Apoptosis and Cell Proliferation (522915), страница 19
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PKC is regulated by a large variety of metabolicpathways involving phospholipids andcalcium levels within a cell. The mainregulator of the pathway is diacylglycerol (DAG) which appears to recruitPKC to the plasma membrane and causeits activation. The activity of DAG ismimicked by the phorbol-ester tumorpromoters. Once activated, PKC canphosphorylate a wide variety of cellularsubstrates that regulate cell proliferationand differentiation. Responses to PKCappear to vary with the types of PKCsexpressed and the types of substratesavailable within a cell. Some evidenceshows that the PKC pathway may interact with and exert effects through theMAPK pathway.P The JAK/STAT pathway is activated bycytokine interaction with a family ofreceptors called the cytokine receptorsuperfamily.
These receptors do notcontain a protein kinase domain themselves, but they associate with and activate a family of protein kinases calledthe JAK (Just Another Kinase or JAnusKinase) family. JAK family members arerecruited to receptor complexes that areformed as a result of ligand binding. Thehigh concentration of JAK in the complex leads to a cross-phosphorylationof JAK and thus activation. JAK thenphosphorylates members of anotherprotein family called STAT (signaltransducers and activators of transcription). These proteins then translocate tothe nucleus and directly modulate transcription.266Control of the Cell CycleOnce the cell is instructed to divide, it enters the active phase of the cell cycle, whichcan be broken down into four segments:P During G1 (G = gap), the cell preparesto synthesize DNA.
In the latter stagesof G1, the cell passes through a restriction point (R) and is then committed tocomplete the cycle.P During S phase the cell undergoes DNAsynthesis and replicates its genome.P During G2 the cell prepares to undergodivision and checks its replication usingDNA repair enzymes.P During M phase, the cell undergoesdivision by mitosis or meiosis and thenre-enters G1 or G0.In most instances, the decision for a cell toundergo division is regulated by the passage of a cell from G1 to S phase. Progression through the cell cycle is controlled bya group of kinases called cyclin-dependentkinases (CDKs), (see Figure 42).
CDKs arethought to phosphorylate cellular substrates, such as the retinoblastoma gene, thatare responsible for progression into each ofthe phases of the cell cycle. CDKs are activated by associating with proteins whoselevels of expression change during differentphases of the cell cycle. These proteins arecalled cyclins.
Once associated with cyclins, CDKs are activated by phosphorylation via CDK-activating kinase (CAKs) orby dephosphorylation via a phosphatasecalled CDC25.D-types cyclins are the primary cyclinsthat respond to external cellular factors.Their levels start off low during G1 andincrease towards the G1/S boundary. Cyclin D regulates CDK4 and CDK6. Cyclin Eis expressed transiently during the G1/Stransition and is rapidly degraded once thecell enters S.
Cyclin E regulates CDK2 andperhaps CDK3. When S phase begins,levels of cyclin A increase and activateCDK2. The cyclin A/CDK2 complex isthought to have a direct role in DNA replication. The progression through mitosisis regulated by the presence of cyclin B.Cyclin B associates with CDC2 and formsthe primary kinase present during mitosis(MPF = “M-phase/maturation promotingfactor”). During anaphase cyclin B is degraded. This degradation of cyclin B appears to regulate the cell’s progression out ofmitosis and into G1.Cell proliferation/viability assay methods2.2 Cell proliferation/viability assay methods12Reproductive assays can be used to determine the number of cells in a culturethat are capable of forming coloniesin vitro.
In these types of experiments,cells are plated at low densities and thenumber of colonies is scored after agrowth period. These clonogenic assaysare the most reliable methods for assessing viable cell number57, 58, 59. Thesemethods, however, are very time-consuming and become impractical whenmany samples have to be analyzed.Permeability assays involve stainingdamaged (leaky) cells with a dye andcounting viable cells that exclude thedye.
Counts can either be performedmanually using a hemocytometer andfor example trypan blue. (Figure 43).This method is quick, inexpensive, andrequires only a small fraction of totalcells from a cell population. Therefore,this method is generally used to determine the cell concentration (cell number/ml) in batch cell cultures.
This ishelpful in ensuring that cell cultureshave reached the optimal level ofgrowth and cell density before routinesub-culture, freezing, or any experiment60.Or counts can be performed mechanicallyusing for example a flow cytometer andpropidium iodide. Alternatively, membrane integrity can be assayed by quantifying the release of substances from cellswhen membrane integrity is lost, e. g. Lactate dehydrogenase (LDH) or 51Cr (described in section 1.3.2.1 starting on page51 of this guide.)3Metabolic activity can be measured byadding tetrazolium salts to cells. Thesesalts are converted by viable cells to colored formazan dyes that are measuredspectrophotometrically.4Direct proliferation assays use DNAsynthesis as an indicator of cell growth.These assays are performed using eitherradioactive or nonradioactive nucleotide analogs.
