[CANCER RESEARCH 41. 306-309. January 1981] 0008-5472/81 /0041-0000$02.00 Responses of Synchronized HeLa Cells to the Tumor-promoting Phorbol Ester 12-O-Tetradecanoylphorbol-13-acetate as Evaluated by Flow Cytometry Volker Kinzel, James Richards, and Michael Stohr Institute ol Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, ABSTRACT A flow cytometric study was carried out on the effects of tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA; 10~8 M) on HeLa cells synchronized by amethopterin for DNA synthesis. Cells treated with TPA at the time of release from the amethopterin block showed a delayed passage through S phase and partially through G? in their immediate life span as measured 24 hr after release. This late G2 delay was not observed when TPA was added to cells during late S or G2 phase. In this case, however, a direct inhibition of cells in G2 became evident as observed about 15 hr after release from block. None of these effects was caused by the nonpromoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10~6 M). These data support observations obtained with asynchronous cultures. The TPA effects resemble those reported after Xirradiation of cell cultures. INTRODUCTION Earlier studies with HeLa cells as a model system for the action of a variety of phorbol esters have shown that these compounds elicit specific responses. Changes in the phospholipid metabolism (2) as well as in the thymidine incorporation (6) were well correlated with the capacity of the chemicals to cause inflammation and promotion in the mouse skin system. In a previous study (3), indications for a variety of transient changes in the HeLa cell cycle caused by small doses of TPA1 Germany Laboratories, Munich, Germany) were obtained from the quoted sources. Cloned HeLa cells were cultivated routinely in Eagle's mini mum essential medium containing Earle's salts supplemented with 10% calf serum as described elsewhere (3). For experi ments, cells were transferred to plastic Petri dishes (Falcon) 2 or 3 days in advance. For synchronization, cells were treated with 1CT6 M amethopterin and 5 x 10~5 M adenosine for 16 hr in complete medium if not otherwise stated (5). The block was released by addition of thymidine (10 /¿g/106cells). At the time points indicated, the medium was replaced by fresh medium containing acetone or DMSO (0.05% final concentration) with or without phorbol ester and with thymidine as above. For cell counting (Coulter counter) and FCM analysis, cells were re moved with trypsin (0.06% in phosphate-buffered saline con taining, per liter, 8g NaCI, 0.02 g KCI, 1.15 g Na2HPO4-2 H2O, and 0.2 g KH2PO4) at 37°. For FCM measurement, the sus pended cells were washed with 0.1 M Tris-CI (pH 7.5) contain ing 0.1 M NaCI, fixed with 70% ethanol, and collected at this stage at 4°. Prior to the measurements, the above buffer containing DAPI (3 /xg/ml) was added to the cells after removal of the ethanol for quantitative fluorescent staining of cellular DNA (1). The suspensions were carefully checked for their single-cell status. The FCM analysis was carried out with a computerized cell sorter FACS II (Becton-Dickinson Co., Moun- were observed. These data obtained with asynchronous cul tures pointed to differential sensitivities and pharmacokinetics dependent on the cycle staging. In order to clarify further the complex situation, experiments with synchronized cultures were carried out, thus increasing target cell compartments for the action of TPA in particular stages of the cell cycle. Analysis was by high-speed FCM. The data support results and inter pretation of experiments with asynchronous HeLa cells (3). MATERIALS AND METHODS The phorbol esters TPA and 4-O-Me-TPA were a generous gift from Prof. Dr. E. Hecker (Institute of Biochemistry, German Cancer Research Center, Heidelberg, Germany). Acetone, DMSO (Merck, Darmstadt, Germany), thymidine, adenosine, DAPI (Serva, Heidelberg, Germany), and amethopterin (Lederle 1 The abbreviations used are: TPA, 12-O-tetradecanoylphorboM 3-acetate; FCM. flow cytometry: 4-O-Me-TPA, 4-O-methyl-12-O-tetradecanoylphorbol-13acetate; DMSO, dimethyl sulfoxide; DAPI, 4',6-diamidino-2-phenylindol. Received February 22, 1980; accepted October 6, 1980. 306 Chart 1. DNA histograms of logarithmically growing HeLa cultures 04), of cultures treated for 16 hr with amethopterin (B). and of cultures 6.5 hr (C) and 24 hr (D) after release by thymidine; abscissa, relative fluorescence intensity equiv alent to DNA content; ordinate, relative cell number. HeLa cells were kept in complete medium containing 10% calf serum. CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1981 American Association for Cancer Research. Effects of TPA on Synchronized HeLa Cells AFTER RELEASE 8hr 10 hr 24 hr TPA 1CT8[M] 4-0-ME-TPA 10'6[M] -16hrAMETHOPTERIN0Tdr 10A hrA A24 RELEASE COMPOUND8 x 106/5-cm dish) by fresh medium containing thymidine (Tdr) and a phorbol Chart 2. Progression of HeLa cells through the cell cycle in the presence of 1CT8 M TPA or 10"" M 4-O-Me-TPA as visualized by DMA histograms. HeLa cells ester (solvent. 