Biomedical subjects

Z Ronai

Publications and source records attributed to Z Ronai.

At least 37 records · Page 2Linked to original sources

PubMed2001

DNA analysis on electrophoretic microchips: effect of operational variables.

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.

DNA↗

PubMed2001

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix.

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.

Adrenal Hyperplasia, Congenital↗

PubMed2001

High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.

Alleles↗

PubMed2001

Gene mutation as a target for early detection in cancer diagnosis.

The increasing number of genetic aberrations implicated in the development of human cancer has prompted a search to detect them at the earliest possible stage of their formation. Of the many such genetic changes identified thus far, relatively few meet the standard for markers in early diagnosis and prognosis, namely that the genetic modifications occur during the early onset phase of cancer development. Parallel to the increasing number of such genes is the growing availability of technologies using more powerful and cost-efficient methods that enable mass screening for genetic alterations. The purpose of this review is to summarize the currently available genes that can serve as markers for early detection of cancers and methods that allow their detection.

DNA Methylation↗

PubMed2001

Cooperation between STAT3 and c-jun suppresses Fas transcription.

Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in melanoma cells efficiently increased Fas expression and sensitized cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibited increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. Our findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize.

Animals↗

PubMed2001

ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation.

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.

Arachidonate 15-Lipoxygenase↗

PubMed2001

Association between Novelty Seeking and the -521 C/T polymorphism in the promoter region of the DRD4 gene.

Association between the human personality trait 'Novelty Seeking' and the polymorphism of the DRD4 gene was first reported by Ebstein and Benjamin in 1996. This was soon followed by replication studies in various ethnic groups and by studying the role of other neurotransmitter receptor and transporter genes in the genetic determination of human temperament. More recently, several polymorphic sites of the upstream regulatory region of the DRD4 gene have been described. Among these the -521 C/T single nucleotide polymorphism (SNP) was shown to be associated with the Novelty Seeking (NS) scores of the Temperament and Character Inventory (TCI) in a Japanese male population. We have investigated the -521 C/T SNP polymorphism in a Caucasian (Hungarian) population, and here we report a replication of the Japanese findings, in an association study involving 109 healthy Hungarian volunteers. We found a weak association between NS and CC vs CT or TT genotypes (P < 0.06). Examination of this relation in male and female sex groups, however, strengthened the association for females (P < 0.01), but showed no genotypic effect for males.

Adolescent↗

PubMed2001

TIP49b, a regulator of activating transcription factor 2 response to stress and DNA damage.

Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control of stress-responsive genes. A yeast two-hybrid screen identified TBP-interacting protein 49b (TIP49b), a component of the INO80 chromatin-remodeling complex, as a novel ATF2-interacting protein. TIP49b's association with ATF2 is phosphorylation dependent and requires amino acids 150 to 248 of ATF2 (ATF2(150-248)), which are implicated in intramolecular inhibition of ATF2 transcriptional activities. Forced expression of TIP49b efficiently attenuated ATF2 transcriptional activities under normal growth conditions as well as after UV treatment, ionizing irradiation, or activation of p38 kinase, all of which induced ATF2 phosphorylation and increased TIP49b-ATF2 association. Constitutive expression of ATF2(150-248) peptide outcompeted TIP49b interaction with ATF2 and alleviated the suppression of ATF2 transcriptional activities. Expression of ATF2(150-248) in fibroblasts or melanoma but not in ATF2-null cells caused a profound G(2)M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2001

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Animals↗

PubMed2001

Cellular thiols and reactive oxygen species in drug-induced apoptosis.

In higher eukaryotes, reactive oxygen species (ROS) are generated during respiration in mitochondria in the course of reduction of molecular oxygen as well as by distinct enzyme systems. ROS have been implicated in the regulation of diverse cellular functions including defense against pathogens, intracellular signaling, transcriptional activation, proliferation, and apoptosis. The reduction-oxidation (redox) state of the cell is primarily a consequence of the precise balance between the levels of ROS and endogenous thiol buffers present in the cell, such as glutathione and thioredoxin, which protect cells from oxidative damage. Dramatic elevation of ROS, exceeding compensatory changes in the level of the endogenous thiol buffers, may result in the sustained activation of signaling pathways and expression of genes that induce apoptosis in affected cells. Many cytotoxic drugs function selectively to kill cancer cells by the abrogation of proliferative signals, leading to cell death, and numerous reports have demonstrated that ROS are generated following treatment with these drugs. In this review, we will summarize recent contributions to our understanding of the importance of cytotoxic drug-induced modulation of cellular redox status for signaling and transcription leading to activation of apoptotic effector mechanisms.

Animals↗

PubMed2001

Activating transcription factor 2-derived peptides alter resistance of human tumor cell lines to ultraviolet irradiation and chemical treatment.

