Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence
of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  Biphasic Calcium Response of Platelet-DerivedGrowth Factor Stimulated Glioblastoma Cells Is a Function of Cell Confluence Gy  € orgy Vereb, 1 *  Burt G. Feuerstein, 2  William C. Hyun, 3 Mack J. Fulwyler, 3 Margit Bal   azs, 4 and J  anos Sz € oll  } osi  1 1 Department of Biophysics and Cell Biology, Research Center for Molecular Medicine,University of Debrecen, Debrecen, Hungary  2 Departments of Lab Medicine and Neurosurgery, and Brain Tumor Research Center, University of California, San Francisco, California 3 Laboratory for Cell Analysis, Department of Laboratory Medicine, University of California, San Francisco, California 4 Department of Preventive Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary  Received 21 May 2005; Revision Received 1 July 2005; Accepted 4 July 2005 Background:  Previous reports have linked the spiking or two-phased character of calcium transients evoked by pla-telet-derived growth factor (PDGF) to the position of cellsin the cell cycle without regard to cell–cell contact andcommunication. Because cell confluence can regulategrowth factor receptor expression and dephosphoryla-tion, we investigated the effect of cell culture confluenceand cell cycle on calcium responses of PDGF-BB–stimu-lated A172 glioblastoma cells. Methods:  Digital imaging cytometry was used to corre-late the peak and duration of calcium response with bro-modeoxyuridine positivity and DNA content and with cul-ture confluence on a cell-by-cell basis. Results:  In serum-starved cultures, complete two-phasecalcium signals and shorter, lower spikes occurred inde-pendent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a completeresponse because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. Conclusion:  Because cell confluence, by virtue of cell–cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in trans-formed cells the ability to produce stronger growth signalsupon reaching confluence and facing contact inhibitioncould provide a proliferative advantage.  q  2005 InternationalSociety for Analytical Cytology  Key terms:  platelet-derived growth factor receptor; cal-cium signaling; intracellular release; store-operated influx;I CRAC ; glioblastoma; cell cycle; cell confluence; cell-cellcontact; bromodeoxyuridine incorporation; digital ima-ging cytometry Platelet-derived growth factor receptor (PDGFR) is areceptor tyrosine kinase that plays an important role inthe development of tumors of the central nervous sys-tem of glial srcin, like multiform glioblastoma (1,2).Ligand binding is followed by autophosphorylation of the receptor (3) and results in activation of variouseffector enzymes on its tyrosine residues, such as phos-pholipase-C g 1 (4), p21ras GTPase activating protein (5),and phosphatidyl-inositol-3-kinase. Activation of phos-pholipase-C g 1 leads to increased inositol-1,4,5-tripho-sphate and diacylglycerol levels, with the former mobi-lizing calcium from intracellular stores (6,7). Another pathway leading to high intracellular Ca 2 1 levels uponPDGFR stimulation is the production of ceramide, sphin-gosine, and sphingosine-1-phosphate upon sphingomyeli-nase activation (1,8). Sphingosine and sphingosine-1-phosphate release Ca 2 1 from the thapsigargin-sensitiveintracellular pools independently of inositol-1,4,5-tri-phosphate (9,10).Ca 2 1 is a second messenger that is critically importantduring cell cycle progression. In many eukaryotic cells,growth factors and hormones that trigger the phosphoino-sitide pathway evoke a biphasic increase in intracellular free Ca 2 1 concentration: an initial transient release of Ca 2 1 Contract grant sponsor: Hungarian Academy of Sciences; Contractgrant numbers: OTKAT037831, OTKAT043061, OTKAT048750; Contractgrant sponsor: Hungarian Ministry of Health; Contract grant number: ETT532/2003, ETT 524/2003; Contract grant sponsor: National Research and Development Program, Hungary; Contract grant number: NKFP-1B-0013/2002; Contract grant sponsor: B  ek   esy Fellowship from the Hun-garian Ministry of Education; Contract grant sponsor: European UnionFP6 Program; Contract grant number: LSHB-CT-2004-503467.