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Biomedical subjects

S Ruzdijic

Publications and source records attributed to S Ruzdijic.

17 recordsLinked to original sources
PubMed2006

Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs.

Exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin resulted in the appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin (96.2-fold) and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin. Slight cross-resistance to two MDR-unrelated drugs 8-Cl-cAMP and sulfinosine was observed. Flow cytometry analysis showed that the accumulation of doxorubicin in the resistant cells was 88.4% lower than in the parental cells. Also, verapamil significantly decreased the efflux rate in NCI-H460 and NCI-H460/R cells, whereas curcumin inhibited the efflux in NCI-H460 cells only. Gene expression data confirmed the induction of mdr1 (P-gp), as judged by the observed 15-fold increase in its mRNA concentration in doxorubicin-resistant NCI-H460/R cells. In contrast, mrp1 and lrp expression was unaffected by the doxorubicin resistance. Further work should develop a rationale for a novel treatment of NSCLC with appropriate modulators of resistance aimed at improving the outcome of the acquired drug resistance.

Antineoplastic Agents↗
PubMed2001

Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43.

The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA.

Amphetamine↗
PubMed2001

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.

Apoptosis↗
PubMed1999

Glucocorticoid receptors in ageing rats.

The role of the glucocorticoid receptor (GR) in senescence was studied in rats of increasing age. Statistically significant changes in the number of GRs from rat liver were detected, whereas the affinity for the ligand triamcinolone acetonide (TA) did not change with increasing age, and was in the range of 1-2 nM. In all cases the number of receptors was lower in rats treated with hormone in vivo relative to untreated animals. In addition, we have found changes in GR activation, as measured by the binding to DNA cellulose in the mentioned age groups. Furthermore, expression of the glucocorticoid hormone (GH)-inducible gene, tyrosine amino transferase (TAT) also showed age-related alterations. We conclude that receptor function shows oscillatory changes during ageing. In addition, response to GH generally declines towards the older age. This specific periodicity in functional characteristics of the GR may reconcile conflicting results about the receptor number and properties during the ageing process, and marks particular age at which individual organism shows the highest or the lowest sensitivity to the actions of GH.

Aging↗
PubMed1998

Using yeast to study glucocorticoid receptor phosphorylation.

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.

Animals↗
PubMed1997

Enhanced serum response element binding activity correlates with down-regulation of c-fos mRNA expression in the rat brain following repeated cortical lesions.

Repeated lesions of rat cerebral cortex result in transient peaks in the level of the c-fos transcript, but after the second lesion, this peak is substantially diminished. Using this lesion paradigm, we have analyzed the participation of the c-fos promoter elements SRE and DSE in the regulation of c-fos transcription. Following a single lesion, SRE/DSE binding activity peaked at 2 h, subsequent to the maximal levels of c-fos mRNA and parallel to the peak of c-Fos protein. After a second lesion (reinduction), 4 h following the initial lesion, SRE/DSE binding activity peaked after only 30 min and was significantly higher than following the first lesion. Once again, this peak occurred after the peak of c-fos mRNA expression and parallel with the second peak of c-Fos protein expression. These results suggested that the SRE and DSE promoter elements participated in the induction and down-regulation of c-fos transcription in vivo and suggested the possible involvement of Fos protein in its own regulation. The ability of Fos/Fra proteins to participate in a transcriptional complex was confirmed in gel-shift experiments with an AP-1 element, and the biphasic trend of binding activity was observed. Supershift experiments were performed to directly determine whether Fos protein was participating in SRE and/or DSE transcriptional complexes. No alterations in the position or intensity of the shifted band were observed using Fos/Fra antiserum suggesting that Fos/Fra proteins could be involved in c-fos down-regulation through mechanisms other than direct participation in the SRE/DSE transcription complex.

Animals↗
PubMed1997

Optimization of in situ hybridization histochemistry for localization of GAP-43 mRNA in fetal brain during human development.

In situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology. The method basically involves the anatomic localization of labeled RNA or DNA molecules that hybridize with complementary target RNA or DNA sequence in the cell. In regard to gene expression, in situ hybridization allows the study of specific mRNA level and distribution between various cell types, or of comparative levels of mRNA throughout development. Major advantages of in situ hybridization are: (a) specific mRNAs can be detected in heterogeneous cell populations where only one of multiple sets of cells present may be transcribing the mRNA of interest; (b) it allows the maximal use of rare tissues that may be in short supply (clinical biopsies, embryos, cultured cells); (c) the location of the mRNA can be identified on conventional microscopic sections without the necessity of electron microscopy; and (d) identification of specific gene without metabolic incorporation of radiolabeled precursor into the endogenous protein in the cells. Because of these advantages, we decided to utilize this approach for specific studies involving the expression and distribution of GAP-43 (growth-associated protein 43) mRNA in early human development. The method we employed for in situ hybridization is basically a modification of the many original protocols developed by others and can be easily applied to many studies of cellular gene expression in the human nervous system.

Autoradiography↗
PubMed1996

GAP-43 mRNA expression in early development of human nervous system.

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Cell Differentiation↗
PubMed1994

Desensitization of c-fos mRNA expression in rat brain following cortical lesions.