Activating transcription factor 2 (ATF2) and its kinase, p38, play an important role in the resistance of melanoma to radiation and chemotherapy. Whereas ATF2 up-regulates the expression of tumor necrosis factor alpha, which serves as a survival factor in late-stage melanoma cells, p38 attenuates Fas expression via inhibition of nuclear factor-kappaB. We investigated whether ATF2-derived peptides could be used to alter the sensitivity of human melanoma cells to radiation and chemical treatment. Of four 50-amino acid peptides tested, the peptide spanning amino acids 50-100 elicited the most efficient increase in the sensitivity of human melanoma cells to UV radiation or treatment by mitomycin C, Adriamycin, and verapamil, or UCN-01, as revealed by apoptosis assays. Sensitization by ATF2 peptide was also observed in the MCF7 human breast cancer cells but not in early-stage melanoma or melanocytes, or in in vitro-transformed 293T cells. When combined with an inhibitor of p38 catalytic activity, cells expressing amino acids 50-100 of ATF2 exhibited an increase in the degree of programmed cell death, indicating that combined targeting of ATF2 and p38 kinases is sufficient to induce apoptosis in late-stage melanoma cells. The ability of the peptide to increase apoptosis coincided with increased cell surface expression of Fas, which is the primary death-signaling cascade in these late-stage melanoma cells. Overall, our studies identified a critical domain of ATF2 that may be used to sensitize tumor cells to radiation and chemical treatment-induced apoptosis and that can induce apoptosis when combined with inhibition of ATF2 kinase, p38.

Activating Transcription Factor 2↗

PubMed2001

Pharmacologic or genetic manipulation of glutathione S-transferase P1-1 (GSTpi) influences cell proliferation pathways.

Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1-1 [glutathione S-transferase P1-1 (isozyme in nonhepatic tissue)] null genotype (GSTpi(-/-)) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTpi(+/+)). Retroviral transfection of GSTP1-1 into GSTpi(-/-) MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTpi(-/-) animals expressed significantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTpi(-/-) mice had higher basal levels of circulating white blood cells compared with GSTpi(+/+). Administration of a peptidomimetic inhibitor of GSTP1-1, TLK199, (gamma-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester), stimulated both lymphocyte production and bone marrow progenitor (colony-forming unit-granulocyte macrophage) proliferation, but only in GSTpi(+/+) and not in GSTpi(-/-) animals. Selection of a resistant clone of an HL60 tumor cell line through chronic exposure to TLK199 resulted in cells with elevated activities of c-Jun NH2 terminal kinase (JNK1) and ERK1/ERK2, and allowed the cells to proliferate under stress conditions that induced high levels of apoptosis in the wild type cells. The in vitro and in vivo data are consistent with the principle that GSTP1-1 influences cell proliferation.

Animals↗

PubMed2000

Glutathione S-transferase p elicits protection against H2O2-induced cell death via coordinated regulation of stress kinases.

To elucidate mechanisms underlying glutathione S-transferase p (GSTp)-mediated cellular protection against oxidative stress-induced cell death, the effect of GSTp on stress signaling pathways was investigated before and after H2O2 treatment. Under nonstressed conditions, increased expression of GSTp via a tet-off-inducible GSTp in NIH 3T3 cells increased the phosphorylation of mitogen-activated protein (MAP) kinase kinase 4, p38, extracellular receptor kinase (ERK), and inhibitor of kappa-kinase (IKK), and reduced phosphorylation of MAP kinase kinase 7 and Jun NH2-terminal kinase (JNK). Whereas H2O2 treatment of cells induced JNK, p38, and IKK activities, in the presence of H2O2 and elevated GSTp expression there was an additional increase in ERK, p38, and IKK activities and a decrease in JNK activity. GSTp-mediated protection from H2O2-induced death was attenuated upon inhibition of p38, nuclear factor KB, or MAP kinase by dominant negative or pharmacological inhibitors. Conversely, expression of a dominant negative JNK protected cells from H2O2-mediated death. These data suggest that the coordinated regulation of stress kinases by GSTp, as reflected by increased p38, ERK, and nuclear factor kappaB activities together with suppression of JNK signaling, contributes to protection of cells against reactive oxygen species-mediated death.

3T3 Cells↗

PubMed2000

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53.

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.

Cell Line, Transformed↗

PubMed2000

p38 protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB activity and Fas expression.

