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Biomedical subjects

M Sikorska

Publications and source records attributed to M Sikorska.

At least 19 recordsLinked to original sources
PubMed2006

Oxidative stress regulated expression of ubiquitin Carboxyl-terminal Hydrolase-L1: role in cell survival.

The ubiquitin Carboxyl-terminal Hydrolase-L1 gene (UCHL1) is a key enzyme in the protein degradation pathway; however, its precise role in protecting cells under stress conditions is unclear. In the present study we investigated the activity of this gene in human NT2/D1 embryonal carcinoma cells subjected to oxygen-glucose deprivation (OGD) and reoxygenation. OGD/reoxygenation cause global metabolic changes due to energy withdrawal and the subsequent generation of reactive oxygen species which initiates either a stress-adaptation-survival response or cell death, depending on the severity of the insult. A bi-phasic change in UCHL1 expression was observed by Q-PCR, Western blotting and flow cytometry. Down regulation of UCHL1 was detected immediately after OGD treatment and its expression was subsequently restored and increased 6 h after OGD treatment as well as during reoxygenation. Furthermore, flow cytometry analysis detected a lower level of UCHL1 only in apoptotic cells that had severe loss of mitochondrial membrane potential. Accordingly, down-regulation of endogenous UCHL1 by antisense cDNA in mouse N2a neuroblastoma cells increased the cell's sensitivity to OGD treatment. This down-regulation of endogenous UCHL1 led to the accumulation of p27, suggesting that UCHL1 is an essential gene to maintain cell homeostasis under normal growth and oxidative stress conditions.

Cell Line, Tumor↗
PubMed2005

Role of mitochondria in neuronal cell death induced by oxidative stress; neuroprotection by Coenzyme Q10.

Neuronal cells depend on mitochondrial oxidative phosphorylation for most of their energy needs and therefore are at a particular risk for oxidative stress. Mitochondria play an important role in energy production and oxidative stress-induced apoptosis. In the present study, we have demonstrated that external oxidative stress induces mitochondrial dysfunction leading to increased ROS generation and ultimately apoptotic cell death in neuronal cells. Furthermore, we have investigated the role of Coenzyme Q10 as a neuroprotective agent. Coenzyme Q10 is a component of the mitochondrial respiratory chain and a potent anti-oxidant. Our results indicate that total cellular ROS generation was inhibited by Coenzyme Q10. Further, pre-treatment with Coenzyme Q10 maintained mitochondrial membrane potential during oxidative stress and reduced the amount of mitochondrial ROS generation. Our study suggests that water-soluble Coenzyme Q10 acts by stabilizing the mitochondrial membrane when neuronal cells are subjected to oxidative stress. Therefore, Coenzyme Q10 has the potential to be used as a therapeutic intervention for neurodegenerative diseases.

Cell Death↗
PubMed2005

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Apoptosis↗
PubMed2004

Paraquat induces oxidative stress and neuronal cell death; neuroprotection by water-soluble Coenzyme Q10.

Neuronal cell death induced by oxidative stress is correlated with numerous neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and stroke. The causes of sporadic forms of age-related neurodegenerative diseases are still unknown. Recently, a correlation between paraquat exposure and neurodegenerative diseases has been observed. Paraquat, a nonselective herbicide, was once widely used in North America and is still routinely used in Taiwan. We have used differentiated Human Neuroblastoma (SHSY-5Y) cells as an in vitro model to study the mechanism of cell death induced by paraquat. We observed that paraquat-induced oxidative stress in differentiated SHSY-5Y cells as indicated by an increase in the production of cellular reactive oxygen species (ROS). Furthermore, apoptosis was evident as indicated by cellular and nuclear morphology and DNA fragmentation. Interestingly, pretreatment of SHSY-5Y cells with water-soluble Coenzyme Q10 (CoQ10) before paraquat exposure inhibited ROS generation. Pretreatment with CoQ10 also significantly reduced the number of apoptotic cells and DNA fragmentation. We also analyzed the effect of paraquat and CoQ10 on isolated mitochondria. Our results indicated that treatment with paraquat induced the generation of ROS from isolated mitochondria and depolarization of the inner mitochondrial membrane. Pretreatment with CoQ10 was able to inhibit ROS generation from isolated mitochondria as well as the collapse of mitochondrial membrane potential. Our results indicate that water-soluble CoQ10 can prevent oxidative stress and neuronal damage induced by paraquat and therefore, can be used for the prevention and therapy of neurodegenerative diseases caused by environmental toxins.

Apoptosis↗
PubMed2004

Regulation of DNaseY activity by actinin-alpha4 during apoptosis.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.

Actinin↗
PubMed2003

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR.

