We have examined the effect of suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and on the expression of 20 apoptosis-related genes. RT-PCR, western blots and flow cytometry were performed to reveal the proteins of apoptosis machinery that were affected to cause cell death. Our data suggest that PBL markedly resisted for approximately 24 h the destructive activity of the agent, but eventually 60% of cells treated with 4 micromol/L SAHA died within 72 h through mitochondrial way of apoptosis. While the expression of the majority of genes remained indifferent against 4 micromol/L SAHA, the cellular levels of BimEL, Bmf-2, Bcl-w and survivin mRNA varied, confirming the pro-apoptotic response of SAHA treated PBL. In addition, the expression of multifunctional proteins c-Myc and p21(WAF1) changed profoundly with the time of SAHA treatment. The Bax activator BimEL increased rapidly, driving cells towards apoptosis likely controlled by c-Myc and p21(WAF1) activities. We suggest that variations in c-Myc and p21(WAF1) expression decelerate the apoptosis in the early period and increase the resistance of resting PBL against SAHA. 2009 John Wiley & Sons, Ltd.
- MeSH
- aktivace enzymů účinky léků MeSH
- buněčná membrána metabolismus účinky léků MeSH
- buněčná smrt účinky léků MeSH
- cytochromy c metabolismus MeSH
- cytoprotekce účinky léků MeSH
- financování organizované MeSH
- fosfatidylseriny metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy metabolismus MeSH
- kaspasy metabolismus MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- lymfocyty cytologie enzymologie metabolismus účinky léků MeSH
- membránové proteiny metabolismus MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie metabolismus účinky léků MeSH
- poly(ADP-ribosa)polymerasy metabolismus MeSH
- proteiny regulující apoptózu metabolismus MeSH
- protoonkogenní proteiny c-myc metabolismus MeSH
- protoonkogenní proteiny metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- transport proteinů účinky léků MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
The present study was undertaken to provide more information on the relationship between the nucleolar size and RNA density. Mature monocytes circulating in human peripheral blood appeared to be very convenient for such study because they contain multiple nucleoli of various sizes in one and the same nucleus. In addition, nucleoli without perinucleolar chromatin represented by nucleolar bodies are easy to be visualized by a simple cytochemical procedure for RNA demonstration. The diameter and density of NoBs in specimens stained for RNA were determined by computer-assisted measurements of individual cells. According to the results, the nucleolar RNA content was apparently related to the nucleolar size because the RNA density of small and large NoBs was practically the same. In addition, the diameter measurements also indicated that one or two of several NoBs in one nucleus were dominant - larger - than the others. It should also be mentioned that the diameter and RNA density of NoBs were studied in monocytes in peripheral blood smears regardless of their limited number. Additional study on lymphocytes indicated that the preparation procedure of smears modified both diameter and density of the measured parameters less than the preparation of cytospins as demonstrated by lower variability of the resulting values. Thus, the observed differences between smears and cytospins also indicated that the specimen preparation procedures should always be considered during evaluation of the nucleolar size or staining intensity of nucleoli or cytoplasm due to the presence of RNA.
The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size.
We have examined the effect of sodium butyrate (SB) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and the effect of this agent on the expression of 20 apoptosis-related genes. Data suggest that PBL treated with 2 mmol L(-1) SB resisted for at least 8 h the destructive activity of the agent, but eventually 30% of cells died within 72 h. As documented by flow cytometry and cytochrome c release study, cells underwent mitochondrial-derived apoptosis. While the expression of the majority of genes examined by RT-PCR and Western blots remained indifferent to 2 mmol L(-1) SB, the cellular levels of BimEL, c-myc, p53, and p21(WAF1) varied profoundly with the time of SB treatment. The Bax activator BimEL increased rapidly, driving cells toward apoptosis likely controlled by c-myc and p21(WAF1) activities. The c-myc, exercising the role of mediator of the function of BimEL and inhibitor of p21(WAF1) expression, decreased significantly for several hours after adding SB but within 48 h it returned to close to its original value. An apoptosis inhibitor and executive caspase substrate p21(WAF1) increased early at the beginning of treatment but subsequently, within a time frame of 72 h, profoundly dropped in terms of both a caspase-dependent and caspase-independent way. We suggest that variations in c-myc and p21(WAF1) expression delay apoptosis making PBL resistant to SB for several hours, and together with fast catabolism of SB in vivo protect PBL against the destructive activity of this anti-cancerous metabolite of colonic bacteria. (c) 2008 John Wiley & Sons, Ltd.