*Correspondence to: Gy  € orgy Vereb, Department of Biophysics and CellBiology, Medical and Health Science Center, University of Debrecen,Nagyerdei krt. 98, H-4012 Debrecen, Hungary.E-mail: vereb@dote.huPublished online 12 September 2005 in Wiley InterScience ( 10.1002/cyto.a.20178 q  2005 International Society for Analytical Cytology Cytometry Part A 67A:172–179 (2005)  from intracellular stores is followed by a sustained phase of Ca 2 1 influx. Blocking Ca 2 1 influx through the cell mem-brane or its mobilization from intracellular stores stops cellsfrom entering G1 and S phase (11). Different patterns of Ca 2 1 signals may be responsible for different ‘‘messages’’transmitted to the cell nucleus (12). Voltage-dependentchannels may contribute to the influx phase of Ca 2 1 signals(13). In mouse fibroblasts PDGFR activation opens T-type voltage-gated Ca 2 1 channels (14). However, in PDGF-BB–stimulated glioblastoma cells blockers of L-type Ca 2 1 chan-nels have no effect on the second, influx-based phase of the Ca 2 1 response (15); rather, depletion of intracellular Ca 2 1 stores seems to trigger Ca 2 1 channel opening (16).This is coherent with the currently accepted concept thatgrowth factor and neurotransmitter-induced calcium influxis generally store dependent and is required for controlling various Ca 2 1 -dependent processes such as synaptic secre-tion or cell division (17,18).The effects of growth factors have been shown todepend on the position of a cell in the cell cycle. In G1phase, the absence of these factors causes reversible transi-tion to G0 (19), and then the cells require growth factorsto re-enter to the cell cycle (20,21). It was previously shown that the expression level of PDGFR is also cell cycledependent and regulates entering into G1 phase from G0(22). However, PDGFR expression levels did not changesignificantly with the cell cycle in other studies (23).Stimulation of PDGFR-expressing transformed oligoden-drocytes and human embryonic kidney 293 cells with PDGF-BB homodimer can induce oscillatory and nonoscil-latory Ca 2 1 responses (24), with the two types being uni-formly distributed in asynchronously proliferating cell cul-tures and being exclusive for any given cell. In G0 arrest,the nonoscillatory Ca 2 1 signal was the most common; inG1 arrest, the oscillatory type occurred in a larger percen-tage of cells. Cells blocked at the G1/S border respondedto PDGF-BB homodimer in a nonoscillatory manner, butupon entering S phase the percentage of oscillatory responses increased significantly. In G2 and M phases, cellcycle had no effect on the pattern of Ca 2 1 response (23).It has been suggested that cell cycle–dependent levels of sphingosine and sphingosine-1-P could be responsible for the oscillatory and nonoscillatory Ca 2 1 responses, respec-tively, of PDGF-stimulated cells (24,25). A control mechanism of cell proliferation in multicellu-lar organisms is the inhibition of cell growth by cellular contact even in the presence of growth factors. PDGFR isa substrate of low-molecular-weight protein tyrosine phos-phatases that control PDGFR-triggered pathways (26) andplay a role in contact inhibition according to experimentsshowing that total protein tyrosine phosphatase activity increases in high-density cell cultures (27). In accordance with this, decreased tyrosine phosphorylation of   b  typePDGFR can be detected in confluent as opposed to sparsecell cultures (28). A172 human glioblastoma cells express relatively largeamounts of PDGF-BB and PDGFR  b  that is constitutively activated by PDGF-BB in an autocrine loop (29). Thesecells, when confluent in culture, respond to PDGF-BB with a two-phase calcium transient consisting of intracel-lular