DNA Agarose Electrophoresis Research Articles

Studies on the Effects of Berbamine on the Human Leukemia Cell Line NB4.

Purpose: To study the effects of berbamine on the human leukemia cell line NB4Methods: Berbamine compound was dissolved to the concentration of 1mg/ml with 0.9% sodium chloride and diluted to desired concentrations (as indicated in figure ledends 0.5,1,2,4,8,12,16ug/mL)with culture medium. Growth of NB4 cells was determined by MTT assay based upon the reduction of tetrazolium dye 3–4,5-dimethylthiazol-2,5-diphenyltetrazolium(MTT). Morphological observation was used to detect the characteristic changes of apoptosis in NB4 cells and DNA agarose electrophoresis were used to detect the characteristic changes of apoptosis in NB4 cells, while apoptosis rate was measured by flow cytometry assay. The _expression of PML/RARα gene was determined by nested-PCR.Result: MTT results showed berbamine could inhibit the growth of NB4 cells significantly after treated with different concentration of berbamine for different time, IC50 was 3.86 ug•mL-1at 48 hours. Typical nuclear condensation and apotosis body were observed as early as 24 hours following a 8μgμg•mL-1 berbamine treatment;while the DNA agarose electrophoresis also show the typical DNA ladder,and the apoptosis rate of NB4 cell was increased from 2.83%to 58.84% after treated with 12ug•mL-1 berbamine for 48 hours. The _expression of PML/RARα gene wasn't changed obviously.Conclusion: Berbamin could inhibit the proliferation of NB4 cells and inducing apoptosis might be its one of mechanisms. The berbamine-induced apoptosis is not related to the _expression of PML/RARα gene.

In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells.

To investigate the effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 in vitro and the possible mechanisms involved. Chitosan nanoparticles were characterized by particle size, zeta potential, and morphology. After treatment with various concentrations of chitosan nanoparticles (25, 50, 75, 100 microg/mL) at various time intervals, cell proliferation, ultrastructural changes, DNA fragmentation, mitochondrial membrane potential (MMP), cell cycle phase distribution and apoptotic peaks of MGC803 cells were analyzed by MTT assay, electron microscopy, DNA agarose gel electrophoresis, and flow cytometry. Chitosan nanoparticles exhibited a small particle size as 65 nm and a high surface charge as 52 mV. Chitosan nanoparticles markedly inhibited cell proliferation of MGC803 cells with an IC50 value of 5.3 microg/mL 48 h after treatment. After treatment with chitosan nanoparticles, the typical necrotic cell morphology was observed by electron microscopy, a typical DNA degradation associated with necrosis was determined by DNA agarose electrophoresis. Flow cytometry showed the loss of MMP and occurrence of apoptosis in chitosan nanoparticles-treated cells. Chitosan nanoparticles effectively inhibit the proliferation of human gastric carcinoma cell line MGC803 in vitro through multiple mechanisms, and may be a beneficial agent against human carcinoma.

Cytotoxicity, apoptosis induction, and mitotic arrest by a novel podophyllotoxin glucoside, 4DPG, in tumor cells1

To define the in vitro cytotoxic activities of 4-demethyl-picropodophyllotoxin 7'-O-beta-D-glucopyranoside (4DPG), a new podophyllotoxin glucoside. Antiproliferation activity was measured in several tumor cell lines by using the microculture tetrazolium MTT assays. Cell cycle distribution was analyzed using flow cytometry and mitosis index assays. Furthermore, transmission electron microscopy, TUNEL, DNA agarose electrophoresis, and activated caspase-3 were used to analyze the induction of apoptotic cell death. Moreover, intracellular changes in the cytoskeleton were detected using immunocytochemistry. 4DPG effectively inhibited the proliferation of cancer cells (HeLa, CNE, SH-SY5Y, and K562 cell lines). For the K562 cell line, the antiproliferation effect of 4DPG was much more potent than that of etoposide (IC50 value: 7.79 x 10(-9) mol/L for 4DPG vs 2.23 x 10(-5) mol/L for etoposide). Further, 4DPG blocked the cell cycle in the mitotic phase. The induction of apoptosis and elevated levels of activated caspase-3 were confirmed in cells treated with 4DPG. The microtubule skeleton of HeLa cells was disrupted immediately after treatment with 4DPG. The cytotoxicity of 4DPG is due to its inhibition of the microtubule assembly of cancer cells at a low concentration, thus inducing apoptosis. These properties qualify 4DPG to be a potential antitumor drug.

