Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of Co-60 gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P lt 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death

Semiquantitative cytochemical method in the evaluation of smoking induced changes of alveolar macrophages glycogen and apoptotic properties

Having in mind hypothesis about discrete changes in alveolar macrophages (AMs') biological markers under the smoking exposure, the study was designed regarding cytological, cytochemical and apoptotic parameters in lung washings. Cell profile and apoptotic capacity (AC) of pulmonary tissue evaluated by bronchoalveolar lavage (BAL) were evaluated by light microscopy in fifteen subjects: 9 non-smokers and 6 smokers. Apoptosis was detected by TUNEL in situ cytochemical method. Semiquantitative indexing and scorring methods were used for AC and evaluation of alveolar macrophages (AMs) glycogen by PAS reaction. Significant increase of macrophages, AC and decrease eosinophils (p<0.05) were revealed in smokers in comparison with nonsmokers. There is significant correlation of AMs' glycogen and smoking exposure (Spearman R= 0.98, p<0.001) as well as eosinophils and AMs' glycogen (Spearman R=0.81, p<0.05). In nonsmokers, percentage of free apoptotic bodies (FAB), correlates with amounts of glycogen in AMs (Spearman R=-0.79, p<0.05). The evidence of smoking induced changes of AMs' metabolic properties supports an idea of cell energy metabolism switching which might be important for programmed cell death regulation in order to avoid harmful influence of noxious agents, including tobacco smoke.Alveolarni makrofazi su uključeni u imuni odgovor u plućnom tkivu kroz niz složenih interreakcija sa drugim imunim ćelijama koje na zahtev migriraju iz krvnih sudova u tkivo. Pušenje utiče na karakteristike imunog odgovora u tkivu pluća i na proces remodeliranja tkiva. U grupama nepušača, (N 9) i pušača, (N 6) urađena je bronhoalveolarna lavaža. Citospin preparati bronhoalveolarnih lavata su bojeni May Grunwald Giemsa om za diferencijalno brojanje ćelija, pomoću svetlosnog mikroskopa. Od citohemijskih tehnika su korišćeni TUNEL, in situ metod za detekciju apoptoze i PAS reakcija za detekciju glikogena u alveolarnim makrofazima. Imajući u vidu fagocitne sposobnosti ovih ćelija, definisan je apoptotski kapacitet kao numerički ekvivalent sposobnosti tkiva da stvara slobodna apoptotska tela i da ih odstranjuje fagocitozom posredstvom neapoptotskih alveolarnih makrofaga. Rezultati ukazuju na povećanje prinosa makrofaga i smanjenje eozinofila u bronhoalveolarnim lavatima pušača u poređenju sa nepušačima (p<0.05). Iako nije nađena statistički značajna razlika sadržaja glikogena u alveolarnim makrofazima pušača u poređenju sa nepušačima, kod pušača skor za Perlsovu reakciju korelira sa ekspozicijom duvanskom dimu (Spearman R= 0.98, p<0.001) i sa prinosom eozinofila u bronhoalveolarnom lavatu (Spearman R=0.81 p<0.05). Kod nepušača, relativni procenat slobodnih apoptotskih tela korelira sa sadržajem glikogena u alveolarnim makrofazima (Spearman R=-0.79 p<0.05). Promené metaboličkih svojstava alveolarnih makrofaga, koje se reflektuju na sadržaju glikogena, mogu biti u osnovi promenjenih imunoloških i apoptotskih osobenosti tkiva pod uticajem pušenja.nul

6-hydroxydopamine lesions of the striatum lead to the alterations of dopamine receptor mrna in parkinsonian rats

The effects of four-site intrastriatal 6-hydroxydopamine (6-OHDA) lesions were examined in adult male rats. Five days after the lesions the animals were checked for specific rotational behavior induced by middle dose of amphetamine and the results confirmed the effectiveness of the lesions. The RNAs from the striatum were isolated at different time points after the lesion, and the RT-PCR analyse were performed for the D1 and D2 receptor mRNA. The results show a decline in the D2 receptor mRNA level (40%) at 6 h and 24 h points while this change was not observed seven days after the lesion. In contrast, no statistically significant changes in the level of the D1 receptor mRNA after the lesion at any time point were found.Ispitivani su efekti četiri ubodne 6-hidroksidopaminske (6-OHDA) lezije striatuma kod odraslih mužjaka pacova. Pet dana nakon lezije, životinje su testirane na specifično rotaciono ponaš anje pod uticajem srednje doze amfetamina i rezultati su potvrdili efikasnost lezije. RNK iz striatuma su izolovane u različitim vremenskim tačkama nakon lezije i urađena je RT-PCR analiza iRNK za D1 i D2 dopaminske receptore. Rezultati pokazuju smanjivanje nivoa iRNK za D2 receptor (40%) 6 h i 24 h nakon lezije, dok sedam dana nakon lezije nema promena. Za razliku od ovih rezultata, u nivou iRNK za D1 receptor ne postoje statistički značajne razlike u bilo kojoj vremenskoj tački