release and store depletion-dependent calcium entry from the extracellular space (15,16). However, even inconfluent cultures, some cells show short spikes of intra-cellular calcium release or no change of calcium concen-tration after PDGF-BB stimulation. These cells appear tobe more separated from their neighbors and are possibly in or entering M phase (15). In the present study, weexamined whether cell cycle actually influences the typeof calcium response exhibited by these cells, thereby caus-ing G2/M cells to produce short calcium spikes or nopeaks at all. Further, in light of the observations thatPDGFR phosphorylation may be regulated by cell conflu-ence, we investigated whether the lack of confluence andconsequential cell–cell communication could be a causeof altered calcium response in cells without neighbors.Our results suggest that in A172 glioblastoma cells thecomplete two-phase calcium signal can occur indepen-dently of the G0/G1 or G2/M cell cycle stages of the cells.However, the confluence of cell culture seems to be anecessary condition for this complete response becausecells in sparse cultures exhibit mostly short spikes with lower peaks or no transients at all. MATERIALS AND METHODSMaterials PDGF-BB homodimer was graciously provided by Dr. Glenn Pierce (AmGen Inc., Thousand Oaks, CA, USA)and used at a final concentration of 20 ng/ml, a doseknown to cause two-phase calcium responses in confluentcell cultures (15,16). Indo-1 free acid and Indo-1-AM werefrom Molecular Probes (Eugene, OR, now Invitrogen,Carlsbad, CA, USA), ionomycin from Calbiochem (SanDiego, CA, USA), and all other reagents, unless otherwiseindicated, were from Sigma-Aldrich (St. Louis, MO, USA). Cell Culture  A172 human glioblastoma cells (30) (American TypeCulture Collection, Rockville, MD, USA; catalog number CRL-1620) were cultured in a humidified atmosphere with 5% CO 2  at 37  C in Dulbecco’s Minimal Essential Medium(Sigma) supplemented with 10% fetal calf serum (Sigma)and without antibiotics. Cells were propagated every 3 to4 days. For experiments, cells were seeded at low density (20,000/cm 2  ) onto coverglass chambers (Nunc, Naper- ville, IL, USA) or onto coverslip slices cut with a diamondpencil and used at a confluency of 70% to 80% (usually after 5 days) or less than 20% (after 2 days). Before stimula-tion experiments, the cultures were starved in serum-freeDulbecco’s Minimal Essential Medium for 8 h. Loading of Cells With Indo-1 Calcium Indicator  Starved cells were incubated with 2  l M Indo-1-AMadded from a 1-mM stock solution of dimethylsulfoxidefor 50 min at 37  C. Cells were then washed three times with warm HEPES buffer (HB; containing 20 mM HEPES,123 mM NaCl, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM CaCl 2 ,5 mM Na-pyruvate, pH 7.2) and incubated for another 173 PDGF-EVOKED CALCIUM RESPONSE IS A FUNCTION OF CELL CONFLUENCE  30 min to allow for complete hydrolysis of the dye, which  was confirmed by complete quenching of the dye fluores-cence by Mn 2 1 in the presence of ionomycin. For measur-ing cells in suspension, Indo-1–loaded cells were trypsi-nized and washed twice with HB using centrifugationat 700  g . Spectrofluorometric Measurement of Intracellular Free Calcium Concentration  Coverslip slices were fitted at a 30   angle to the excita-tion beam in 10  3  10-mm polystirol cuvettes and mea-sured in a Perkin-Elmer MPF 44-B spectrofluorometer. Alternatively, cells in suspension at a concentration of 10 5  /cm 3 (in 2.5 cm 3  ) were placed into the cuvettes. Excita-tion was at 360  6  8 nm, emission was measured using acustom-built T-setup at 405 6 8 nm through a monochro-mator and at 485 6 20 nm through a bandpass filter. Theratio of emission intensities was saved with a time resolu-tion of 0.5 Hz. PDGF-BB was added at 20 ng/ml final con-centration and gently mixed into the cuvette by pipettinga few cycles. Image Cytometric Measurement of Intracellular Free Calcium Concentration  Intracellular