Identifying mechanisms that underlie the resistance of human melanoma to radiation and chemotherapy is expected to assist in developing new strategies for the treatment of this tumor type. We recently demonstrated that through up-regulation of TNFalpha, ATF2 increases the resistance of late stage melanoma cells to apoptosis induced by UV-irradiation. In elucidating the role of ATF2 kinases, we now demonstrate that ASK1/MKK6/p38 elicits suppression of Fas expression. ASK1/p38 downregulates the expression of a Fas via NF-kappaB/SP1 site on the Fas promoter. Deletion or mutation of NF-kappaB/SP1 within the Fas promoter abrogates p38 effect. ASK1/p38 silences the Fas promoter by inhibition of IkappaBalpha phosphorylation - thereby limiting NF-kappaB activity. Forced expression of a dominant negative form of p38 (p38-ASP) or treatment with p38 pharmacological inhibitor, SB203580, increases NF-kappaB activity, Fas expression and the levels of UVC-induced apoptosis in late stage melanoma cells. Inhibition of p38 activity also restored NF-kappaB activity and Fas expression in early-phase melanoma cells, suggesting that p38 elicited suppression of Fas expression is not restricted to late phase melanoma. Identifying p38-mediated down-regulation of Fas expression illustrates a novel regulatory pathway by which ASK1/MKK6/p38 alters the degree and nature of the UV-induced apoptosis of melanoma cells. Oncogene (2000).

Apoptosis↗

PubMed2000

Amino-terminal-derived JNK fragment alters expression and activity of c-Jun, ATF2, and p53 and increases H2O2-induced cell death.

The stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1-206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities of p21(Waf1/Cip1)-Luc, AP1-Luc, and Jun2-Luc, respectively. Expression of N-JNK in cells that were treated with H(2)O(2) impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53-transcriptional activities. N-JNK elicited an increase in H(2)O(2)-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNK-derived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress.

3T3 Cells↗

PubMed2000

Stability of the ATF2 transcription factor is regulated by phosphorylation and dephosphorylation.

Trans-activation of the activating transcription factor-2 (ATF2) in response to cellular stress requires the N-terminal phosphorylation of ATF2 by stress-activated protein kinases (SAPK). In this study, we investigated the role of ATF2 phosphorylation in the maintenance of ATF2 stability. Activation of SAPK by forced expression of DeltaMEKK1 increased overall ATF2 ubiquitination, presumably because of the enhanced dimerization of ATF2. Treatment of DeltaMEKK1-expressing cells with okadaic acid led to the increase in N-terminal phosphorylation, protection from ubiquitination, and accumulation of exogenously expressed ATF2, indicating the role of protein phosphatases in balancing the effects of stress kinases. Analysis of ubiquitination and degradation of the constitutively dimerized ATF2 mutant (ATF2(Delta150-248)) showed that activation of JNK or p38 kinase renders ATF2 resistant to ubiquitination and degradation. This effect is mediated by JNK/p38-dependent phosphorylation of ATF2 at Thr-69 and Thr-71, because the phosphorylation-deficient mutant (ATF2(Delta150-248-T69A,T71A)) was not protected from ubiquitination and degradation by the activation of SAPK. Treatment of cells with okadaic acid elevated the tumor necrosis factor alpha-induced ATF2 level and the extent of its specific N-terminal phosphorylation. Cycloheximide, which activates SAPK, while inhibiting protein synthesis, stabilized endogenous ATF2. However, treatment of cells with the high dose of SB203580, which inhibits JNK and p38 kinase, resulted in efficient degradation of ATF2 in cells exposed to cycloheximide. This degradation was abrogated by co-treatment with the proteasome inhibitor MG132. Our findings suggest that N-terminal phosphorylation of ATF2 dimers protect ATF2 from ubiquitination and degradation. We propose the hypothesis that the balance between SAPK and protein phosphatases affects the duration and magnitude of ATF2 transcriptional output because of the effect on substrate recognition for ubiquitination and degradation.

Activating Transcription Factor 2↗

PubMed2000

Role of TRAF2/GCK in melanoma sensitivity to UV-induced apoptosis.

Radiation resistance is a hallmark of human melanoma, and yet mechanisms underlying this resistance are not well understood. We recently established the role of ATF2 in this process, suggesting that stress kinases, which contribute to regulation of ATF2 stability and activity, play an important role in the acquisition of such resistance. Here we demonstrate that changes in the expression and respective activities of TRAF2/GCK occur during melanoma development and regulate its sensitivity to UV-induced apoptosis. Comparing early- and late-stage melanoma cells revealed low expression of TRAF2 and GCK in early-stage melanoma, which coincided with poor resistance to UV-induced, TNF-mediated apoptosis; forced expression of GCK alone or in combination with TRAF2 efficiently increased JNK and NF-kappaB activities, which coincided with increased protection against apoptosis. Conversely, forced expression of the dominant negative form of TRAF2 or GCK in late-stage melanoma cells reduced NF-kappaB activity and decreased Fas expression, resulting in a lower degree of UV-induced, Fas-mediated cell death. Our results illustrate a mechanism in which protection from, or promotion of, UV-induced melanoma cell death depends on the nature of the apoptotic cascade (TNF or Fas) and on the availability of TRAF2/GCK, whose expression increases during melanoma progression. Oncogene (2000) 19, 933 - 942.

Apoptosis↗