Apoptosis and Cell Proliferation (522915), страница 18
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During thisperiod, cytoplasmic LDH is released into the culture SN due toplasma membrane damage.P The LDH activity in the culture SN is measured by a substratereaction and quantitated with an ELISA plate reader.P Constitutively expressed ubiquitous protein: assay generally notP LDH activity in serum: special assay medium (reduced serumrestricted by target cell typeconcentrations or BSA instead of serum) requiredP Does not require prelabeling and extensive washing of the target cells P Spontaneous release of LDH by target cells and effector cells: assaylimited to cytotoxic events causing high cell lysis over short period oftime (2–8 h)page 52 ofthis guideBrdUReduced metabolicactivityP Dye solution is added to cells cultured in MTP and cells areincubated. Viable (metabolically active) cells cleave tetrazoliumsalts to colored formazan compounds; dead cells do not.P Amount of formazan is quantitated with an ELISA plate reader.P No cell type restrictionP Does not require prelabeling and washing of the cellsP Optimal for microplate formatpages73–75 ofthis guideCellular DNA Fragmentation ELISALDH Release Assay51Cytotoxicity Detection Kit, LDHMetabolic activity assaysCell Proliferation Kit I (MTT), Kit II (XTT),Reagent (WST-1)P Increased metabolic activity of effector cells may mask target celldeath during cell-mediated cytotoxicityP No changes in the metabolic activity during the early phases ofapoptosisFor productinformation, see1page 54of this guideG Table 13: Methods for studying cytotoxicity.60Cell Death – Apoptosis and NecrosisCell Death – Apoptosis and Necrosis1Apoptosis Assay MethodsApoptosis Assay Methods6122.1Introduction2.1.12.1.2Terminology of cell proliferation and viabilityCell cycle2.2Cell proliferation/viability assay methods2.2.12.2.1.12.2.1.22.2.1.3Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityAssays that measure DNA synthesisSummary of methods for studying cell proliferation and cell viability incell populationsSingle reagents for the measurement of DNA synthesisMethods for studying cell proliferation and viability inindividual cellsAssays that measure DNA synthesisAssays that monitor expression of cell cycle-associated antigensSummary of methods for studying cell proliferation and viability inindividual cells2.2.1.42.2.22.2.2.12.2.2.22.2.2.3646464677070778484868696100Chapter 2Cell Proliferationand ViabilityIntroductionTerminology of cell proliferation and viability2.1 IntroductionCell Proliferation and ViabilityRapid and accurate assessment of viable cellnumber and cell proliferation is an important requirement in many experimentalsituations involving in vitro and in vivostudies.
Examples of where determinationof cell number is useful include the analysisof growth factor activity, serum batchtesting, drug screening, and the determination of the cytostatic potential of anticancer compounds in toxicology testing.In such toxicological studies, in vitro testing techniques are very useful to evaluatethe cytotoxic, mutagenic, and carcinogeniceffects of chemical compounds on humancells.2.1.1 Terminology of cell proliferationand viabilityUsually, one of two parameters is used tomeasure the health of cells: cell viability orcell proliferation.
In almost all cases, theseparameters are measured by assaying for“vital functions” that are characteristic ofhealthy cells.Cell ViabilityCell viability can be defined as the numberof healthy cells in a sample. Whether thecells are actively dividing or are quiescent isnot distinguished. Cell viability assays areoften useful when non-dividing cells (suchas primary cells) are isolated and maintained in culture to determine optimal culture conditions for cell populations.2The most straightforward method for determining viable cell number is a directcounting of the cells in a hemocytometer.Sometimes viable cells are scored based onmorphology alone; however, it is more helpful to stain the cells with a dye such as trypan blue. In this case, viability is measuredby the ability of cells with uncompromisedmembrane integrity to exclude the dye.Alternatively, metabolic activity can beassayed as an indication of cell viability.Usually metabolic activity is measured inpopulations of cells by incubating the cellswith a tetrazolium salt (MTT, XTT, Wst-1)that is cleaved into a colored formazanproduct by metabolic activity.64Cell ProliferationCell proliferation is the measurement of thenumber of cells that are dividing in a culture.
One way of measuring this parameteris by performing clonogenic assays. Inthese assays, a defined number of cells areplated onto the appropriate matrix and thenumber of colonies that are formed after aperiod of growth are enumerated. Drawbacks to this type of technique are that it istedious and it is not practical for large numbers of samples.
In addition, if cells divideonly a few times and then become quiescent, colonies may be too small to becounted and the number of dividing cellsmay be underestimated. Alternatively,growth curves could be established, whichis also time-consuming and laborious.Another way to analyze cell proliferation isthe measurement of DNA synthesis as amarker for proliferation.