Indo-1 fluorescence was digitally imagedusing an ACAS 570 stage-scanning laser fluorescence cyto-meter (Meridian Instruments Inc., Okemos, MI, USA) in ahumidified thermostated atmosphere. Excitation using the350- to 360-nm band of an Ar-ion laser was done through a380-nm dichroic mirror and an Olympus DAPO 40 3  /1.3numerical aperture oil-immersion objective. Emission wassplit with a 450-nm dichroic mirror and detected usingtwo photomultiplier tubes behind 405 6 20 and 485 6 20nm bandpass filters. The ratio of background subtractedemissions was used to calculate free calcium concentra-tion.Using two-dimensional scans with 0.5- l m pixel resolu-tion, fluorescence images of both emission wavelengths in240- 3 220- l m areas were taken at a frequency of 0.1 Hz(Fig. 2a). The emission ratio was converted into absolutecalcium concentration using a calibration curve andplotted as free Ca 2 1 concentration versus time after inte-grating inside each region of interest (ROI) correspondingto a cell of interest (Figs. 2a and 2b). PDGF-BB was addedat 20 ng/ml final concentration in 0.25 ml volume to allow for quick mixing with the buffer already over the cells. Calibration of Calcium Measurements To be able to convert Indo-1 fluorescence ratios intoabsolute free calcium concentrations (31), 5  l M Indo-1(pentapotassium salt) was added to buffers simulating theintracellular environment in terms of ion concentration,pH, and viscosity/polarity and containing various concen-trations of free calcium. The buffer composition was10 mM 2-(N-morpholino)ethane sulfonic acid, 115 mMKCl, 20 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA (ethylene gly-col bis [beta-aminoethylether]-N,N 0 -tetraacetic acid), and40% glycerol, pH 7.4. Calcium concentration spanning a10- to 2,000-nM range was set using 0.1 to 1.5 mM CaCl 2 .The solutions were imaged with instrument settings iden-tical to cell imaging and averaged, background-subtractedemission ratios were matched to free calcium concentra-tions calculated using a K  d  of 105 nM for Ca 2 1 and EGTA in the buffer used. Bromodeoxyuridine Incorporation andMeasurement of Cellular DNA Content  Before calcium measurement, cells were incubated with 10  l M 5-bromo-2 0 -deoxyuridine (BrdU) for 1 h at 37  C. After having measured the calcium response to PDGF,cells were washed twice in HB, once in phosphate buf-fered saline (PBS), and fixed in 70% ethanol (in PBS) at4  C for 20 min, followed by a 1-h incubation in denaturingsolution (2.5 M HCl/0.5% Triton X-100), two washes inPBS/0.1% Triton X-100, blocking with 1.5% nonfat dry milk in PBS/0.1% Triton X-100, and two washes again.Monoclonal mouse anti-BrdU at 20  l g/ml in PBS was thenused overnight at 4  C, followed by washes (PBS/0.1% Tri-ton X-100), incubation with 10  l g/ml FITC-anti-mousegoat immunoglobulin G for 2 h at 25  C, two washes, incu-bation with 1  l g/ml propidium iodide (PI) for 10 min, two washes, and mounting in Mowiol (0.1 M Tris-HCl, pH 8.5,25 w/v% glycerol, and 10% Mowiol 4-88, Hoechst Pharma-ceuticals, Frankfurt, Germany) (32). Samples were imaged with an ACAS 570 cytometer using 488-nm laser excita-tion, 490-nm dichroic mirror, and the standard FITC andPI filter sets in front of the two emission photomultiplier tubes. Image Analysis Calcium responses of individual cells were plotted as afunction of the average free calcium concentration withincell boundaries against time. For each cell, peak and dura-tion of the calcium signal was recorded, as well as its posi-tion on the coverslip chamber. After labeling for BrdU andPI, the coverslip chamber was relocated to the same posi-tion and images of anti-BrdU indirect fluorescence and PIfluorescence were recorded for the very same cells. BrdUand PI signals were integrated within each cell boundary and correlated with the peak and duration of calciumresponse. Statistical Analysis Because peak and duration values of calcium responsedid not follow a normal distribution, comparison