The effect of cortical lesions on the c-fos mRNA expression in adult rat brain was studied using in situ hybridization and Northern blot analysis. It was observed that the single cortical lesion evoked a rapid increase in the content of c-fos mRNA in whole brain. However, when rats received a second and third cortical injury 4 and 8 h after the first one, there was reduced elevation of c-fos mRNA after the second (60%) and third (75%) injuries in comparison with those seen after a single cortical lesion. These results represent the desensitization period during which the induction of c-fos mRNA by repeated cortical lesions was decreased. On the contrary, when animals were treated with a fourth cortical lesion 24 h after the first injury, with recovery for 16 h after the third one, the levels of c-fos mRNA were again elevated to a degree almost comparable to that seen after the single injury alone. The anatomical distribution of c-fos mRNA-expressing cells after cortical lesions revealed a large increase of hybridization to a heterogeneous population of hippocampal neurons including small cells of stratum granulosum in the dentate gyrus and larger cells within hippocampal stratum pyramidale. A dramatic labeling of neurons with c-fos riboprobe in frontoparietal and piriform cortex was also observed but with predominantly localized hybridization to c-fos mRNA on the damaged side of the cortex following the first and second cortical injury. However, hippocampal distribution of c-fos mRNA-containing cells was bilateral as a result of polysynaptic potentials evoked by cortical lesions.

Animals↗
PubMed1993

Temporal and spatial preferences of c-fos mRNA expression in the rat brain following cortical lesion.

The expression of the proto-oncogene c-fos is increased in neuronal cells by a number of stimuli and the usefulness of this gene as a marker of neuronal activity has been demonstrated. The temporal and spatial expression of c-fos mRNA following the induction of a unilateral cortical lesion have been investigated in the rat brain by Northern blot analysis and in situ hybridization histochemistry. It was observed that the lesion evoked a rapid increase (20-fold) in the content of c-fos mRNA in the ipsilateral cortex, whereas in the contralateral cortex c-fos mRNA expression was more modest (7-fold). In the whole hippocampus a large and very rapid increase (17-fold) of c-fos mRNA expression was detected. The effect of a cortical lesion on Ca2+ uptake and membrane potential was also investigated. Using synaptosomes as a model system, we have provided evidence that Ca2+ entry via membrane depolarization increases in coordination with c-fos gene expression in neuronal cells. The principal conclusions from this study are that cortical lesions induce transient expression of the c-fos gene in specific neuronal cells of the rat brain.

Animals↗
PubMed1987

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs.

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

Animals↗
PubMed1984

Cross-linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells.

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr = 49 000 and 52 500 were the major components. In contrast polypeptides of Mr = 43 000 and 57 000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Inhibition of cell growth and proliferation in human glioma cells and normal human astrocytes induced by 8-Cl-cAMP and tiazofurin.

8-Cl-cAMP and tiazofurin (TR) are anti-tumor agents that besides their antiproliferative effect, also induce differentiation of tumor cells. Although, these agents exert a profound effect on the same events of tumor cell life, it is thought that 8-Cl-cAMP and TR act by modulating the signal transduction pathway through distinct mechanisms. We have compared their effect on two human glioma cell lines (U87 MG and U251 MG) and examined if there is selectivity in their action toward normal human astrocytes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMedDate not supplied

Semiquantitative cytochemistry in evaluation of apoptotic capacity in bronchoalveolar lavage of smokers and patients with non-small-cell lung cancer.

Apoptotic capacity of pulmonary tissue to produce or remove apoptotic cells by alveolar macrophages (ALMs) was investigated in three groups: healthy volunteers, smokers and patients with non-small-cell lung cancer (NSCLC). Differential cell counting of bronchoalveolar lavage (BAL) specimens revealed significantly higher percentages of neutrophils and eosinophils and decreased percentage of macrophages in BAL of patients with NSCLC in comparison with nonsmokers and smokers. Proportion of lymphocytes was significantly higher in patients with NSCLC than in smokers. These changes in the BAL cell profile may reflect immunology of the lung in pulmonary malignancies. BAL eosinophils were significantly lower and AMs increased in smokers in comparison with nonsmokers. This result might be understood as a consequence of changed tissue architecture of pulmonary tissue in situ, influenced by smoking. Apoptotic detection in cytocentrifuge preparations of BAL cell suspensions was evaluated by TUNEL method. Subsequent steps, adsorption, internalization and digestion of apoptotic cells by alveolar macrophages (AMs) were estimated by semiquantitative cytochemical scoring and indexing method and correlated with percent of free apoptotic cells. Significant increase of apoptotic capacity of pulmonary tissue in control smokers (289.55+/-50.77) in comparison with that of non-smokers (218.29+/-56.24) could be a consequence of stimulated digestion inside the AMs; decreased apoptotic capacity of pulmonary tissue in NSCLC (150.30+/-40.61; p<0.05), in comparison with non-smokers and smokers is in relation to a reduced phagocytosis of the apoptotic remnants, which might be either the cause or the consequence of the oncogenic process.

Apoptosis↗
PubMedDate not supplied

Reversible inhibition of IL-8 receptor B mRNA expression and proliferation in non-small cell lung cancer by antisense oligonucleotides.

We examined the importance of IL-8 receptor B mRNA expression in the growth of non-small cell lung cancer (NSCLC). Using antisense oligonucleotide ICN 197, we were able to inhibit IL-8 R B mRNA expression in vitro. The sequence specific effect of antisense oligonucleotide and down-regulation of IL-8 R B mRNA was shown by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Southern blot analysis. The proliferation of treated cells was measured by 3H thymidine incorporation. We found that treatment of NSCLC cells caused reversible growth inhibition and reversible down regulation of IL-8 R B mRNA. Furthermore, we observed that the treatment of nude mice with oligonucleotide ICN 197 inhibited the growth of tumors developed from NSCLC cells injected subcutaneously. Our data in vitro suggest that IL-8 receptor B mRNA expression is required to maintain the proliferative rate of NSCLC. Based on the data in vivo. oligonucleotide ICN 197 may be considered for the development of novel therapeutic treatment for lung cancer.

Animals↗