Apoptotic DNA degradation could be initiated by the accumulation of single-strand (ss) breaks in vulnerable chromatin regions, such as base unpairing regions (BURs), which might be preferentially targeted for degradation by both proteases and nucleases. We tested this hypothesis in anti-Fas-treated apoptotic Jurkat cells. Several nuclear proteins known for their association with both MARs and the nuclear matrix, that is, PARP, NuMA, lamin B and SATB1, were degraded, but the morphological rearrangement of the BUR-binding SATB1 protein was one of the earliest detected changes. Subsequently, we have identified several genes containing sequences homologous to the 25 bp BUR element of the IgH gene, a known SATB1-binding site, and examined the integrity of genomic DNA in their vicinity. Multiple ss breaks were found in close proximity to these sites relative to adjacent regions of DNA. Consistent with our prediction, the results indicated that the initiation of DNA cleavage in anti-Fas-treated Jurkat cells occurred within the BUR sites, which likely became accessible to endonucleases due to the degradation of BUR-binding proteins.

Base Sequence↗
PubMed2001

SATB1 cleavage by caspase 6 disrupts PDZ domain-mediated dimerization, causing detachment from chromatin early in T-cell apoptosis.

SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ~50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

Amino Acid Sequence↗
PubMed2000

Effects of neoplastic transformation and teniposide (VM26) on protein kinase C isoform expression in rodent fibroblasts.

This study examined changes in protein kinase C (PKC) isoforms in rodent fibroblasts (rat F111 and mouse NIH3T3), transformed by the polyoma virus middle T antigen (mT) and undergoing apoptosis in response to teniposide (VM26). The mT-transformed cells up-regulated PKC delta and down-regulated both PKC epsilon and PKC lambda expression, and were more sensitive to the drug than their non-transformed counterparts. The drug treatment further lowered the expression of PKC epsilon, triggered nuclear translocation of PKC delta and its site-specific proteolysis, consistent with the notion that changes in specific PKC isoforms play a role not only in the neoplastic transformation of fibroblasts, but also in their apoptotic response.

3T3 Cells↗
PubMed2000

Caspase-dependent and independent cell death in rat hepatoma 5123tc cells.

The objective of this study was to establish whether apoptosis in 5123tc rat hepatoma cells required the caspase-3 dependent pathway. Apoptosis was induced by either growth factor deprivation or treatment with a topoisomerase II inhibitor, VM26, in the absence or presence of caspase inhibitors (DEVD-fmk, z-VAD-fmk and BAF). The results indicated that, although these inhibitors at 10 microM concentration completely blocked caspase-3 activity, they had no effect on either the rate of cell death or on any other apoptotic features, e.g., chromatin condensation, DNA fragmentation, protein cleavage, suggesting that caspase-3 was not required to mediate nuclear destruction in these hepatoma cells. At higher concentrations, up to 100 microM, z-VAD-fmk and BAF, but not DEVD-fmk, did block apoptosis, however, they also caused cell swelling and membrane permeabilization, which are the hallmarks of necrotic cell death. Clearly, high concentrations of these inhibitors must have interfered non-specifically with other metabolic pathways, e.g., z-VAD-fmk at a high concentration blocked protein phosphorylation, and caused cell death by a different mechanism.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Detection of DNA fragmentation and endonucleases in apoptosis.

DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented.

Animals↗
PubMed1999

Neither caspase-3 nor DNA fragmentation factor is required for high molecular weight DNA degradation in apoptosis.

In this paper, we show that there is a two-step process of DNA fragmentation in apoptosis; DNA is first cleaved to large fragments of 50-300 kb that are subsequently cleaved to smaller oligonucleosomes in some, but not all cells. Significantly, only the first stage is considered essential for cell death since some cells, for example human MCF7 breast carcinoma cells and human NT2 neuronal cells, do not show this behavior but still display normal nuclear morphological apoptotic changes. In cells that usually produce small fragments blocking the second (internucleosomal) stage of DNA fragmentation prevents neither nuclear condensation nor apoptosis. We are beginning to understand why the extent of DNA fragmentation during apoptosis varies enormously and why it appears to be a function of the cell type not the inducer. Presumably, this reflects the content of not only endonuclease activit(ies) but also on the ability of the cells to activate caspases, particularly caspase-3, and other proteases that may be involved in endonuclease activation. Since NT2 cells activate caspase-3, but do not correctly process DFF45, other factors must also impinge on the inevitability of that process.

Adenocarcinoma↗
PubMed1999

Apoptosis-related functional features of the DNaseI-like family of nucleases.

Rat DNaseYb and its human homolog DHP2 are members of a new family of DNaseI-like endonucleases. They contain all the conserved amino acid residues to engage a DNaseI-like catalytic activity and the same molecular mechanisms of DNA hydrolysis. The sequence similarity can be extended to other families of nucleases, such as FEN-1, DNA polymerases, RNaseH and exonuclease III, involved in the ion-dependent hydrolysis of nucleic acids. Their unique features include the NLS signals that place them in the nuclei and a high content of positively charged amino acid residues that results in their high affinity for the substrate. Their properties are consistent with a role in the early stage DNA degradation during apoptosis. The caspase-DFF45/CIDE-CPAN pathway is most likely involved in the second stage of internucleosomal DNA degradation. However, cells express constitutively multiple transcripts encoding DNA degrading enzymes and related molecules, hence they have the molecular diversity to engage the self-destructive pathway appropriate to a given trigger.