of these values in cell subsets was done using the Mann-Whitney rank sum test or, when more than two categories wereestablished, the Kruskal-Wallis one-way analysis of var-iance on ranks, followed by Dunn’s pairwise multiplecomparison procedure adjusted for ties. Correlation of thepeak and duration of calcium response with each other and DNA or BrdU content was tested using Spearman’srank correlation test. Correlation of peak and duration with confluence and cell cycle data (BrdU positivity,phase) was checked using binary or multinomial logisticregression procedures. To test whether the different out-comes in any given classification category (DNA, BrdU,174  VEREB ET AL.  confluence) were randomly distributed over the outcomesof the other categories, chi-square test was used. RESULTS AND DISCUSSIONSuspending Otherwise Adherent Cells ShortensCalcium Response to PDGF To test the effect of cell–cell communication on largepopulations, a trivial approach is to use spectrofluorome-try for measuring free cytoplasmic calcium concentration.Figure 1 shows typical traces of fluorescence ratios (F485/ F405). Cell populations adhering to the coverslip (solidline) responded after a lag to PDGF-BB stimulus by a riseof ratio (corresponding to a rise of free Ca 2 1 concentra-tion) (31), followed by a slow fall to a concentration main-tained above resting levels for at least 700 s. EGTA addedat the end of the measurement caused a drop in ratio tobelow resting levels, indicating that membrane Ca 2 1 chan-nels are still open to maintain free calcium concentrationabove resting levels. Restoring extracellular Ca 2 1 caused theelevation of intracellular concentration again. In the case of cells from a matching culture, but trypsinized and sus-pended (dotted line), the lag phase appeared to be longer,and the rate of rise slower, which possibly refers to the lack of synchronization caused by the loss of cell–cell contacts. Also, after reaching a peak, the fall in concentration is faster than in the case of adherent cells, and resting level isreached by 300 s. Probing with EGTA and re-addition of cal-cium revealed that membrane calcium channels wereclosed at this last stage. Although these observations sup-port the notion that cell–cell contact provided for by cellconfluence is necessary to achieve a complete, long, two-phase calcium response in these cells, one needs to con-sider the possibility that it is the adherence of cells to sub-strate, rather than their contacts with each other, that isneeded for the complete response (33). Furthermore,although spectrofluorometric measurements provide dataon large cell populations, they do not permit the investiga-tion of individual adherent cells and correlating their response to stimuli with other properties, such as their position in the cell cycle. Digital Image Cytometry Allows for Correlating theDynamics of Individual Calcium Responses with other, More Static Cellular Parameters To correlate the type of calcium response with cellconfluence and cell cycle, we spiked cells with BrdUbefore measuring intracellular calcium concentration inconfluent and sparse cultures. Indo-1 fluorescence wasthen imaged (Fig. 2a) at 485-nm (detector 1 data) and405-nm (detector 2 data) emission wavelengths to obtainimage pairs at a rate of 0.1 Hz. Image sequences werepostprocessed by converting ratio images to free cal-cium concentration maps, marking cell boundaries for individual cells, and averaging the calcium concentrationinside the boundaries for each time point. As apparentin Figure 2b, each cell produced a characteristicresponse to PDGF stimulus, which could be character-ized by its peak value and duration. Cells were then pro-cessed to label the incorporated BrdU with indirectimmunofluorescence and nuclear DNA with PI. Cover-slips were removed from the chamber and mounted onmicroscopic slides. The area imaged for calcium concen-tration was relocated in the cytometer and PI and FITCimages were