Comparative analysis of expression of angiogenic factors and CD44 gene in human glioma and neuroblastoma cell lines in vitro

Angiogenesis is essential for tumor growth and relies on the production of angiogenic factors. By comparative analysis using RT-PCR method of angiogenic growth factors: VEGF, bFGF, PDGF-A, angiogenin- 1 and IL-8 we established the level of expression of these genes necessary for angiogenesis in glioma and neuroblastoma cell lines. Our analyses were also extended to CD44 gene, which plays an important role in cascade of metastasis and progression of brain tumors. Significant differences in the level of gene expression of angiogenic factors and CD44 gene between the two cell lines observed throughout this study can be used as a prognostic marker for predicting clinical outcome in human brain tumors at the time of the initial staging.Angiogeneza je neophodna za rast tumora i zahteva proizvodnju angiogenih trofičkih faktora koji učestvuju u tumorogenezi. Uporednom analizom angiogenih trofičkih faktora: VEGF, bFGF, PDGF-A, angiogenina-1 i IL-8 pomoću metode RT-PCR utvrdili smo nivo ekspresije ovih gena uključenih u proces angiogeneze u ćelijskim linijama glioma i neuroblastoma. Takođe smo proširili analize i na CD44 gen koji igra važnu ulogu u kaskadi nastanka i progresiji metastaza tumora mozga. Dobijeni rezultati ukazuju na značajnu razliku u nivou genske ekspresije angiogenih faktora i CD44 gena u ove dve ćelijske linije čije se poreklo razlikuje ne samo po nastanku već i po mestu rasejavnja metastaza. Rezultati bi mogli da posluže kao prognostički faktor u prekliničkim i kliničkim istraživanjima tumora mozga od inicijalnih do terminalnih stupnjeva nastanka i terapije

Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p < 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.nul

Cell death in irradiated prostate cancer cells assessed by flow cytometry

Despite the significant advances in cancer chemotherapy, radiotherapy still remains a method of choice for treatment of metastatic human prostate cancer. This study presents quantitative analysis of 60Co gamma-radiation effects on cell growth and cell death of metastatic human prostate cancer PC-3 cell line, performed in time (24-72h) and dose (2-20 Gy) dependent manner. The irradiated PC-3 cells were mostly dying by necrosis at late time intervals (72h), while apoptotic cell death was negligible. The EC50 or 50% of cytotoxicity was not achieved within the radiation doses used (2-20 Gy), but significant cell growth inhibition with IC50 of 10.4 Gy was observed. It is concluded that the increase in the radiation dose may have an important cytostatic effect, but for the complete eradication of metastatic prostate cancer novel cytotoxic drugs and radiosensitizers should be introduced as adjuvant.Uprkos značajnom napretku u hemoterapiji kancera, radioterapija ostaje metod izbora u tretmanu metastaziranog kancera prostate. Ovaj rad predstavlja kvantitativnu analizu efekata 60Co gama zračenja na ćelijski rasti i ćelijsku smrt PC-3 ćelijske linije humanog kancera prostate, pri čemu je praćena vremenska (2-72h) i dozna zavisnost (2-20 Gy). Ozračene PC-3 ćelije su uglavnom umirale nekrozom u kasnijem vremenskom intervalu (72h), dok je apoptoza bila zanemarljiva. Vrednost EC50 odnosno 50% citotoksičnosti nije dostignuta primenjenim dozama, ali je ustanovljena značajna inhibicija ćelijskog rasta, sa vrednošću IC50 od 10.4 Gy. Zaključeno je da povećanje doze može imati značajan citostatički efekat ali da je za kompletno odstranjivanje metastaziranog kancera prostate neophodno uvođenje novih citotoksičnih agenasa ili radiosenzitera kao adjuvanata.nul