Their incorporation intoDNA is then measured.Figure 43: Measurement of proliferation by counting the cells with ahemocytometer. The addition of trypanblue helps to distinguish viable,unstained cells (p) from non-viable,blue-stained cells (P). H1 mmCell Proliferation and ViabilityA variety of methods have been devisedthat measure the viability or proliferationof cells in vitro and in vivo. These can besubdivided into four groups:0.25 mmThe first section describes those assays designed to study cell proliferation in wholepopulations of cells, followed by a sectioncovering proliferation assays designed tomeasure proliferation in individual cells (insitu).2For a discussion of the advantages and limitations of all types of cell proliferation assays, read Sections 2.2.1.3 and 2.2.2.3 of thisguide.For discussions of particular assays, turnto the pages indicated in the followingmethod selection guide:67Cell proliferation/viability assay methodsCell proliferation/viability assay methodsStartlMonoclonalantibodies(see page 97)nsatioIndividualcellsCell cycleassociatedantigensDo you want to measureDNA synthesis or thepresence of cell cyleassociated antigens?In vivoDo you want to studyproliferation in vitro or in vivoIn vitroDo you want to studyproliferation in cell populations or in individual cells?IndividualcellsCell Proliferation and ViabilityLightMicroscopyFluorescenseIn Situ CellProliferation Kit, AP(See page 90)BrdU Labeling andDetection Kit II(See page 87)Do you analyze data by flowcytometry or microscopy?CellpopulationDo you analyze data by flowcytometry or microscopy?FlowcytometryMicroscopyMonoclonal antibodies(See page 97)FlowcytometryIn Situ Cell Proliferation Kit,FLUOS(See page 88)BrdU Labeling andDetection Kit I(See page 87)DNA synthesisCell cycleassociatedantigensDNAsynthesisDo you use a fluorescenceor light microscope?Do you want to measureDNA synthesis or thepresence of cell cycleassociated antigens?Do you use a fluorescence orlight microscope?In Situ Cell Proliferation Kit,FLUOS(See page 88)BrdU Labeling andDetection Kit I(See page 87)FluorescenceLightCell Proliferation and ViabilityCelulpopDo you want to measureproliferation in individualcells or in cell populationsIn Situ CellProliferation Kit, AP(See page 90)BrdU Labeling andDetection Kit II(See page 87)Cell cycleassociated antigensDo you wantto measure DNA synthesis orother cellular changes?OthercellularchangesDo you want to measuremetabolic activity of livingcells or levels of cell cycleassociated antigens?MetabolicactivityCell Proliferation Kit I (MTT)Cell Proliferation Kit II (XTT)Cell Proliferation ReagentWST-1(See pages 73–75)DNAsynthesis2Do you want to use acolorimetric or chemiluminescent assay?Chemiluminescent2Cell Proliferation ELISA,BrdU, chemiluminescent(See page 81)ColorimetricCell Proliferation ELISA,BrdU, colorimetric(See page 81)BrdU Labeling andDetection Kit III(See page 79)G Figure 44: Method/Product selection guide6869Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activity2.2.1 Methods for studyingcell proliferation and viability incell populationsCell Proliferation and ViabilityA number of methods have been developedto study cell viability and proliferation incell populations.
The most convenient modern assays have been developed in a microtiterplate (MTP) format (96-well plates).This miniaturization allows many samplesto be analyzed rapidly and simultaneously.The MTP format also reduces the amountof culture medium and cells required aswell as cost of plasticware.
Colorimetric assays allow samples to be measured directlyin the microtiterplate with an ELISA platereader.MTP assays have been developed based ondifferent parameters associated with cellviability and cell proliferation. The mostimportant parameters used are metabolicactivity and DNA synthesis for MTP format.P Cellular damage will inevitably result inloss of the ability of the cell to maintainand provide energy for metabolic cellfunction and growth. Metabolic activity assays are based on this premise.Usually they measure mitochondrialactivity.
The cells are incubated witha colorometric substrate (MTT, XTT,WST-1) (described on pages 73–75 ofthis guide.P As outlined above, during the S phasethe cell undergoes DNA synthesis andreplicates its genome. If labeled DNAprecursors, in our case BrdU, are addedto the cell culture, cells that are aboutto divide incorporate BrdU into theirDNA. The incorporated BrdU can thenbe detected by a quantitative cellular enzyme immunoassay using monoclonalantibodies against BrdU (described onpage 81 of this guide).2In the following sections we will describedetails of each of these cell viability andproliferation assays.Besides MTP assays, cell populations canbe analyzed for expression of cell cycleassociated antigens by Western blotting,described in section 2.2.2.2 on page 97 ofthis guide.702.2.1.1 Assays that measure metabolicactivityOne parameter used as the basis for colorimetric assays is the metabolic activity ofviable cells.
For example, a microtiter plateassay which uses the tetrazolium salt MTTis now widely used to quantitate cell proliferation and cytotoxicity53, 65.Because tetrazolium salts are reduced to acolored formazan only by metabolicallyactive cells, these assays detect viable cellsexclusively. For instance, in the MTT assay,MTT is reduced by viable cells to a colored,water-insoluble formazan salt. After it issolubilized, the formazan formed can easilyand rapidly be quantitated in a conventional ELISA plate reader at 570 nm (maximumabsorbance).[Author’s note: MTT is cleaved to formazanby the “succinate-tetrazolium reductase”system (EC 1.3.99.1) which belongs to themitochondrial respiratory chain and is active only in viable cells.
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