In these assays, labeled DNA precursors (3H-thymidine orbromodeoxyuridine) are added to cells andtheir incorporation into DNA is quantifiedafter incubation. The amount of labeledprecursor incorporated into DNA is quantified either by measuring the total amountof labeled DNA in a population, or by detecting the labeled nuclei microscopically.Incorporation of the labeled precursor into DNA is directly proportional to theamount of cell division occurring in theculture.Cell proliferation can also be measuredusing more indirect parameters. In thesetechniques, molecules that regulate the cellcycle are measured either by their activity(e.g.
CDK kinase assays) or by quantifyingtheir amounts (e.g. Western blots, ELISA,or immunohistochemistry).2.1.2 Cell CycleIn an organism, the rate of cell division is atightly regulated process that is intimatelyassociated with growth, differentiation andtissue turnover. Generally, cells do not undergo division unless they receive signalsthat instruct them to enter the active segments of the cell cycle. Resting cells are saidto be in the G0 phase (quiescence) of thecell cycle (Figure 42). The signals that induce cells to divide are diverse and trigger alarge number of signal transduction cascades.IntroductionCell CycleFigure 42: Cell cycle: A schematic overview.
EG0CycBDegradationCycBCycADegradationCycDcdc2G1Mcdk4,6Cell cycleCycEcdc25CG2A thorough discussion of the types ofsignals and the variety of responses theycan elicit are beyond the scope of this guide(Table 14). Generally, signals that directcells to enter the cell cycle are called growthfactors, cytokines, or mitogens.cdk2Scdc2CycAcdk2cdc25ACycEDegradationAbbreviationDescriptionReferenceRTKReceptor Tyrosine KinaseMarshall, (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185.RASGTP exchange proteinWhite, M. A. et al. (1995) Multiple Ras functions can contribute to mammalian cell transformation.Cell 80: 533–541.RAFMAP kinase kinase kinaseAvruch, J.
et al. (1994) Raf meets Ras-Completing the framework of a signal transduction pathway.Trends Biochem. Sci. 19: 279–283.MEKMAP kinase kinase orMAPk/Erk kinaseMarshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase. Curr. Opin.Genet. Dev. 4: 82–89.MAPKMitogen activated protein kinaseor ErkMarshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase. Curr. Opin.Genet. Dev. 4: 82–89.PKCProtein Kinase CBlobe, G. et al. (1996) Protein Kinase C isoenzymes: regulation and function.
Cancer Surveys27: 213–248.JAKJust Another Kinase or Janus Kinase Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks and STATs. TIBS19: 222–227.STATSignal Transducers and Activatorsof Transcription2Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks and STATs. TIBS19: 222–227.Marx, J. (1994) How cells cycle toward cancer. Science 263: 319–321.CyclinsCDKCyclin Dependent KinaseMacLachlan, T.
K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-dependent kinases and cdkinhibitors: implications in cell cycle control and cancer. Crit. Rev. Eukaryot. Gene Expr. 5: 127–156.CDC2Cell division cycle mutantMacLachlan, T. K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-dependent kinases and cdkinhibitors: implications in cell cycle control and cancer. Crit. Rev.
Eukaryot. Gene Expr. 5: 127–156.CAKCDK Activating KinaseMorgan, D. O. (1995) Principles of CDK Regulation. Nature 374: 131–134.Signal Transduction PathwaysThree major types of signal transductionpathways are activated in cells in responseto growth factors or mitogenic stimuli. Theresponse to these stimuli varies from celltype to cell type and the pathways continueto grow more and more complex. Thesetypes of pathways continue to be the focusof a great deal of research and, consideringthe importance of cell cycle regulation inbiology, the pathways will continue togrow in complexity for some time to come.P The MAP kinase (MAPK) type of pathways are triggered through a cascade ofphosphorylation events that begins witha growth factor binding to a tyrosine kinase receptor at the cell surface. Thiscauses dimerization of the receptor andan intermolecular cross-phosphorylation of the two receptor molecules.
Thephosphorylated receptors then interactwith adaptor molecules that triggerdownstream events in the cascade. Thecascade works through the GTP ex-Cell Proliferation and ViabilityCycAG Table 14: Published sources thatcontain more information about cellproliferation.65IntroductionCell Cyclechange protein RAS, the protein kinaseRAF (MAPKKK), the protein kinaseMEK (MAPKK), and MAP kinase(Erk). MAPK then phosphorylates avariety of substrates that control transcription, the cell cycle, or rearrangements of the cytoskeleton.Cell Proliferation and ViabilityP The protein kinase C (PKC) pathwaysconsist of a family of phospholipid dependent protein kinases.
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