- MeSH
- aktivace enzymů MeSH
- apoptóza účinky léků MeSH
- cytochromy c metabolismus sekrece MeSH
- financování organizované MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika metabolismus MeSH
- intracelulární membrány účinky léků MeSH
- kaspasy metabolismus MeSH
- kolagen typ XI metabolismus MeSH
- kultivované buňky MeSH
- kyselina máselná farmakologie MeSH
- lidé MeSH
- lymfocyty metabolismus sekrece účinky léků MeSH
- membránové proteiny genetika metabolismus MeSH
- mitochondrie účinky léků MeSH
- proteiny regulující apoptózu genetika metabolismus MeSH
- protoonkogenní proteiny c-myc genetika metabolismus MeSH
- protoonkogenní proteiny genetika metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- Check Tag
- lidé MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
70 l. : il. ; 30 cm
Charakterizace, kvantifikace a buněčná selektivita antiproliferativních procesů cílených do nukleolu nádorových buněk bude studována zkoumáním změn exprese rRNA a ex-prese, modifikace a integrity proteinových faktorů ribozomálního transkripčního aparátu.Budou užity buněčné modely reprezentované leukemickými liniemi na různé úrovni hematopoetické diferenciace. Represe proliferace a buněčná smrt bude navozena vnějšími efektory, zejména terapeuticky účinnými činidly.; A linkage between the antiproliferative and proapoptotic efect of therapeutic agents and down regulation of rRNA synthesis in leukemic cells will be studied by in-vestigation of nucleolar transcription factors expression, modification and integrity. Leukemic cell lines representing various states of haematopoetic differenti-ation and chemical and physical repressors of proliferation and inductors of program-med cell death will be used.
- MeSH
- apoptóza MeSH
- geny rRNA MeSH
- imunologické faktory MeSH
- leukemie MeSH
- organizátor jadérka MeSH
- proliferační antigen buněčného jádra MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- hematologie a transfuzní lékařství
- onkologie
- biologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
The present study was undertaken to provide more information on the heterochromatin density in central and peripheral nuclear regions during “cell dedifferentiation“ represented by blastic transformation of mature T lymphocytes. Heterochromatin was visualized using a simple cytochemical method for the demonstration of DNA followed by computer-assisted densitometry of digitised images. The results indicated that the blastic transformation was accompanied by a marked and significant decrease in the heterochromatin density at the nuclear membrane. Thus, this nuclear peripheral region seems to be important not only for cell differentiation but also dedifferentiation events. It is also interesting that the non-stimulated resting mature cells in the present study were characterized by less condensed heterochromatin at the nuclear membrane than differentiated granulocytic or erythrocytic precursors and apoptotic myeloblasts or leukemic B lymphocytes described in the previous study. However, in contrast to these cells, resting and mature T lymphocytes in the present study are known to revert to cycling blastic cells after PHA treatment. In addition, it is also known that nuclear peripheral regions with heterochromatin represent sites of chromosomal attachments as well as “together crowded replicons“ and silent genes.
- MeSH
- aktivace lymfocytů genetika imunologie účinky léků MeSH
- dediferenciace buněk genetika imunologie účinky léků MeSH
- denzitometrie metody využití MeSH
- financování organizované využití MeSH
- fytohemaglutininy imunologie krev účinky léků MeSH
- heterochromatin genetika patologie MeSH
- interpretace statistických dat MeSH
We have examined the ability of actinomycin D to induce apoptosis in human peripheral blood lymphocytes. Run-On assays were performed to specify the primary molecular damage, reverse transcription-PCR, Western blots and flow cytometry studies were performed to ascertain which proteins of the apoptosis machinery were affected to cause actinomycin D-induced cell death. Expression of 23 apoptosis-related genes was investigated. The down-regulation of ribosomal RNA synthesis caused by actinomycin D induced a mitochondria-dependent apoptosis. Although the expression of the majority of examined genes remained indifferent against actinomycin D activity, the cellular level of p53 protein increased, subsequently upregulating both Puma mRNA and protein. Puma-mediated mitochondrial apoptosis was accompanied by nucleolin cleavage and Bcl-2 mRNA destabilization. The stability of the cellular level of Bcl-2 protein independent of a mRNA decrease suggests that protection of Bcl-2 protein against proteasomal degradation can moderate the apoptotic process. In peripheral blood lymphocytes cultured in vitro, the apoptosis induced by a low concentration of actinomycin D (10 nmol/l) is dependent on p53 and Puma activation. This apoptotic pathway is demonstrated in peripheral blood lymphocytes for the first time. A different apoptotic pathway induced in peripheral blood lymphocytes using this drug has, however, been previously revealed by other authors. The combination of cell specificity and dose-dependent effects can likely play a decisive role in apoptosis observed in peripheral blood lymphocytes after genotoxic drug application.
