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Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences. (experimentell patologi)
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences. (experimentell patologi)
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences. (experimentell patologi)
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics. (experimentell patologi)ORCID iD: 0000-0003-4075-159X
2004 (English)In: Methods in Cell Science, ISSN 1381-5741, Vol. 25, no 3-4, 185-194 p.Article in journal (Refereed) Published
Abstract [en]

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.

Place, publisher, year, edition, pages
2004. Vol. 25, no 3-4, 185-194 p.
Keyword [en]
Apoptosis, Flow cytometry, Lysosomes, pH measurement
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-15134DOI: 10.1007/s11022-004-8228-3OAI: oai:DiVA.org:liu-15134DiVA: diva2:54551
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-08-30Bibliographically approved
In thesis
1. Impact of Lysosomal Function in Cancer and Apoptosis
Open this publication in new window or tab >>Impact of Lysosomal Function in Cancer and Apoptosis
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lysosomes, the recycling units of the cell, participate in the signaling pathway to apoptosis, which has stimulated the search for anti-cancer drugs targeting the lysosomal compartment. Lysosomes are, however, often altered in cancer cells. The aim of this thesis was to investigate the involvement of lysosomes during apoptosis in normal and cancer cells. We developed and used flow cytometric methods to measure cytosolic and lysosomal pH in cells. The cytosolic pH of U937 cells decreased, in a caspase-independent way, by 1.4 pH-units during apoptosis. Concomitantly, the lysosomal pH increased from 4.3 to 5.2, suggesting that proton release from lysosomes might be responsible for cytosolic acidification. When studying the lysosomal pH of head and neck squamous cell carcinoma (HNSCC) cell lines and normal oral keratinocytes (NOKs), the pH was significantly increased in three of five HNSCC cell lines, as compared to NOKs. Moreover, high lysosomal pH correlated to low expression of the B subunit of the vacuolar V0/V1-ATPase, a necessary component of the proton pump responsible for lysosomal acidification, and to reduced intrinsic cisplatin sensitivity. Cisplatin-induced apoptosis was, at least partly, dependent on lysosomal cathepsins. When investigating the colony formation ability of the two HNSCC cell lines LK0412 and SqCC/Y1, both were found to give rise to holoclones, indicating the presence of cells with cancer stem cell properties. Holoclone cells from the LK0412 cell line were less sensitive to cisplatin compared to more differentiated paraclone cells. Moreover, we detected differences in intracellular localization of the lysosomal compartment and expression of cathepsins between holo- and paraclone cells.

This thesis shows that changes found in the lysosomal compartment of cancer cells, such as alteration of lysosomal pH, might influence the outcome of a drug treatment. In addition, differences in drug sensitivity between subpopulations of tumor cells may affect the outcome of an anticancer therapy.

Abstract [sv]

Programmerad celldöd eller apoptos är en viktig mekanism för att upprätthålla balans mellan kroppens celler. Vid exempelvis cancer fungerar inte styrningen av denna process, vilket leder till att för få celler dör och en tumör kan växa ohämmat. Denna avhandling fokuserar på lysosomen, en mycket sur organell i cellen som är ansvarig för nedbrytning av cellmaterial. Hos cancerceller är lysosomerna ofta förändrade. Vi har undersökt lysosomernas roll under apoptos hos normala celler och hos cancerceller. För att kunna undersöka pH-förändringar under apoptos har vi utvecklat metoder att mäta cytosoliskt och lysosomalt pH med hjälp av en teknik som kallas flödescytometri. I apoptotiska celler ser vi att det cytosoliska pH:t sjunker med 1.4 pH-enheter till pH 5.7 samtidigt som det lysosomala pH:t ökar från 4.3 till 5.5. Detta tyder på att läckage av vätejoner från lysosomerna kan orsaka en försurning av cytosolen under apoptos. Genom att studera normala orala keratinocyter och jämföra dessa mot fem olika cellinjer eeablerade från skivepitelcancer från munhåla har vi också funnit ett samband mellan det lysosomala pH:t och känsligheten för cellgiftet cisplatin. Cisplatinbehandling leder till apoptos hos alla celler men en högre dos krävs hos celler som har ett högt lysosomalt pH. Tumörer tros innehålla ett litet antal sk cancerstamceller, som har förmåga att kontinuerligt kopiera sig själva utan att åldras. Överlevnad av dessa celler tros vara orsaken till att en tumör återkommer efter en behandling. Vi visar i denna avhandling att cellinjer från skivepitelcancer innehåller celler som har cancerstamcellsegenskaper, och att dessa celler kan ha en lägre känslighet mot cisplatin jämfört med mer utvecklade cancerceller.

Lysosomerna utgör ett intressant framtida mål för nya cancerläkemedel. I denna avhandling visar vi att förändringar i det lysosomala systemet kan påverka effekten av ett läkemedel och att skillnader mellan olika sub-populationer av celler från samma tumör kan påverka resultatet av en behandling.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 112 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1080
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-15138 (URN)978-91-7393-794-8 (ISBN)
Public defence
2008-10-24, Linden, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-08-30Bibliographically approved

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Nilsson, CathrineKågedal, KatarinaJohansson, UnoÖllinger, Karin

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