taken. In Figure 2c, these two fluorescence values are displayed on a pseudocolor scale in one com-mon image. The ROIs for each cell of interest wereretrieved and FITC and PI fluorescences above threshold were integrated within each ROI. On rare occasions,such as cell 5 in the example, cells were lost during thepreparation process and had to be omitted from the ana-lysis. Peak value and duration of calcium response for each cell were then analyzed as a function of DNA con-tent (PI fluorescence), BrdU positivity, and cell cultureconfluence. Quality of Calcium Transients is Independent of G0/G1 and G2/M Phases Figure 3a shows the plot of DNA content (PI fluores-cence) versus BrdU immunofluorescence signal for allcells investigated. The threshold for BrdU positivity is at230 (dotted line), clearly separating G1/G0 cells (BrdUsignal < 200) on the left of the line from the others thatexhibit BrdU signal above 250. This latter population isdivided into three categories; early S with PI fluores-cence centered around 260, late S with PI fluorescencecentered around 520, and mid-S phase for DNA contentin between. The frequency distribution histogram in Fig-ure 3b confirms the peak values of DNA content charac-teristic of G1 and G2 to be 260 and 525, respectively.These values were used to normalize DNA contentshown in Figures 3c and 3d. The one cell with high DNA content and no BrdU incorporation is in G2/M thatgot to that point before BrdU pulsing. For analysis pur-poses, cells with 4n DNA content, whether BrdU posi-tive or not, were considered G2/M because immediately after measuring calcium response their PI fluorescenceis maximal, whereas BrdU pulsing happened 1.5 to 2 h before that measurement. Cells that are BrdU positivebut still below 1.1 times the diploid DNA content are F IG . 1. Calcium response of A172 cells in spectrofluorometry. Cellsadherent on coverslip (solid line) or in suspension (dotted line) were sti-mulated with 20 ng/ml PDGF-BB where indicated (arrow). Ratio of 485-to 405-nm fluorescence emission, proportional to free intracellular cal-cium concentration, was measured and plotted over time. EGTA and Ca 2 1  were added at 3 mM final concentration as indicated (arrows). 175 PDGF-EVOKED CALCIUM RESPONSE IS A FUNCTION OF CELL CONFLUENCE  F IG . 3. Correlation of cell cycle, and culture confluence with calciumresponse parameters. Triangles represent cells from confluent cultures, whereas circles represent cells from sparse cultures throughout.  a:  PIfluorescence versus BrdU content. The dotted line marks separation of BrdU-negative G1/G0 and G2/M cells from BrdU-positive early S, S phaseand late S/G2/M cells.  b:  Frequency distribution histogram of DNA con-tent in the overall population. The peaks for 2n and 4n DNA content formthe basis of normalizing DNA in c and d.  c, d:  Duration of response andpeak of response plotted against normalized DNA content for each cell.Open symbols represent BrdU-negative cells, and closed symbols repre-sent BrdU-positive cells. Peak values at or below 0.2  l M mean that freecalcium concentration has remained at the resting level.F IG . 2. Calcium response, DNA content, and BrdU positivity correlatedon a cell-by-cell basis.  a:  Fluorescence map of confluent cell cultureobtained with the ACAS 570 laser scanning cytometer. Detector 1 data are485  6  20 nm and detector 2 data are 405  6  20 nm emission intensitieson the pseudocolor scale indicated in the image. Field of view is 240  3 220  l m at 0.5  l m/pixel resolution.  b:  Intracellular calcium concentrationaveraged over individual cells (numbered 1 through 6 in a) is plottedagainst time. Resolution is 0.1 Hz.  c:  Dual pseudocolor map of the samecells as in  a  . PI fluorescence intensity is plotted on a black-to-red, FITCfluorescence from indirect immunolabeling of incorporated BrdU on ablack-to-green scale. Dual positive cells are yellow. 176  VEREB ET AL.
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks