LEUKEMIA HL-60 CELLS
Authors Jurate Savickiene, Grazina Treigyte, Veronika-Viktorija Borutinskaite and Ruta Navakauskiene*
Abstract DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all-trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.
Keywords HL-60, Differentiation, RG108, Zebularine, HDAC inhibitors, E-cadherin, Histones
Address and Contact Information Department of Molecular Cell Biology, Institute of Biochemistry, Vilnius University, Mokslininku 12, Vilnius, LT 08662, Lithuania
* Author for correspondence. e-mail: ruta.navakauskiene@bchi.vu.lt, tel.: +370 5272 9327, fax: +370 5272 9196
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0025-4 Volume 17 (2012) pp 526-541
Title INTRASPECIFIC POLYMORPHISM OF RIBOSOMAL DNA LOCI NUMBER AND MORPHOLOGY IN Brachypodium pinnatum AND Brachypodium sylvaticum
Authors Ewa Breda, Elzbieta Wolny and Robert Hasterok*
Abstract The genus Brachypodium has become the target of extensive cytomolecular studies since one of its representatives, B. distachyon, has been accepted as a model plant for temperate cereals and forage grasses. Recent preliminary studies suggested that intraspecific rDNA polymorphism can occur in at least two members of the genus, B. sylvaticum and B. pinnatum, so the aim of this study was to further analyse this phenomenon. FISH with 25S rDNA and 5S rDNA probes was performed on somatic metaphase chromosomes, supplemented by the silver staining technique which distinguishes transcriptionally active from inactive 18S-5.8S-25S rDNA loci. The number, size and chromosomal distribution of 5S rDNA loci were very constant: two loci were invariably observed in all studied diploid accessions of both species, while four 5S rDNA loci were present in the tetraploid B. pinnatum. In contrast to 5S rDNA loci, those of the 35S rDNA were more variable. Two or three loci were observed in the diploid B. pinnatum and four in tetraploid accessions. In chromosome complements of B. sylvaticum accessions from two to six 35S rDNA sites were detected. Regardless of total rDNA locus number, only two were transcriptionally active in diploid accessions of both species, while two or four were active in the tetraploid B. pinnatum. Additionally, the fluorescent CMA/DAPI banding method was used to identify the relation between rDNA sites and CMA+ bands. It was revealed that the number and chromosomal distribution of CMA+ bands are in congruence only with 35S rDNA loci which gave strong FISH signals.
Keywords Brachypodium, Chromomycin A3, DAPI, FISH, NOR, Polymorphism, rDNA, rRNA genes
Address and Contact Information Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
* Author for correspondence. e-mail: hasterok@us.edu.pl, tel.: +48-32-2009571, fax: +48-32-2562434
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0026-3 Volume 17 (2012) pp 542-548
Title DYNAMIC INSTABILITY - A COMMON DENOMINATOR IN PROKARYOTIC AND EUKARYOTIC DNA SEGREGATION AND CELL DIVISION
Authors John A. Fuesler* and Hsin-Jung (Sophia) Li
Abstract Dynamic instability is an essential phenomenon in eukaryotic nuclear division and prokaryotic plasmid R1 segregation. Although the molecular machines used in both systems differ greatly in composition, strong similarities and requisite nuances in dynamics and segregation mechanisms are observed. This brief examination of the current literature provides a functional comparison between prokaryotic and eukaryotic dynamically unstable filaments, specifically ParM and microtubules. Additionally, this mini-review should support the notion that any dynamically unstable filament could serve as the molecular machine driving DNA segregation, but these machines possess auxiliary features to adapt to temporal and spatial disparities in either system.
Keywords Dynamic instability, Microtubules, ParM filaments, R1 plasmid, Mitosis, Mitotic spindle, Brownian ratchet, Cytoskeleton evolution, Catastrophe/recovery
Address and Contact Information Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
* Author for correspondence. e-mail: jfuesler@princeton.edu
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0030-7 Volume 17 (2012) pp 549-558
Title SULPHAMOYLATED ESTRADIOL ANALOGUE INDUCES ANTIPROLIFERATIVE ACTIVITY AND APOPTOSIS IN BREAST CELL LINES
Authors Michelle Visagie, Thandi Mqoco and Anna Joubert*
Abstract Research into potential anticancer agents has shown that 2-methoxyestradiol exerts antiproliferative activity in vitro and in vivo in an estrogen receptor-independent manner. Due to its limited biological accessibility and rapid metabolic degradation, several new analogues have been developed in recent years. This study investigated the in vitro effects of a novel in silico designed compound (C16) in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7), an estrogen receptor-negative breast adenocarcinoma epithelial cell line (MDA-MB-231) and a nontumorigenic breast cell line (MCF-12A). Light microscopy revealed decreased cell density, cells blocked in metaphase and the presence of apoptotic characteristics in all three cell lines after exposure to C16 for 24 h. Polarizationoptical transmitted light differential interference contrast revealed the presence of several rounded cells and decreased cell density. The xCELLigence real-time label-independent approach revealed that C16 exerted antiproliferative activity. Significant inhibition of cell growth was demonstrated after 24 h of exposure to 0.2 µM C16 in all three cell lines. However, the non-tumorigenic MCF-12A cell line recovered extremely well after 48 h when compared to the tumorigenic cell lines. This indicates that C16 acts as an antiproliferative agent, possesses antimitotic activity and induces apoptosis in vitro. These features warrant further investigation.
Keywords C16, Cancer, Proliferation, MCF-7, MCF-12A, MDA-MB-231, Metaphase, Apoptosis, Xcelligence, Tumorigenic
Address and Contact Information Department of Physiology, University of Pretoria, Pretoria, South Africa
* Author for correspondence. annie.joubert@up.ac.za, tel.: +27 12 319 2246, fax: +2712 321 1679
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0029-0 Volume 17 (2012) pp 559-570
Title THE INFLUENCE OF 8-PRENYLNARINGENIN ON THE ACTIVITY OF VOLTAGE-GATED Kv1.3 POTASSIUM CHANNELS IN HUMAN JURKAT T CELLS
Authors Justyna Gąsiorowska, Andrzej Teisseyre*, Anna Uryga and Krystyna Michalak
Abstract Using the whole-cell patch-clamp technique, we investigated the influence of 8-prenylnaringenin on the activity of the voltage-gated Kv1.3 potassium channels in the human leukemic T lymphocyte cell line Jurkat. 8-prenylnaringenin is a potent plant-derived phytoestrogen that has been found to inhibit cancer cell proliferation. The results show that it inhibited the Kv1.3 channels in a concentration-dependent manner. Complete inhibition occurred at concentrations higher than 10 µM. The inhibitory effect of 8-prenylnaringenin was reversible. It was accompanied by a significant acceleration of channel inactivation without any pronounced change in the activation rate. Of the naringenin derivatives tested to date, 8-prenylnaringenin is the most potent inhibitor of the Kv1.3 channels. The potency of the inhibition may be due to the presence of a prenyl group in the molecule of this flavonoid. The inhibition of the Kv1.3 channels might be involved in the antiproliferative and pro-apoptotic effects of 8-prenylnaringenin that have been observed in cancer cell lines expressing these channels.
Keywords 8-prenylnaringenin, Kv1.3 channel, Jurkat T lymphocytes, Patchclamp, Cancer cell proliferation, Cancer cell apoptosis
Address and Contact Information Wrocław Medical University, Department of Biophysics, ul. Chałubińskiego 10, 50-368 Wrocław, Poland
* Author for correspondence. e-mail: andrzej.teisseyre@am.wroc.pl, tel.: +48 717841414, fax: +48 717840088
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0028-1 Volume 17 (2012) pp 571-585
Title CLONING OF THE HUMAN ACTIVATED LEUKOCYTE CELL ADHESION MOLECULE PROMOTER AND IDENTIFICATION OF ITS TISSUE-INDEPENDENT TRANSCRIPTIONAL ACTIVATION BY Sp1
Authors Fang Tan1, Flaubert Mbunkui2 and Solomon F. Ofori-Acquah1,*
Abstract Activated leukocyte cell adhesion molecule (ALCAM) belongs to the immunoglobulin cell adhesion molecule super family. ALCAM is implicated in tumor progression, inflammation, and the differentiation of hematopoietic stem cells. Hitherto, the identity of regulatory DNA elements and cognate transcription factors responsible for ALCAM gene expression remained unknown. In this report, the human ALCAM promoter was cloned and its transcriptional mechanisms elucidated. The promoter is TATA-less and contains multiple GC-boxes. A proximal 650-bp promoter fragment conferred tissueindependent activation, whereas two contiguous regions upstream of this region negatively influenced promoter activity in a tissue-specific manner. The positive regulatory promoter region was mapped to a core 50 base pair sequence containing a conical Sp1 element. Mutation analysis revealed that this element alone or in tandem with elements immediately upstream was required for maximal promoter activity. Chromatin analysis revealed that Sp1 binds exclusively to the canonical binding sequence in vivo, but not to DNA sequence immediately upstream. Finally, we showed that over-expression of Sp1 significantly increased the basal promoter activity. Thus, Sp1 activated the ALCAM promoter in most cells. These findings have important ramifications for unraveling the roles of ALCAM in inflammation and tumorigenesis.
Keywords ALCAM, Cis elements, Endothelial cells, Epithelial cells, Hematopoietic cells, Promoter activity, Sp1, Transcriptional regulation
Address and Contact Information 1Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA,
2Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA
* Author for correspondence: Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322, USA, e-mail: soforia@emory.edu, tel: +1 404 727 2293, fax +1 404 727 4455
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0027-2 Volume 17 (2012) pp 586-597
Title EFFECTS OF LEAD CHLORIDE ON HUMAN ERYTHROCYTE MEMBRANES AND ON KINETIC ANION SULPHATE AND GLUTATHIONE CONCENTRATIONS
Authors Tiziana Gugliotta, Grazia De Luca, Pietro Romano, Caterina Rigano, Adriana Scuteri and Leonardo Romano*
Abstract Our study concerns the effects of exposure to lead chloride on the morphology, K+ efflux, SO4 - influx and GSH levels of the human erythrocyte. Blood was collected in heparinized tubes and washed three times. The cells were suspended at 3% hematocrit and incubated for 1 h at 25oC in a medium containing increasing concentrations of lead chloride (0, 0.3, 0.5 and 1 µM). After incubation, the suspensions were centrifuged and the erythrocyte pellets were divided into three aliquots for testing. The results show: an increase in the permeability of erythrocytes treated with lead chloride with consequent damage and cellular death, especially in the presence of high concentrations; an increase in potassium ion efflux; alterations in the morphology and membrane structure of the red blood cells; and a decrease in sulphate uptake, due either to the oxidative effect of this compound on the band 3 protein, which loses its biological valence as a carrier of sulphate ions, or to a decrease in the ATP erythrocyte concentration. In conclusion, the exposure of erythrocytes to Pb2+ ions leads to a reduction in the average lifetime of the erythrocytes and the subsequent development of anemia. These data are discussed in terms of the possible effect of lead on the reduction-oxidation systems of the cell. Oxidant agents, such as lead, are known to cross-link integral membrane proteins, leading to K/Cl-cotransport. The increased K+ efflux affects the altered redox state.
Keywords Anion transport, Band 3 protein, Erythrocytes, Erythrocyte membrane, GSH, GSSG, Lead chloride, Scanning electron microscopy, Anemia, Adenosine 5’triphosphate, Sulphate influx
Address and Contact Information Dipartimento di Scienze della Vita “M. Malpighi”, Facolta di Scienze MM.FF.NN., Universita degli Studi di Messina, Italy
* Author for correspondence: Dipartimento di Scienze della Vita “M. Malpighi” - Sezione di Fisiologia generale e Farmacologia - Facolta di Scienze MM.FF.NN.Via Salita Sperone n° 31, Ctr. Papardo, 98166 Papardo, Messina, Italy. e-mail: tiziana_gugliot@yahoo.it, tel: 0039 090 393979, fax: 0039 090 394030
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0031-6 Volume 17 (2012) pp 598-615
Title THE HUMAN TELOMERASE CATALYTIC SUBUNIT AND VIRAL TELOMERASE RNA RECONSTITUTE A FUNCTIONAL TELOMERASE COMPLEX IN A CELL-FREE SYSTEM, BUT NOT IN HUMAN CELLS
Authors Laetitia Trapp-Fragnet1, *, Delphine T. Marie-Egyptienne2, 3, 4, #, Johans Fakhoury2, 5, 6, #, Denis Rasschaert7, § and Chantal Autexier2, 3, 5, §
Abstract The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.
Keywords Telomerase, Holoenzyme, hTERT, vTR, Dyskerin, Marek’s disease virus
Address and Contact Information 1INRA, UR1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France
2Lady Davis Institute for Medical Research, Jewish General Hospital Montréal, QC H3T 1E2, Canada,
3Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada,
4Ontario Cancer Institute, Princess Margaret Hospital, 610 University Toronto, Ontario, Canada M5G 2M9 (current address)
5Department of Medicine, Experimental Medicine Division, McGill University, Montréal, QC, Canada,
6Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, QC, Canada H3A 2K6 (current address),
7UFR Sciences et Techniques, Université François Rabelais de Tours, Avenue Monge, F-37000 Tours, France
* Author for correspondence. e-mail: fragnet@tours.inra.fr
#, § These authors contributed equally to this paper
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0033-4 Volume 17 (2012) pp 616-632
Title EVALUATION OF MELANOGENESIS IN A-375 MELANOMA CELLS TREATED WITH 5,7-DIMETHOXYCOUMARIN AND VALPROIC ACID
Authors Ewa Chodurek1*, Arkadiusz Orchel1, Joanna Orchel2, Sławomir Kurkiewicz3, Natalia Gawlik4, Zofia Dzierżewicz1 and Krystyna Stępień3
Abstract Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10-150 µM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.
Keywords A-375 cell line, Malignant melanoma, Valproic acid, Tyrosinase, Gene expression, 5,7-dimethoxycoumarin, Melanin, Pyrolysis-gas chromatography/mass spectrometry
Address and Contact Information 1Department of Biopharmacy, 2Department of Molecular Biology, 3Department of Instrumental Analysis, 4Department of Biotechnology and Genetic Engineering, Medical University of Silesia, Narcyzów 1, Sosnowiec 41-200, Poland
* Author for correspondence. e-mail: echodurek@sum.edu.pl, tel.: +48 32 3641064, fax: +48 32 3641060
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0034-3 Volume 17 (2012) pp 633-645
Title THE STRONGEST RESISTANCE OF Staphylococcus aureus TO ERYTHROMYCIN IS CAUSED BY DECREASING UPTAKE OF THE ANTIBIOTIC INTO THE CELLS
Authors Elżbieta Pi±tkowska1, Jerzy Pi±tkowski2* and Anna Przondo-Mordarska1
Abstract The consequence of excessive use of macrolides is a high occurrence of mechanisms responsible for resistance to these drugs. Of 97 erythromycinresistant bacterial strains gathered in the Wrocław area in Poland, 60% exhibited very high resistance, and those with the inducible MLSB (macrolidelincosamide- streptogramin B) resistance phenotype predominated. Direct genetic investigation revealed that the erm genes coding for ribosomal methylases are the most frequently occurring erythromycin resistance-determining genes. No genetic resistance determinant was detected in 13% of the erythromycin-resistant strains. The efflux mechanism occurs in strains isolated from the nasopharyngeal cavity twice as often as in those isolated from other material, where the mechanism connected with target site modification predominates. Measurements of radiolabelled antibiotic accumulation inside bacterial cells revealed that in highly resistant strains (MIC > 1024 g/ml), an important factor responsible for the resistance is the permeability barrier at the cell wall level. This would be a hitherto unknown mechanism of resistance to erythromycin in Staphylococcus aureus.
Keywords Transmembrane transporters, Antibiotic efflux, Resistance to erythromycin, Drug accumulation, Staphylococcus aureus, Ribosomal methylases, ermA, ermC, msr(A), MLSB
Address and Contact Information 1Department of Microbiology, Silesian Piasts University of Medicine in Wrocław, Poland,
2Institute of Genetics and Microbiology, University of Wrocław, Poland
* Author for correspondence. Institute of Genetics and Microbiology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148 Wrocław, tel. +48 603 194 282, e-mail: jerzy.piatkowski@microb.uni.wroc.pl
[Rozmiar: 1332 bajtÄ‚ı‚w]

DOI: 10.2478/s11658-012-0032-5 Volume 17 (2012) pp 646-669
Title PUMA, A CRITICAL MEDIATOR OF CELL DEATH – ONE DECADE ON FROM ITS DISCOVERY
Authors Paweł Hikisz; and Zofia M. Kiliańska2,§,*
Abstract PUMA (p53 upregulated modulator of apoptosis) is a pro-apoptotic member of the BH3-only subgroup of the Bcl-2 family. It is a key mediator of p53-dependent and p53-independent apoptosis and was identified 10 years ago. The PUMA gene is mapped to the long arm of chromosome 19, a region that is frequently deleted in a large number of human cancers. PUMA mediates apoptosis thanks to its ability to directly bind known anti-apoptotic members of the Bcl-2 family. It mainly localizes to the mitochondria. The binding of PUMA to the inhibitory members of the Bcl-2 family (Bcl-2-like proteins) via its BH3 domain seems to be a critical regulatory step in the induction of apoptosis. It results in the displacement of the proteins Bax and/or Bak. This is followed by their activation and the formation of pore-like structures on the mitochondrial membrane, which permeabilizes the outer mitochondrial membrane, leading to mitochondrial dysfunction and caspase activation. PUMA is involved in a large number of physiological and pathological processes, including the immune response, cancer, neurodegenerative diseases and bacterial and viral infections.
Keywords Apoptosis, BH3-only proteins, Carcinogenesis, Inhibitory members of the Bcl-2 family, Intrinsic apoptosis pathway, p53, Pro-apoptotic members of Bcl-2 family, PUMA, Post-translational regulation, Transcription factors
Address and Contact Information 1Department of Thermobiology, 2Department of Cytobiochemistry, University of ŁódĄ, ul. Pomorska 141/143, 90-236 ŁódĄ, Poland
§These authors contributed equally to this paper
* Author for correspondence. e-mail: zkilian@biol.uni.lodz.pl
[Rozmiar: 1332 bajtÄ‚ı‚w]