- MeSH
- apoptóza účinky léků MeSH
- daktinomycin aplikace a dávkování farmakologie MeSH
- down regulace MeSH
- financování organizované MeSH
- lidé MeSH
- lymfocyty účinky léků MeSH
- messenger RNA metabolismus MeSH
- mitochondrie metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- poškození DNA účinky léků MeSH
- proteiny regulující apoptózu metabolismus MeSH
- protinádorová antibiotika aplikace a dávkování farmakologie MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus MeSH
- protoonkogenní proteiny metabolismus MeSH
- průtoková cytometrie MeSH
- ribozomální DNA účinky léků MeSH
- upregulace účinky léků MeSH
- viabilita buněk MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
Poměrně mladým členem rodiny lipokalinů, malých sekrečních proteinů účastnících se převážně transmembránového přenosu lipofilních substancí je neutrofilní, s gelatinázou asociovaný lipokalin. Původně izolován ze specifických granul neutrofilů byl později nalezen v kostní dřeni a výstelkových tkáních některých orgánů. Exprese neutrofilního lipokalinu ve výstelkových tkáních a tělních tekutinách mohutně stoupá při zánětlivých a nádorových onemocněních. Jeho úloha je prokázána v modulaci imunitní odpovědi a pozoruhodné je jeho bakteriostatické působení, zprostředkované účinnou kompeticí neutrofilního lipokalinu s mikroorganizmy o chelátory železa. Schopností vytvářet protektivní komplexy s gelatinázou B zasahuje neutrofilní lipokalin do regulace procesů fyziologické i patologické přestavby tkání, zejména angiogeneze. Diagnosticky významná jsou vyšetření hladin neutrofilního lipokalinu v tělních tekutinách, způsobilá rozlišit bakteriální a virové infekce. Stanovení neutrofilního lipokalinu v krevním séru a moči pacientů ohrožených selháním funkce ledvin nabízí velmi časný marker akutního selhání či marker nefrotoxicity při podávání některých léčiv. Neutrofilní lipokalin reprezentuje citlivý neinvazivní marker ledvinné ischémie a u pacientů s cystickou fibrózou marker akutní plicní exacerbace. Aktuální jsou diskuze o úloze neutrofilního lipokalinu jako časného markeru karcinogeneze slinivky břišní a markeru neutrofilní aktivace při vážných střevních onemocněních. Spektrum poznatků o funkci neutrofilního lipokalinu se rychle rozrůstá a s ním i nabídka klinických aplikací.
Neutrophil gelatinase associated lipocalin belongs to a family of small proteins, lipocalins, engaged in the transmembrane transportation of lipophylic substances. Originally isolated from specific granules of neutrophils, it was later located in bone marrow cells as well as lung, bronchial and colon epithelial cells. The expression of neutrophil lipocalin in epithelial cells and in body fluids considerably augments during the occurrence of inflammations and some cancers. A modulation of immunity response was thus suggested to be the main function of neutrophil lipocalin as well as the bacteriostatic effect originating from competition between neutrophil lipocalin and bacteria for siderophoric iron. Forming protective complexes with gelatinase B, the neutrophil lipocalin is implicated in regulatory processes of physiological and pathological rebuilding of tissues, mainly in the angiogenesis. The determination of neutrophil lipocalin levels in body fluids able to discriminate between bacterial and viral infections provides a powerful diagnostic tool. The examination of neutrophil lipocalin in the sera and urine of patients at risk of renal failure offers a very early marker of this acute state. Neutrophil lipocalin represents a sensitive non-invasive marker of renal ischemia and in patients with cystic fibrosis the marker of acute pulmonary exacerbation. Discussions have been conducted regarding the role of neutrophil lipocalin as an early marker of pancreatic cancer or of neutrophilic activation in severe cases of bowel diseases.