Ultra-Fast High-Resolution Agarose Electrophoresis of DNA and RNA using Low-Molarity Conductive Media

Current DNA electrophoretic solutions employ high ionic concentrations and require long electrophoretic run times. Here we demonstrate that high and low molecular weight double-stranded DNA, single-stranded DNA (ssDNA), and RNA can be separated rapidly in agarose-based low-molarity conductive media. Separation of small DNA fragments was optimized by substituting 1-mM solutions of alkali metals or a nonbiological amine that distributed voltage with a minute current. These ultra-dilute solutions can separate DNA at least 15-fold faster Low-molarity media at 5-10 mM adequately separated RNA and larger DNA fragments as well. These novel media reduce the Joule heating of the electrophoretic system and allow for easy-to-use, ultra-fast separation of DNA fragments.

Molecular mechanisms in C-Phycocyanin induced apoptosis in human chronic myeloid leukemia cell line-K562

C-Phycocyanin (C-PC), the major light harvesting biliprotein from Spirulina platensis is of greater importance because of its various biological and pharmacological properties. It is a water soluble, non-toxic fluorescent protein pigment with potent anti-oxidant, anti-inflammatory and anti-cancer properties. In the present study the effect of highly purified C-PC was tested on growth and multiplication of human chronic myeloid leukemia cell line (K562). The results indicate significant decrease (49%) in the proliferation of K562 cells treated with 50 μM C-PC up to 48 h. Further studies involving fluorescence and electron microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing and nuclear condensation. Agarose electrophoresis of genomic DNA of cells treated with C-PC showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 25 and 50 μM C-PC for 48 h showed 14.11 and 20.93% cells in sub-G0/G1 phase, respectively. C-PC treatment of K562 cells also resulted in release of cytochrome c into the cytosol and poly(ADP) ribose polymerase (PARP) cleavage. These studies also showed down regulation of anti-apoptotic Bcl-2 but without any changes in pro-apoptotic Bax and thereby tilting the Bcl-2/Bax ratio towards apoptosis. These effects of C-PC appear to be mediated through entry of C-PC into the cytosol by an unknown mechanism. The present study thus demonstrates that C-PC induces apoptosis in K562 cells by cytochrome c release from mitochondria into the cytosol, PARP cleavage and down regulation of Bcl-2.

Capillary electrophoretic determination of apoptosis of rat cerebellar granule cells induced by 1-methyl-4-phenyl pyridium ion.

A capillary electrophoresis method for the direct detection of whole cell apoptosis is described. We successfully used this method to detect the apoptosis of rat cerebellar granule cells, induced by 1-methyl-4-phenyl pyridium ion (MPP(+)). The conditions for the detection were optimized: including the effects of running buffer pH, the voltage, and the ID of the capillaries. The effects of MPP(+) concentration and apoptosis time on the relative content of the apoptotic cells were studied. The relative standard deviations of the migration time and the absorbance of the apoptotic cells were found to be 10.8 and 8.6%, respectively. The results correlated well with those obtained by using the methyl green-pironin stained experiment and DNA agarose electrophoresis.

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone . In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.

Induction of Apoptosis and Growth Inhibition of Cultured Human Endometrial Adenocarcinoma Cells (Sawano) by an Antitumor Lipoprotein Fraction of Rice Bran

Objective. A lipoprotein fraction extracted from rice bran (RBF) has been proved to be nontoxic to normal cells and to possess the ability to derange energy metabolism and induce apoptosis in Ehrlich ascites tumor cells. This study is designed to investigate the therapeutic effects of RBF as an antitumor substance on endometrial adenocarcinoma cells (Sawano).Methods. Cultured human endometrial adenocarcinoma cells were treated with medium only (untreated), DMSO, or RBF at 100, 200, and 300 μg/ml. Mitotic indexes were counted by Giemsa stain and apoptotic index by fluorescent TUNEL stain for confocal laser scanning microscopy. DNA fragments were examined by agarose gel electrophoresis.Results. Characteristic apoptotic morphology was observed in Giemsa-stained cells and further confirmed by confocal laser scanning microscopy in RBF-treated cells. Growth of Sawano cells was inhibited by RBF in a dose- and time-dependent manner. Apoptotic indexes (AI) showed an increasing tendency, and mitotic indexes (MI) showed a downward tendency. In 300 μg/ml RBF-treated Sawano cells, AI even reached 41.62 ± 2.23% and MI decreased to 0.79 ± 0.08% at 96 h. Both showed significant differences from to controls and other RBF-treated groups. DNA agarose electrophoresis revealed DNA fragmentation in a ladder pattern.Conclusions. RBF can induce apoptosis of cultured human endometrial adenocarcinoma cells (Sawano) and inhibit its cell proliferation as an antitumor substance.

Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.

Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.

5-Lipoxygenase inhibitors reduce PC-3 cell proliferation and initiate nonnecrotic cell death.

Products of the arachidonic acid-metabolizing enzyme, 5-lipoxygenase, stimulate the growth of several cell types. Selective inhibitors of the enzyme, including SC41661A and MK886, reduce PC-3 prostate cell proliferation. With continued culture, cells die, but the mode of death, necrotic or nonnecrotic, has not been established. Flow cytometry, laddering after agarose electrophoresis of DNA from inhibitor-treated cells, and light and electron microscopy were employed to examine the type of death in PC-3 prostate cells cultured with either 5-lipoxygenase inhibitor. The inhibitors induced nonnecrotic, programmed cell death. SC41661A-treated cells exhibited "foamy," vacuolated cytoplasm and mitochondria with disrupted cristae and limiting membranes, while some cells contained numerous polysomes and extended hypertrophic Golgi and secretory cisternal networks. A proportion of the treated cells detached and the nuclei of these cells were characteristic of type 1 "apoptotic" programmed cell death. MK886, a 5-lipoxygenase-inhibitor with a different mechanism of action, induced nonnecrotic changes largely confined to the cytoplasm, most consistent with type 2 "autophagic" programmed cell death. In preliminary studies of mechanism, we demonstrated that PC-3 cells express mRNA for 5-lipoxygenase and for 5-lipoxygenase-activating protein. The less active inhibitor, SC45662 neither reduced proliferation nor induced DNA laddering. The antioxidant, N-acetyl-l-cysteine but not butylated hydroxy toluene or alpha tocopherol, partially reduced the inhibition of proliferation from SC41661A. SC41661A and MK886 inhibit PC-3 cell proliferation and induce a form of type 1 or type 2 programmed cell death, respectively. PC-3 cells contain messenger RNA for 5-lipoxygenase and 5-lipoxygenase-activating proteins. Drug-induced changes included altered redox potential, inferred from the increased survival due to the antioxidant and glutathione precursor, N-acetyl-l-cysteine. PC-3 cells are an appropriate model for studying the mechanism responsible for 5-lipoxygenase inhibitor-induced cellular suicide.

Apoptosis of Epithelial Cells and Macrophages Due to Infection with the Obligate Intracellular PathogenChlamydia psittaci

AbstractWe have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.

Identification, purification and characterization of a putative novel growth-inhibitory and/or apoptotic protein from rat liver.

The existence of endogenous growth inhibitors was postulated in 1914 by Boveri. However, most regretfully, progress in the isolation, characterization and mechanisms of actions of endogenous growth-inhibitory proteins is scanty compared to the information available on growth-stimulatory proteins. Accordingly, the major purpose of the present study was to isolate and characterize an endogenous growth-inhibitory protein from normal rat liver so that its role during liver carcinogenesis could be evaluated. For protein purification, a combination of alcohol precipitation, gel permeation chromatography and ion exchange chromatography techniques was utilized. For characterization and mechanisms, the methods utilized were DNA synthesis, immunoblotting, immunohistochemistry, protein sequencing, DNA-agarose electrophoresis and Hoechst staining. The purified protein inhibited the growth of several cell lines in culture as measured by the rate of DNA synthesis using 3H-thymidine. In SDS-PAGE stained by the silver staining method, the molecular weight of the polypeptide was found to be 14 kD. Polyclonal antiserum was raised against this 14 kD polypeptide in rabbit. Immunoblotting experiments showed that the antibody recognizes specifically the 14 kD polypeptide and immunolocalization studies showed that the polypeptide is predominantly a cytoplasmic protein. Addition of antibody and inhibitory polypeptide simultaneously to the cultures more or less abolished the inhibitory activity of the polypeptide. Sequencing of the N-terminal 17 amino acids of the growth-inhibitory polypeptide showed Val-Leu-Leu-Ala-Glu-Ala-Glu-Thr-Ala-Ile-Val-Asn-Gly-Leu-Asp-Lys-Ile. Comparing this sequence using a BLAST protein data base indicated that there was no significant homology between the sequence of the growth-inhibitory polypeptide and protein sequences deposited with the data bank, suggesting that this could be a novel growth-inhibitory polypeptide. The mechanisms of growth inhibition appeared to be apoptosis as determined by electrophoretic analysis of DNA fragmentation and staining of the cells with the dye Hoechst 33342. A growth-inhibitory protein of 14 kD can be isolated from normal rat liver. The physiologic role of the protein in liver appears to be either growth regulatory or apoptotic.

Characterization of the Cell Death Process Induced by Staurosporine in Human Neuroblastoma Cell Lines

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population ( ec 50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal. © 1997 Elsevier Science Ltd.

Analysis of neuronal death in the central nervous system using a new apoptosis model

To examine in detail neural apoptosis in the central nervous system (CNS) for the establishment of new therapies, we have developed an experimental in vitro model of neuronal death and analyzed the mechanism of apoptosis. Septal nuclei were dissected from embryonic brains (E16) of Wistar rats and cultured in chemically defined medium. Highly enriched neurons were obtained from the cultures at 4 days. Exposure to heat shock (43.0°C, 60 min) between 24 and 36 h later, resulted in the death of approximately 70% of cells. Morphologically, dying neurons showed disruption of neurites, nuclear condensation, multiple nuclear fragments, condensation of cytoplasm and multiple cellular fragmentation. Agarose electrophoresis of chromosomal DNA revealed a typical ladder-pattern of fragmentation. Following heat treatment, incubation at 37°C was necessary to detect DNA fragmentation. Quantitative analysis by in situ terminal deoxynucleotidyl transferase assay, revealed that the percentage of apoptotic cells markedly increased 8 h after heat treatment, and continued to gradually increase up until 30 h. Neuronal death and DNA fragmentation were prevented by inhibiting RNA and protein synthesis. Cell death and the DNA cleavage were also inhibited by cultivation in calcium-free medium. The addition of homogenous basic fibroblast growth factor to the medium markedly enhanced cell survival under these pathogenic conditions. These results suggest that neural cell death after mild heat treatment has apoptotic characteristics and may be useful for analyzing the mechanism of apoptosis. The clinical application of drugs acting against molecular components and as well as neurotrophic factors, may in the future prevent apoptosis in neural disease.

Promotion of Heat-Induced Apoptosis in FM3A Cells by Protease Inhibitors

Although it has been shown that proteases may play a positive role in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells. Cysteine protease inhibitor, trans-Epoxy-succinyl-l-leucylamido-(4-guanidino)butane (E-64, 100 μg/ml) and aspartate protease inhibitor, pepstatin-A (100 μg/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 °C. The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis. Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock. E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat. The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.