Amino Acid Sequence↗
PubMed1998

DNaseY: a rat DNaseI-like gene coding for a constitutively expressed chromatin-bound endonuclease.

A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was identified and cloned. Sequence analysis of the cDNA showed it to be the rat homologue of human DNAS1L3. The DNaseY gene product had 42% identity to DNaseI, including conserved critical active site residues, the essential disulfide bridge, the calcium binding domain, and a signal peptide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNaseI (pI 4.8). The DNaseY gene contained a number of exons similar to that of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene product was catalytically inactive but was converted to an active 33 kDa endonuclease following processing of the hydrophobic signal peptide. Antibody generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which possessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Zn2+, and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it was not transcriptionally up-regulated in apoptotic cells. All these features were consistent with a role in the early stages of apoptotic DNA fragmentation.

Amino Acid Sequence↗
PubMed1997

Jun and JNK kinase are activated in thymocytes in response to VM26 and radiation but not glucocorticoids.

In order to establish what role members of the activating protein-1 (AP-1) gene families, i.e., c-fos, c-jun, junB, and junD, play in thymic apoptosis, we have analyzed changes in their expression in response to three different agents: a glucocorticoid analog dexamethasone, an inhibitor of topoisomerase II teniposide VM26, and gamma radiation. All three agents induced thymic death at a similar rate and with the same morphological and biochemical features. There was a rapid and transient increase in the steady-state mRNA level of junB and c-fos genes in all treatments, including control cultures, reminiscent rather of cellular stress response to the environmental changes than to the apoptotic stimuli. On the other hand, treatments with the DNA-damaging agents, VM26 and gamma radiation, resulted in superinduction of the c-jun mRNA and in the activation of the stress response signaling pathway of c-Jun N-terminal kinase. Gene transcription ceased completely in cells with fragmented DNA and the down-regulation of genes such as junD and tubulin was reflective of the thymocytes' commitment to apoptosis. The DNA-binding activities of the serum response factors, cyclic AMP response element binding proteins, and AP-1 factors, indicative of their transcriptional competence, were compromised shortly after induction of apoptosis regardless of the agent employed, consistent with previously reported enhancement in cellular proteolysis which is an essential component of the apoptotic cell death.

Animals↗
PubMed1997

Identification of a novel 97 kDa endonuclease capable of internucleosomal DNA cleavage.

The two steps of DNA digestion seen in apoptotic cells were recreated in nuclei isolated from 5123tc rat hepatoma cells. The initial DNA cleavage, into high molecular weight fragments (300-50 kb), was stimulated by magnesium ions alone, whereas the second step required both calcium and magnesium ions and produced the ladder of oligonucleosomes. Endonucleolytic activities involved in both steps of DNA cleavage could be separated under appropriate conditions since the magnesium-modulated activity was tightly bound to the chromatin whereas the calcium/magnesium-dependent internucleosomal cleaving activity was easily extractable with a low ionic strength buffer. This calcium/ magnesium-dependent activity was attributed to a novel 97 kDa endonuclease which was also activated by manganese and cobalt and inhibited by millimolar concentrations of zinc, consistent with the properties ascribed to the apoptotic endonuclease. Furthermore, this activity became resistant to extraction with a low salt buffer in nuclei of apoptotic cells. Isoelectrofocusing revealed that the p97 protein existed in multiple forms of different isoelectric points (pI range 4.6-5.0), indicative of its postranslational modification. The p97 enzyme was present constitutively in a variety of cultured cells and rat tissues. It was active over a broad range of pH (6-9), but it was inactivated by reducing agents. In vitro, it displayed both endo- and exonucleolytic activities, and it was capable of both single- and double-stranded DNA cleavage. Rabbit polyclonal anti-p97 antibodies were generated and used to further distinguish this protein from other known cellular nucleases, namely, DNases I and II.

Animals↗
PubMed1997

Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands of Manduca sexta.

Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae, homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4-day period during larva-to-pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end-labeling of single strand breaks, and pulsed-field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coupled with synthesis of a glycine-rich protein, reminiscent of silk-like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell-independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. It is at this period that unequivocal nucleosomal ladders are seen and end-labeling in situ or electrophoretic techniques document single on double-strand breaks, respectively. DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instor, raising the possibility of potential limitations built into the cells before their final collapse.

Animals↗
PubMed1997

Induction of apoptosis in neoplastic cells by depletion of vitamin B12.

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.

Journal Article↗