Method of RNA purification from endothelial cells for DNA array experiments.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
N N Khodarev
Publications and source records attributed to N N Khodarev.
Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts.
U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown in vitro were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation. The xenografts were harvested at 1, 5, or 24 h after irradiation and subjected to the same analyses. We observed and report on (i) cell-type common and cell-type specific responses, (ii) genes induced at low levels of irradiation but not at higher doses, (iii) temporal patterns of gene response in U87 xenografts that varied depending on radiation dose and temporal patterns of response that were similar at all doses tested, (iv) significantly higher up-regulation of cells in xenografts than in in vitro cultures, and (v) genes highly up-regulated by radiation. The responding genes could be grouped into nine functional clusters. The representation of the nine clusters was to some extent dependent on dose and time after irradiation. The results suggest that clinical outcome of ionizing radiation treatment may benefit significantly by taking into account both cell-type and radiation-dose specificity of cellular responses.
Animals↗
Angiostatin effects on endothelial cells mediated by ceramide and RhoA.
Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.
Angiostatins↗
Quantitative analysis of DNA array autoradiographs.
DNA arrays and chips are powerful new tools for gene expression profiling. Current arrays contain hundreds or thousands of probes and large scale sequencing and screening projects will likely lead to the creation of global genomic arrays. DNA arrays and chips will be key in understanding how genes respond to specific changes of environment and will also greatly assist in drug discovery and molecular diagnostics. To facilitate widespread realization of the quantitative potential of this approach, we have designed procedures and software which facilitate analysis of autoradiography films with accuracy comparable to phosphorimaging devices. Algorithms designed for analysis of DNA array autoradiographs incorporate 3-D peak fitting of features on films and estimation of local backgrounds. This software has a flexible grid geometry and can be applied to different types of DNA arrays, including custom arrays.
Algorithms↗
LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation.
Using a directional cloning strategy, DNA sequence information was obtained corresponding to the site of early radiation-induced apoptotic DNA fragmentation within the human lymphoblastoid cell line TK6. Data were obtained from 88 distinct clones comprising approximately 65 kbp of sequenced material. Analysis of all cloned material showed that sequences in the 10 bp immediately adjacent to the cleavage sites were enriched in short oligoT tracts. The proportion of repetitive DNA within the entire cloned material was found to be within the normal range. However the distribution of Alu and LINE repetitive DNA were biased to positions at or adjacent to the apoptotic cleavage site. In particular, a non-random distribution of five cleavage sites was found clustered within the second ORF of the LINE L1 that partially overlapped with two binding sites for the nuclear matrix-associated protein SATB1. Three other clones, containing alpha satellite elements, were also linked to a DNA matrix binding function. These data indicate that the site of chromatin loop formation at the nuclear matrix may be a specific target for early DNA fragmentation events during apoptosis.
Apoptosis↗
Accumulation of specific RNAs encoding transcriptional factors and stress response proteins against a background of severe depletion of cellular RNAs in cells infected with herpes simplex virus 1.
Herpes simplex virus 1 encodes several functions to preclude the shutoff of host response to infection, including degradation of mRNA immediately after infection. To determine whether any cellular mRNAs accumulate in infected cells against a background of severe loss of host RNA, we hybridized cDNAs derived from three different cell lines infected with wild type and a mutant virus to a DNA array containing probes for 588 human genes representing different functional groups. The results were that (i) infected cells accumulated at levels above those of mock-infected cells, a small number of transcripts representing transcriptional factors that could regulate gene expression both positively and negatively, and one stress response protein (GADD45), (ii) the amount and nature of the accumulated transcripts showed limited variability depending on the cell and virus, and (iii) at least some of the proteins encoded by the accumulated transcripts could benefit either the virus or the host.
Cell Line↗
Abortive apoptosis as an initiator of chromosomal translocations.
Apoptosis is a well-recognized regulator of a cell populations size and structure. Irreversible stages of apoptosis lead to activation of different enzymatic cascades, changes in cell morphology and DNA fragmentation. However, little is known about nuclear events which accompany the initial stages of apoptosis. These events are connected with introduction of limited amounts of double strand breaks into genomic DNA, some of which may be subsequently rejoined. We hypothesize here that the initial stages of apoptotic DNA fragmentation may be reversible and connected with the initiation of recombinational events and certain chromosomal translocations. The factors influencing apoptosis reversibility and cell survival after delivery of apoptotic stimuli may provide new insights into mechanisms of lymphocyte development and tumorigenesis.
Apoptosis↗
Association between DNA cleavage during apoptosis and regions of chromatin replication.
We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5-1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.
Apoptosis↗
Mycoplasma infection can sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s).
Mycoplasma infection may lead to various pathologies in a broad range of hosts. It has been shown that Mycoplasma may trigger cell death in cell cultures; however, the mechanism remains unknown. In the present paper we show that Mycoplasma infection of different lymphocyte and epithelial tumour cell lines leads to the inhibition of proliferation, and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis. We also showed that this infection leads to an increased sensitivity of cells to various inducers of apoptosis targeting different signalling pathways. Finally, we show that increased apoptosis is associated with overexpression of an endonuclease produced by Mycoplasma. This endonuclease is recovered in the nuclear fraction of host cells, introduces mostly DSB and is active at neutral pH in the presence of divalent cations. Activation of this endonuclease is connected with limited proteolysis, which may be reproduced in vitro by snake venom serine proteinase.
Animals↗
Mechanisms of induction of apoptotic DNA fragmentation.
PURPOSE: Despite its common use as an indicator of apoptosis, little is known about the mechanisms controlling apoptotic DNA fragmentation in irradiated cells. This review discusses the pathways of chromatin fragmentation, and the role of both nucleases and chromatin structure in this process. DEFINITIONS: DNA fragmentation linked to apoptosis is a combination of cleavage events excising both large DNA fragments within the range 0.4-1.0 Mbp and 50 kbp followed by random cuts within internucleosomal regions (i.e. DNA laddering). The first two cleavage steps can be detected in virtually all apoptotic cells, but DNA laddering is not ubiquitously observed. Endonucleases that mediate this cleavage of chromatin may be classified by substrate specificity, mode of DNA cleavage and their cofactor requirements. CONCLUSIONS: Three major pathways of DNA fragmentation are proposed and discussed: (1) upregulation of endonucleases, (2) their intranuclear/intracellular redistribution and (3) primary changes of chromatin structure.
Animals↗
Topologically constrained domains of supercoiled DNA in eukaryotic cells.
The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51+/-0.17Mbp and 1.34+/-0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.
Carcinoma, Squamous Cell↗
An inducible lymphocyte nuclear Ca2+/Mg(2+)-dependent endonuclease associated with apoptosis.
Apoptotic cell death is typically accompanied by internucleosomal chromatin fragmentation. Although a number of candidate enzymes have been proposed, there is as yet no direct evidence for the involvement of any particular endonuclease in this process. Here we demonstrate the existence of an endonuclease(s) that is up-regulated during apoptotic T cell death. The endonuclease(s) is located in the nucleus, and its activity is increased up to eightfold by a variety of stimuli or conditions that induce apoptosis in T cell hybridomas and thymocytes. Treatments that prevent TCR-mediated apoptosis, such as cyclosporin A or concomitant administration of glucocorticoids, also prevent the induction of enzyme activity. The endonuclease activity is associated with three molecular forms, designated A, B, and C, with apparent M(r) of 49K, 47K, and 45K, respectively, and constitutes the major endonuclease activity in T hybridoma cells. From A exists in resting cells, and its activity is increased threefold after the induction of apoptosis. Forms B and C are absent in resting cells and are induced up to 20-fold after stimuli that lead to apoptosis. All three forms are Ca2+/Mg2+ dependent and are inhibited by Zn2+. This enzyme(s) introduces double strand breaks and single strand nicks into supercoiled plasmid DNA, demonstrating the mode of DNA fragmentation characteristic of products of apoptotic chromatin degradation. The enzyme(s) produces DNA fragments with 5'-P and 3'-OH terminals, also consistent with apoptotic chromatin degradation. Finally, enzyme solubilized from cells activated to die cleaves chromatin in nuclei isolated from unstimulated T hybridoma cells, yielding the classic DNA ladder. Because of its biologic properties, we named this enzyme(s) inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease, or ILCME. Because inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease possesses the key features predicted for an apoptosis-specific enzyme, it is a new candidate for an enzyme(s) that participates in DNA cleavage in apoptotic T cells.
Animals↗
[Comparative characteristics of some parameters of the cell nucleus in the series myeloma-hybridoma-lymphocyte].
A comparative study of several parameters of the cell nuclei of hybridoma MLC-1c and its parent cells--myeloma X-63.Ag8.653 and spleen lymphocytes of Balb/c mice, has been carried out. The results of cytogenetic studies suggest that although the hybridoma and myeloma cell lines used in this study are rather stable, they contain some proportion of the altered chromosomal material. Two-dimensional electrophoresis performed according to O'Farrell revealed that the similarity between the relative presentation and reciprocal location of the nuclear proteins expressed by the myeloma and the hybridoma was greater than that between these cell lines and lymphocytes. Probing of the chromatin structure by micrococcal nuclease showed no significant differences in the degree of nuclease resistance of chromatin between myeloma, hybridoma and lymphoid cells. A comparative study of the Ca/Mg-dependent endonuclease activity of the nuclei in situ and in nuclear extracts demonstrated that whereas its content in lymphocytes was rather high, in myeloma and hybridoma it was practically absent. At the same time, cell nucleus extracts of the myeloma and the hybridoma contained high amounts of DNA-binding proteins which were undetectable in mouse spleen lymphocytes.
Animals↗
Internucleosomal chromatin degradation in myeloma and B-hybridoma cell cultures.
The activity of Ca/Mg-dependent endonuclease (CME) is strongly inhibited in myeloma X-63.Ag8.653 and B-hybridoma MLC-1c as compared with mouse splenocytes. Nevertheless, pronounced internucleosomal chromatin degradation occurs in both cell lines during long-term cultivation without passing. In isolated cell nuclei of X-63 the activation of CME, which precedes chromatin fragmentation in vivo and loss of cell viability, is revealed. The time-course of CME activation is opposite to cell proliferation and is not accompanied by alterations in enzyme quantity. The results suggest that cell death of X-63 and MLC-1c occurs via apoptosis, and involves the mechanisms controlling the activation and/or interaction of CME with chromatin.
Animals↗
Intermolecular homologies of human interferon-alpha.
Human interferon-alpha 2 (IFN) was analyzed by homology search computer program with the use of protein primary structures data bases. Results indicate that four domains with heightened ability to form homology pairs with different proteins exist in the IFN molecule. These domains occupy regions 35-56, 72-85, 97-110 and 124-136, mainly between the alpha-helical cylinders on the tertiary structure models. Additionally, results show in IFN structure the presence of amino-acid motifs that create the opportunity for this cytokine to influence directly the processes of DNA functioning in cell nuclei.
Amino Acid Sequence↗
[Cholescintigraphy in clinical practice].
Explore the source record for details and available documents.
Biliary Tract Diseases↗
[Changes in Ca, Mg-dependent endonuclease of DNA in isolated nuclei of human lymphocytes in lymphoproliferative diseases].
Ca, Mg-dependent endonuclease is one of the main DNAses of lymphocyte chromatin. It's activity is known to increase in the immune response and to decrease in spontaneous and experimental CLL. These observations became a basis for analysis of possible clinical meaning of it's enzymatic activity assay. Donors' peripheral blood lymphocytes being tested, normal level of endonucleolysis for men and children was defined. Except that patients with different clinical forms of lymphoproliferative diseases such as chronic lympholeukemia, non-Hodgkin lymphomas, Hodgkin's disease were observed. The results showed that Ca, Mg-dependent endonucleolysis activity was decreased in comparison to donors' one. Ca, Mg-dependent endonucleolysis activity was the same in the group of patients with non-malignant pathology and in donors' one. Successful treatment and remission state of our patients was associated with alteration of the Ca, Mg-dependent endonucleolysis activity to normal level as well as immunological parameters. That is why the activity of Ca, Mg-dependent endonucleolysis is suggested to be a new criterion of immune state and lymphocyte malignant transformation.
Adolescent↗
Early postirradiation chromatin degradation in thymocytes.
The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.
Animals↗