Changes in expression of GFAP, ApoE and APP mRNA and protein levels in the adult rat brain following cortical injury

The recovery period following cortical injury (CI) is characterized by a dynamic and highly complex interplay between beneficial and detrimental events. The aim of this study was to examine the expressions of Glial Fibrillary Acidic Protein (GFAP), Apolipoprotein E (ApoE) and Amyloid Precursor Protein (APP), all of which are involved in brain plasticity and neurodegeneration. Our results reveal that CI strongly influenced GFAP, ApoE and APP mRNA expression, as well as GFAP and ApoE protein expression. Considering the pivotal role of these proteins in the brain, the obtained results point to their potential contribution in neurodegeneration and consequent Alzheimer's disease development.Projekat ministarstva br. 17305

Inactivation of melanoma cells irradiated with gamma rays and low energy protons

13th Balkan biochemical Biophysical Days and Meeting on Metabolic Disorders, October 12.-15., 2003, Kusadasi, Turke

Apoptotic clearance in rabbits with experimental pulmonary emphysema

In order to better understand pathogenesis of pulmonary emphysema, the model of experimentally induced pulmonary emphysema in Chinchilla rabbits was used for the estimation of apoptotic clearance of pulmonary tissue. Bronchoalveolar lavage was performed in three groups of animals: experimental group-E on hypercholesterolemic diet (4% edible oil solution of crystalline cholesterol), control group-C1 on standard diet for that animal species and animals on oily diet-C2. Apoptotic detection in cytocentrifuge preparations of lung washings was evaluated by in situ TUNEL. The property of alveolar macrophages to engulf apoptotic cells was estimated by light microscopy including 300 features (related subsequent steps: adsorption, internalization and intracellular processing of free apoptotic bodies) and was evaluated by scoring and indexing method. Internalization of apoptotic bodies by alveolar macrophages, as well as free apoptotic bodies were decreased in E compared to both C1 and C2 group (p<0.01 and p<0.05 respectively). Intracellular processing of apoptotic bodies by alveolar macrophages is significantly decreased in C2 in comparison with E (p<0.05) and C1 group (p<0.01). Apoptotic capacity of pulmonary tissue is significantly decreased in C2 in comparison with C1 group (p<0.01). The results implicate that immuno-metabolic competence of pulmonary tissue might be essentially associated with tissue remodeling in pulmonary emphysema.U cilju boljeg razumevanja patogeneze plućnog emfizema, u radu je korišć en eksperimentalni model emfizema pluća na činčila kunićima za procenu apoptotskog kapaciteta plućnoga tkiva. Bronholaveolarna lavaža je urađena na tri grupe životinja: eksperimentalnoj grupi-E na hiperholesterolskoj dijeti (4% uljani rastvor kristalnog holesterola), kontrolnoj grupi-C1 na standardnoj dijeti za tu životinjsku vrstu i grupi životinja na uljanoj dijeti-C2. Određivanje apoptotskih parametara cito-centrifužnih preparata bronhoalveolarnog lavata vršeno je posle bojenja preparata TUNEL in situ citohemijskim metodom. Sposobnost alveolarnih makrofaga da odstrane apoptotske ćelije fagocitozom procenjivana je svetlosnom mikroskopijom na 300 prikaza po preparatu (prikazi uključuju: adsorpciju, internalizaciju i intracelularno procesiranje apoptotskih tela) i evaluirana metodom indeksiranja i skora. Internalizacija apoptotskih tela alveolarnim makrofazima, kao i relativni procenat slobodnih apoptotskih tela, bili su signifikantno smanjeni u E grupi poredeći sa C1 (p<0.01) i C2 grupom (p<0.05). Intracelularno procesiranje apoptotskih tela alveolarnim makrofazima bilo je signifikantno smanjeno u C2 u odnosu na E (p<0.05) i C1 grupu (p<0.01). Apoptotski kapacitet tkiva pluća bio je signifikantno smanjen u C2 u poređenju sa C1 grupom (p<0.01). Ovi rezultati ukazuju da imuno-metabolička kompetentnost plućnog tkiva može biti suštinski povezana sa remodelovanjem tkiva pluća u eksperimentalnom emfizemu pluća.nul

Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p < 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.nul