Publications by authors named "D Spitkovsky"

56 Publications

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation.

Cell Tissue Bank 2021 Jan 24. Epub 2021 Jan 24.

Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n = 16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n = 16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.
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http://dx.doi.org/10.1007/s10561-020-09898-1DOI Listing
January 2021

and Analysis of Adhesive, Anti-Inflammatory, and Proangiogenic Properties of Novel 3D Printed Hyaluronic Acid Glycidyl Methacrylate Hydrogel Scaffolds for Tissue Engineering.

ACS Biomater Sci Eng 2020 10 8;6(10):5744-5757. Epub 2020 Sep 8.

Institute of Photon Technologies of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences, 108840 Moscow, Russia.

In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusion three-dimensional (3D) printing with photocured (λ = 445 nm) hyaluronic acid glycidyl methacrylate (HAGM). The developed product was compared with the polylactic--glycolic acid (PLGA) scaffolds generated by means of the original antisolvent 3D printing methodology. The cytotoxicity and cytocompatibility of the scaffolds were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests, flow cytometry, and scanning electron microscopy. Anti-inflammatory and proangiogenic properties of the scaffolds were evaluated in the dorsal skinfold chamber mouse model by means of intravital fluorescence microscopy, histology, and immunohistochemistry throughout an observation period of 14 days. , none of the scaffolds revealed cytotoxicity on days 1, 2, and 5 after seeding with umbilical cord-derived multipotent stromal cells, and the primary cell adhesion to the surface of HAGM scaffolds was low. , implanted HAGM scaffolds showed enhanced vascularization and host tissue ingrowth, and the inflammatory response to them was less pronounced compared with PLGA scaffolds. The results indicate excellent biocompatibility and vascularization capacity of the developed 3D printed HAGM scaffolds and position them as strong candidates for advanced tissue engineering applications.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00741DOI Listing
October 2020

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

Cell Physiol Biochem 2019 ;52(6):1309-1324

Center for Physiology and Pathophysiology, Institute of Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.

Background/aims: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice.

Methods: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections.

Results: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging.

Conclusion: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.
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http://dx.doi.org/10.33594/000000092DOI Listing
May 2019

Generation of Cardiomyocytes in Pipe-Based Microbioreactor Under Segmented Flow.

Cell Physiol Biochem 2016 9;38(5):1883-96. Epub 2016 May 9.

Background/aims: Embryonic stem (ES) cells have got a broad range differentiation potential. The differentiation is initiated via aggregation of non-differentiated ES cells into embryoid body (EB) capable of multi-lineage development. However experimental variables present in standard differentiation techniques lead to high EB heterogeneity, affecting development into the cells of desired lineage, and do not support the process automatization and scalability.

Methods: Here we present a novel pipe based microbioreactor (PBM) setup based on segmented flow, designed for spatial maintenance of temperature, nutrition supply, gas supply and sterility.

Results: We verified PBM feasibility for continuous process generating cardiac cells starting from single ES cell suspension followed by EB formation for up to 10 days. The ES cells used in the study were genetically modified for cardiac-specific EGFP expression allowing optical monitoring of cardiomyocytes while EBs remained within PBM for up to 10 days. Efficiency of cardiac cells formation within PBM was similar compared to a standard hanging drop based protocol.

Conclusion: Our findings ensure further development of microfluidic bioreactor technology to enable robust cardiomyocytes production for needs of drug screening, tissue engineering and other applications.
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http://dx.doi.org/10.1159/000445550DOI Listing
February 2017

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

Arch Toxicol 2016 Jun 29;90(6):1529-1539. Epub 2016 Apr 29.

Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.
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http://dx.doi.org/10.1007/s00204-016-1719-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435100PMC
June 2016

Toxicogenomics directory of chemically exposed human hepatocytes.

Arch Toxicol 2014 Dec 16;88(12):2261-87. Epub 2014 Nov 16.

Department of Statistics, TU Dortmund University, Dortmund, Germany.

A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.
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http://dx.doi.org/10.1007/s00204-014-1400-xDOI Listing
December 2014

Evidence for self-maintaining pluripotent murine stem cells in embryoid bodies.

Stem Cell Rev Rep 2014 Feb;10(1):1-15

Pluripotent stem cells have great potential for regenerative medicine; however, their clinical use is associated with a risk of tumor formation. We utilized pluripotent cells expressing green fluorescent protein and puromycin resistance under control of the Oct4 promoter to study the persistence of potential pluripotent cells under embryoid body (EB) culture conditions, which are commonly used to obtain organotypic cells. We found that i.) OCT4-expressing cells dramatically decrease during the first week of differentiation, ii.) the number of OCT4-expressing cells recovers from day 7 on, iii.) the OCT4-expressing cells are similar to embryonic stem cells grown in the presence of leukemia inhibitory factor LIF but express several markers associated with germ cell formation, such as DAZL and STRA-8 and iv.) the persistence of potentially pluripotent cells is independent of supportive cells in EBs. Finally, OCT4-expressing cells, isolated from EBs after 2-month of culture, were further maintained under feeder-free conditions in absence of LIF and continued to express OCT4 in 95 % of the population for at least 36 days. These findings point to an alternative state of stable OCT4 expression. In the frame of the landscape model of differentiation two attractors of pluripotency might be defined based on their different characteristics.
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http://dx.doi.org/10.1007/s12015-013-9472-1DOI Listing
February 2014

Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing.

Stem Cell Rev Rep 2011 Sep;7(3):506-17

Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, Germany.

Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing. We therefore studied the effects of cryopreservation on the viability, proliferation potential, and the pluripotency status of hESCs by combining cellular readouts and transcriptomics. We identified biological processes and pathways affected by cryopreservation in order to understand the limited survival rate of hESCs by comparing transcriptomes of hESCs at different time points after thawing with cells that did not undergo cryopreservation. While the transcriptomes of cells post thawing were very similar to those of control non-frozen hESCs for the early time points, we observed increased expression of genes involved in apoptosis, embryonic morphogenesis, ossification, tissue morphogenesis, regeneration, vasculature development and cell death at later time points. Our data suggest that inhibition of anoikis apoptosis and the stress-induced differentiation pathways are promising targets for improving the survival rate and maintaining pluripotency of hESCs after cryopreservation.
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http://dx.doi.org/10.1007/s12015-011-9230-1DOI Listing
September 2011

Fibroblasts support functional integration of purified embryonic stem cell-derived cardiomyocytes into avital myocardial tissue.

Stem Cells Dev 2011 May 23;20(5):821-30. Epub 2011 Jan 23.

Institute for Neurophysiology, University of Cologne, Cologne, Germany.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.
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http://dx.doi.org/10.1089/scd.2010.0398DOI Listing
May 2011

Direct contact of umbilical cord blood endothelial progenitors with living cardiac tissue is a requirement for vascular tube-like structures formation.

J Cell Mol Med 2011 Sep;15(9):1914-26

Institute of Cellular Biology and Pathology 'Nicolae Simionescu', Bucharest, Romania Institute for Neurophysiology, University of Cologne, Cologne, Germany.

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro-angiogenic growth factor cocktail consisting of VEGF, IGF-1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti-human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube-like structures in direct contact with the living ventricular slices. Furthermore, the pro-angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube-like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro-angiogenic cocktail. In conclusion, EPCs form vascular tube-like structures in contact with living cardiac tissue and the direct cell-to-cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro-environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.
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http://dx.doi.org/10.1111/j.1582-4934.2010.01197.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918047PMC
September 2011

A Cre-based double fluorescence indicator system for monitoring cell fusion events and selection of fused cells.

Biotechniques 2010 Feb;48(2):113-20

Institute for Neurophysiology, University of Cologne, Cologne, Germany.

We have established an in vitro Cre/loxP-based assay for monitoring cell fusion events that specifically traces the transport of cytoplasm from one cell to its fusion partner. Cells with a double fluorescence vector indicate fusion with cells expressing Cre recombinase by switching expression from red to green fluorescent protein through a Cre-mediated recombination event that simultaneously activates puromycin-acetyltransferase expression. This strategy allows for both the observation and puromycin selection of indicator cells that have undergone fusion with a Cre recombinase-expressing partner. A fusion protein of Cre with estrogen receptor (ER) can be used to control Cre recombinase activity through the tamoxifen-induced translocation of the Cre-ER fusion protein to the nucleus. Here we have established a new methodology that not only allows the monitoring of the transport of cellular contents, but also enables the purification of fused cells using puromycin.
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http://dx.doi.org/10.2144/000113352DOI Listing
February 2010

"The good into the pot, the bad into the crop!"--a new technology to free stem cells from feeder cells.

PLoS One 2008 21;3(11):e3788. Epub 2008 Nov 21.

Institute for Research in Operative Medicine (IFOM), Faculty of Medicine, University of Witten/Herdecke, Witten, Germany.

A variety of embryonic and adult stem cell lines require an initial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003788PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582950PMC
June 2009

Generation of a double-fluorescent double-selectable Cre/loxP indicator vector for monitoring of intracellular recombination events.

Nat Protoc 2008 ;3(9):1510-26

Institute for Neurophysiology, Robert Koch Str. 39, 50931 Cologne, Germany.

Here we describe the generation of a double-fluorescent Cre/loxP indicator system. This protocol involves (i) all cloning steps to generate the plasmid vector (3-5 months); (ii) a guide to prepare high-efficiency transformation competent E. coli; (iii) generation of double-fluorescent reporter cell lines (3-4 weeks); and (iv) the functional testing of the indicator cell lines by application of cell-permeable Cre recombinase. The indicator is designed to monitor recombination events by switching the fluorescence light from red to green. The red fluorescence, indicating the nonrecombined state, is accompanied by the expression of a resistance gene against the antibiotic blasticidin. Appearance of green fluorescence concomitantly with the activation of puromycin-acetyltransferase monitors the recombination of the indicator construct by the Cre recombinase. In summary, we have developed a plasmid vector allowing a fast, stable and straightforward generation of transgenic clones. The expression of red fluorescent protein enables the selection of positive clones upon transfection and significantly shortens the time for identification of stable clones. This feature and the option to select for recombined cells by puromycin application are advantages compared with other alternative methods. Moreover, we developed a method utilizing cell-permeable Cre protein to validate the transgenic clones. Ultimately, this powerful methodology facilitates Cre/loxP-based applications such as cell lineage tracking or monitoring of cell fusion.
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http://dx.doi.org/10.1038/nprot.2008.143DOI Listing
December 2008

Adult mesenchymal stromal stem cells for therapeutic applications.

Minim Invasive Ther Allied Technol 2008 ;17(2):79-90

Institute of Neurophysiology, University of Cologne, Cologne, Germany.

Mesenchymal stromal stem cells (MSC) can be found in almost any adult organ. They can be isolated and expanded within several weeks up to hundreds of millions of cells. The cell isolation based on the surface antigen expression may significantly enrich for the desired cell population and reduce the time required for cell expansion. MSC display a unique molecular signature which clearly discriminates them from other stem cell types. MSC can be differentiated into the cells of several lineages. Additionally, the unique biological properties of MSC are mediated by strong immunomodulatory activity and by paracrine mechanisms. Potential therapeutic applications of the cells require clinically compliant protocols for cell isolation and expansion. The therapeutic utility of MSC has been evaluated and found to be useful in several pre-clinical animal models as well as in clinical trials.
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http://dx.doi.org/10.1080/13645700801969758DOI Listing
June 2008

Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers.

Hepatology 2007 Sep;46(3):861-70

Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany.

Unlabelled: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage.

Conclusion: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.
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http://dx.doi.org/10.1002/hep.21745DOI Listing
September 2007

Reproducible and complete early separation of stem and feeder cells does not attenuate the differentiation potential of feeder-freed stem cells.

J Stem Cells Regen Med 2007 16;2(1):7-8. Epub 2007 May 16.

Institute for Research in Operative Medicine, University Witten/Herdecke , Germany.

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June 2014

Respiratory chain deficiency slows down cell-cycle progression via reduced ROS generation and is associated with a reduction of p21CIP1/WAF1.

J Cell Physiol 2006 Oct;209(1):103-12

Institute of Vegetative Physiology, University of Köln, Köln, Germany.

We have used HeLa cells without mitochondrial DNA (rho0-cells) and transient rho0-phenocopies, obtained from wild-type cells by short-term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron-sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in rho0-cells and in rho0-phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell-cycle regulators were reduced in rho0-cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the rho0-phenocopies, the expression pattern did not fully duplicate the complex response observed in rho0-cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK-signaling pathways did not correlate with proliferation rates of rho0-cells and rho0-phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell-cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1.
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http://dx.doi.org/10.1002/jcp.20711DOI Listing
October 2006

Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model.

Circulation 2005 Aug;112(9 Suppl):I96-104

Department of Surgery, Toronto General Research Institute, Toronto General Hospital, Toronto, Ontario, Canada.

Background: Cell transplantation offers the promise in the restoration of ventricular function after an extensive myocardial infarction, but the optimal cell type remains controversial. Human unrestricted somatic stem cells (USSCs) isolated from umbilical cord blood have great potential to differentiate into myogenic cells and induce angiogenesis. The present study evaluated the effect of USSCs on myocardial regeneration and improvement of heart function after myocardial infarction in a porcine model.

Method And Results: The distal left anterior descending artery of Yorkshire pigs (30 to 35 kg) was occluded by endovascular implantation of a coil. Four weeks after infarction, single-photon emission computed tomography technetium 99m sestamibi scans (MIBI) and echocardiography were performed. USSCs (100 x 10(6)) or culture media were then directly injected into the infarcted region (n=8 per group). Pigs were immunosuppressed by daily administration of cyclosporin A. At 4 weeks after transplantation, MIBI and echocardiography were repeated and heart function was also assessed with a pressure-volume catheter. The infarcted myocardium and implanted cells were studied histologically. MIBI showed improved regional perfusion (P<0.05) and wall motion (P<0.05) of the infarct region in the transplant group compared with the control. Ejection fraction evaluated by both MIBI and echocardiography decreased in the control group but increased in the transplant group (P<0.01). Scar thickness of the transplant group was higher than the control. The grafted cells were detected 4 weeks after transplantation by both immunohistochemistry and in situ hybridization.

Conclusions: Engrafted USSCs were detected in the infarct region 4 weeks after cell transplantation, and the implanted cells improved regional and global function of the porcine heart after a myocardial infarction. This study suggests that the USSC implantation will be efficacious for cellular cardiomyoplasty.
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http://dx.doi.org/10.1161/01.CIRCULATIONAHA.105.524678DOI Listing
August 2005

Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells.

BMC Cancer 2004 Aug 31;4:58. Epub 2004 Aug 31.

Institute of Carcinogenesis, N. N. Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow 115478, Russia.

Background: Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test (cytological smear analysis). However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody.

Methods: Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4 (MTM Laboratories, Germany) was used.

Results: In control samples we did not find any p16INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I - CIN II - CIN III - invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma (out of 21 analyzed) were negative.

Conclusions: Overexpression of the protein p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to exclude a patient from the high risk group. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary test for early diagnostics of cervical cancer.
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http://dx.doi.org/10.1186/1471-2407-4-58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517716PMC
August 2004

Activity of complex III of the mitochondrial electron transport chain is essential for early heart muscle cell differentiation.

FASEB J 2004 Aug 4;18(11):1300-2. Epub 2004 Jun 4.

Institute of Vegetative Physiology, Center of Physiology and Pathophysiology, The University of Köln, Germany.

During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the alpha-myosin heavy chain promoter (alpha-MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that specifically complex III function rather than mitochondrial ATP production is necessary for early heart cell development. When the underlying mechanism was examined, we noticed that antimycin A but not KCN lead to inhibition of spontaneous intracellular Ca++ oscillations, whereas both substances decreased mitochondrial membrane potential, as expected. We postulate that mitochondrial complex III activity is necessary for these Ca++ oscillations, which in turn are a prerequisite for cardiomyocyte differentiation.
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http://dx.doi.org/10.1096/fj.03-0520fjeDOI Listing
August 2004

Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells.

Free Radic Biol Med 2004 Mar;36(6):765-73

Institute of Pharmacy, University of Mainz, D-55099 Mainz, Germany.

The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.
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http://dx.doi.org/10.1016/j.freeradbiomed.2003.12.019DOI Listing
March 2004

Interphase chromosome locus displacement induced by low-doses of radiation.

Radiats Biol Radioecol 2002 Nov-Dec;42(6):604-7

State Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia.

The most important stage in the making of mutations is a reparation of different DNA damage, including the more deleterious double-strand DNA breaks (DSB). The first stage of adaptive response--fundamental antimutagenic cell reaction, purposeful to reparation for induced DSB repair--is investigated in present work. Non-radioactive in situ hybridization of biotin-labeled DNA probe was used to mark chromosome 1 pericentromeric regions (PR) in G0 human lymphocytes. It was shown that under 3-10 cGy (X-radiation, 160 kV) PR become displaced from a nucleus periphery to inner territory of a nucleus. The moving process realizes during several hours after an irradiation. As far as some non-specific gene repressors are co-localized with chromosome centromeric regions it is possible hypothesizes that the displacement cause changing expression of some genes. It is possible to propose that an absence of radiation induced chromosome locus displacement may be one of causes DSB repair disturbance. This hypothesis was tested by the model. It is assumed that one consequence of the underlying defect may be inappropriate involvement of cell's recombination machinery in the repair of DSB. We studied lymphocytes of patients with hereditary BRCA2 mutation. It is thought that this gene takes part in DSB repair. The significant differences of the PR moving between control samples and the cases were revealed under 10 cGy. Similar results were observed on lymphocytes of patients with Fanconi syndrome. Thus, abnormal moving of interphase nucleus chromosomes conditioned by low-dose irradiation may suggest on imperfect machinery of DSB repair, i.e. genetic risk. We realize that further investigations are needed for definitive conclusion.
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January 2003

The human papillomavirus oncoprotein E7 attenuates NF-kappa B activation by targeting the Ikappa B kinase complex.

J Biol Chem 2002 Jul 1;277(28):25576-82. Epub 2002 May 1.

Institute for Vegetative Physiology, University of Cologne, Robert-Koch-Strasse 39, Germany.

Infection with high-risk human papillomaviruses (HPV) can lead to the development of cervical carcinomas. This process critically depends on the virus-encoded E6 and E7 oncoproteins, which stimulate proliferation by manipulating the function of a variety of host key regulatory proteins. Here we show that both viral proteins dose-dependently interfere with the transcriptional activity of NF-kappaB. A variety of experimental approaches revealed that a fraction of the E7 proteins is found in association with the IkappaB kinase complex and attenuates induced kinase activity of IkappaB kinase alpha (IKKalpha) and IKKbeta, thus resulting in impaired IkappaBalpha phosphorylation and degradation. Indirect immunofluorescence shows that E7 impairs TNFalpha-induced nuclear translocation of NF-kappaB, thus preventing NF-kappaB from binding to its cognate DNA. While E7 obviates IKK activation in the cytoplasm, the E6 protein reduces NF-kappaB p65-dependent transcriptional activity within the nucleus. We suggest that HPV oncogene-mediated suppression of NF-kappaB activity contributes to HPV escape from the immune system.
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http://dx.doi.org/10.1074/jbc.M201884200DOI Listing
July 2002

Elevated expression of c-myc in lymphoblastoid cells does not support an Epstein-Barr virus latency III-to-I switch.

J Gen Virol 2001 Dec;82(Pt 12):3051-3055

GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Klinische Molekularbiologie und Tumorgenetik, Marchioninistr. 25, D-81377 München, Germany1.

Epstein-Barr virus (EBV) transforms primary B cells in vitro. Established cell lines adopt a lymphoblastoid phenotype (LCL). In contrast, EBV-positive Burkitt's lymphoma (BL) cells, in which the proto-oncogene c-myc is constitutively activated, do not express a lymphoblastoid phenotype in vivo. The two different phenotypes are paralleled by two distinct programmes of EBV latent gene expression termed latency type I in BL cells and type III in LCL. Human B cell lines were established from a conditional LCL (EREB2-5) by overexpression of c-myc and inactivation of EBV nuclear protein 2 (EBNA2). These cells (A1 and P493-6) adopted a BL phenotype in the absence of EBNA2. However, the EBV latency I promoter Qp was not activated. Instead, the latency III promoter Cp remained active. These data suggest that the induction of a BL phenotype by overexpression of c-myc in an LCL is not necessarily paralleled by an EBV latency III-to-I switch.
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http://dx.doi.org/10.1099/0022-1317-82-12-3051DOI Listing
December 2001

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells.

Int J Cancer 2001 Sep;93(6):810-6

Institut für Klinische Molekularbiologie und Tumorgenetik, Hämatologikum der GSF, München, Germany.

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.
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http://dx.doi.org/10.1002/ijc.1404DOI Listing
September 2001

Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri.

Int J Cancer 2001 Apr;92(2):276-84

Division of Molecular Diagnostics and Therapy, Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany.

Cytological screening for cervical cancer or its precursors using Papanicolaou's smear test (Pap test) has been highly efficient to reduce the morbidity and mortality of cervical cancer. However, evaluation of the Pap test relies on subjective diagnostic parameters and is affected by a high rate of false-positive and false-negative results. More objective diagnostic parameters to identify truly dysplastic or neoplastic cells in cervical smears as well as in cervical biopsy samples would therefore avoid insecurity for many patients and the high screening costs associated with repeated testing. Cervical dysplasia is induced by persistent infections through high-risk types of human papillomaviruses (HPVs). Outgrowth of dysplastic lesions is triggered by increasing expression of two viral oncogenes, E6 and E7, which both interact with various cell cycle-regulating proteins. Among these is the retinoblastoma gene product pRB, which is inactivated by E7. pRB inhibits transcription of the cyclin-dependent kinase inhibitor gene p16(INK4a). Increasing expression of the viral oncogenes in dysplastic cervical cells might thus be reflected by increased expression of p16(INK4a). In line with this hypothesis, we observed marked overexpression of p16(INK4a) in all cervical intraepithelial neoplasm (CIN) I lesions (n = 47) except those associated with low-risk HPV types (n = 7), all CIN II lesions (n = 32), all CIN III lesions (n = 60) and 58 of 60 invasive cervical cancers. In contrast, no detectable expression of p16(INK4a) was observed in normal cervical epithelium (n = 42), inflammatory lesions (n = 48) and low-grade cervical lesions (CIN I) associated with low-risk HPV types (n = 7). Dysplastic cells could also be identified in cervical smears using a specific p16(INK4a) monoclonal antibody. These data demonstrate that p16(INK4a) is a specific biomarker to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears.
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http://dx.doi.org/10.1002/ijc.1174DOI Listing
April 2001

Cell cycle activation by c-myc in a burkitt lymphoma model cell line.

Int J Cancer 2000 Sep;87(6):787-93

Institute for Clinical Molecular Biology and Tumor Genetics, GSF-Research Center for Environment and Health, Munich, Germany.

The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.
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http://dx.doi.org/10.1002/1097-0215(20000915)87:6<787::aid-ijc4>3.0.co;2-6DOI Listing
September 2000

Telomere DNA sequences and the concept of ontogenetic reserve cells.

Authors:
D M Spitkovsky

Biochemistry (Mosc) 1997 Nov;62(11):1285-90

Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia.

Dynamics of telomere sequences are considered in normal and immortalized cells. Immortalized cells are suggested to be derived mainly from a special subpopulation of ontogenetic reserve cells. Their epigenetic program consists of autoregeneration during external stimulus for genome reorganization and corresponding appearance of genetic variants of cells. It is suggested that cells surviving the crisis stage contain a special signal sequence integrated into telomere DNA. Its elimination during shortening of DNA telomere sequences in dividing ontogenetic reserve cells is a signal for the cooperative transition of chromatin into a new steady state that corresponds to the epigenotype of immortalized cells. Localization of telomere DNA sequences in intrachromosomal "hot spots" reflects phylogenetic rearrangement of the genome.
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November 1997

Opposite transcriptional effects of cyclic AMP-responsive elements in confluent or p27KIP-overexpressing cells versus serum-starved or growing cells.

Mol Cell Biol 1998 Jan;18(1):409-19

Applied Tumor Virology, Abteilung 0610 and Institut National de la Santé et de la Recherche Médicale U 375, Heidelberg, Germany.

The minute virus of mice, an autonomous parvovirus, requires entry of host cells into the S phase of the cell cycle for its DNA to be amplified and its genes expressed. This work focuses on the P4 promoter of this parvovirus, which directs expression of the transcription unit encoding the parvoviral nonstructural polypeptides. These notably include protein NS1, necessary for the S-phase-dependent burst of parvoviral DNA amplification and gene expression. The activity of the P4 promoter is shown to be regulated in a cell cycle-dependent manner. At the G1/S-phase transition, the promoter is activated via a cis-acting DNA element which interacts with phase-specific complexes containing the cellular transcription factor E2F. It is inhibited, on the other hand, in cells arrested in G1 due to contact inhibition. This inhibitory effect is not observed in serum-starved cells. It is mediated in cis by cyclic AMP response elements (CREs). Unlike serum-starved cells, confluent cells accumulate the cyclin-dependent kinase inhibitor p27, suggesting that the switch from CRE-mediated activation to CRE-mediated repression involves the p27 protein. Accordingly, plasmid-driven overexpression of p27 causes down-modulation of promoter P4 in growing cells, depending on the presence of at least two functional CREs. No such effect is observed with two other cyclin-dependent kinase inhibitors, p16 and p21. Given the importance of P4-driven synthesis of protein NS1 in parvoviral DNA amplification and gene expression, the stringent S-phase dependency of promoter P4 is likely a major determinant of the absolute requirement of the minute virus of mice for host cell proliferation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC121511PMC
http://dx.doi.org/10.1128/mcb.18.1.409DOI Listing
January 1998

Down-regulation of cyclin A gene expression upon genotoxic stress correlates with reduced binding of free E2F to the promoter.

Cell Growth Differ 1997 Jun;8(6):699-710

Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg, Germany.

Treatment of mammalian cells by DNA-damaging agents leads to various cellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for its widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G1 phase of the cell cycle, most likely due to a signal generated by the stabilization of p53 and the subsequent induction of p21WAF-1/Cip1. We show here that cisplatin-treated mammalian cells accumulate normal levels of cyclin D1 and cyclin E but fail to produce cyclin A. The block to cyclin A gene expression occurs at the level of transcription and is mediated by an E2F binding site in the cyclin A promoter. It is shown here that, upon cisplatin treatment, transcriptionally active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Immunoprecipitation experiments suggest that the loss of free E2F results, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107. A role for the kinase inhibitor p21WAF-1/Cip1 in repression of the cyclin A promoter is supported by our finding that ectopic expression of p21WAF-1/Cip1 is sufficient to inhibit transcription from the cyclin A gene, dependent on the E2F site. The data establish the E2F site in the human cyclin A promoter as a key target for the signaling pathway leading to G1 arrest in response to DNA damage by cisplatin and potentially other genotoxic agents.
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June 1997