0.05% DMSO). Cells were prepared for and analyzed by FCM measurements; abscissa, relative fluorescence intensity equivalent to DNA con were grown for 2 days in the presence of 10% calf serum before addition of tent; ordinate, relative cell number. amethopterin. The medium was replaced 16 hr later (cell number at this time, 1.2 AFTER RELEASE 24 hr 10 hr 8hr TPA 4-0-ME-TPA KT6[M] -16hr r AMETHOPTERIN -r -t 6.5 8 « A 24 hr A Tdr COMPOUND RELEASE Chart 3. Progression of HeLa cells through the cell cycle with 10 8 M TPA or 10~6 M 4-O-Me-TPA added with fresh medium 6.5 hr after the amethopterin block had been released by direct addition of thymidine (Tdr). For further details, see Chart 2. tain View, Calif.) which was equipped with a UV laser beam. For fluorescent excitation of DAPI, the laser was tuned to 363 nm at 500 milliwatts. The fluorescent pulses were detected above 390 nm. In order to check the G2-M fraction during the measurements for cell doublets, probes were sorted directly on glass slides and controlled by fluorescence microscopy. JANUARY 1981 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1981 American Association for Cancer Research. 307 V. Kinzel et al. 2t HR 15 HR -16 HR ANETHOPTERIN 15 HR 6 HR 0 HR RELEASE TPA V 2t HR nTPA 8 HR 00vvu TU D TPA 10 HR TDa Chart 4. Progression of HeLa cells through the cell cycle in the presence of 1CT8 M TPA and/or 0.05% acetone added with a medium change 6, 8, or 10 hr after release from the amethopterin block. HeLa cells were grown for 2 days in the presence of 0.5% calf serum prior to starting synchronization (cell number at this time, 1.4 x 106 cells/5-cm dish). The block was released by directly adding thymidine. For further details, see Chart 2. RESULTS AND DISCUSSION A preceding study (3) with asynchronous HeLa cells indi cated differential sensitivities of HeLa cells to TPA depending on the cell cycle staging. It seemed to be important in which phase the cells were first exposed to the phorbol ester. In order to amplify the number of cells, particularly in S phase and the subsequent phases, synchronization at very early S phase by amethopterin (5) was chosen. TPA, 4-O-Me-TPA, or the solvent were added in fresh medium at time of release of the block or later as indicated. At certain intervals, a set of cultures was checked microscopically, counted, and prepared for FCM anal ysis. The coincidence of results obtained with different methods had indicated already (3) that TPA itself does not interfere with 308 the binding of the fluorochrome to DMA. The synchronization procedure itself is documented by DMA histograms in Chart 1. Cultures in logarithmic growth (A) have accumulated mainly in early S phase 16 hr after addition of amethopterin (ß).After release from the block by thymidine, cells have moved to late S phase after 6.5 hr (C) and showed 24 hr after release the distribution of an almost random culture (D). Chart 2 shows results of the addition of TPA (10"8 M) or 4O-Me-TPA (10~6 M) in fresh medium together with thymidine for release of the amethopterin block. It can be noted that TPA does not interfere with the release of the block by thymidine. This result is in agreement with the first experiment ever done and published on the action of TPA in cell cultures (4). There fore, the Gìblock seen in asynchronous HeLa cultures (3) occurs earlier in the cycle. The DNA histograms taken 8 and 10 hr after release show, however, a delayed traverse of the synchronized fraction through S phase only for TPA, whereas cultures with DMSO (data not shown) or 4-O-Me-TPA accu mulate DNA faster (compare, e.g., the small valley between the G, fraction and the right DNA peak of 8-hr TPA with the broader one of 8-hr 4-O-Me-TPA, or compare the shape of the right DNA peak of 10-hr TPA with 10-hr 4-O-Me-TPA). These data support the observation obtained from asynchronous cultures that, under the influence of TPA, cells accumulate DNA or traverse through S phase more slowly than those in control cultures in the presence of DMSO or 4-O-Me-TPA. The TPA group shows in addition a rather large G2-M peak 24 hr after release from the amethopterin block. The microscopic exami nation of these cultures revealed only a relatively small number of cells in mitosis at this time in the TPA group, indicating that the peak represents mainly G2 cells. The addition of TPA 6.5 hr after the cells have been released from the amethopterin block (Chart 3) also causes a delayed traverse of the cells through the rest of S phase as evident at 10 hr (compare, e.g., the broad base of the right DNA peak of 10-hr TPA with the small base of the corresponding peak of 10-hr 4-O-Me-TPA). The decreased mitotic activity, i.e., the immediate G2 block in the presence of TPA, became evident 11 to 12 hr after release from the amethopterin block. In the DMSO and 4-O-Me-TPA groups, more cells had divided than in the TPA group and appeared therefore in the Gìfraction. Unlike the preceding experiment (Chart 2), the 24-hr TPA group does not show a pronounced G2-M peak. This supports the data obtained with asynchronous cultures. Cells first exposed to TPA in the early part of S phase exhibit an accumulation in G2 in their immediate life span. This must probably be interpreted as a delayed traverse through G2 of a part of these cells. This TPA effect is different from the immediate G2 block seen in asynchronous or here in synchronous cells. For further analysis of these effects, experiments were car ried out in medium containing 0.5% calf serum. Under these conditions, cells pass more slowly through S phase after re lease from the amethopterin block. TPA (10~8 M) together with fresh medium was added either 6, 8, or 10 hr after release by thymidine (Chart 4). The 24-hr values reveal a steady decrease of the G2 fraction the later TPA was added, thus confirming the above interpretation. The immediate TPA effect on G2, how ever, is more pronounced the later TPA was added (compare, e.g., 15-hr acetone values with 15-hr TPA values), in other words, when most cells were in the sensitive phase at the addition of TPA. Although no pulse treatment with TPA was CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1981 American Association for Cancer Research. VOL. 41 Effects of TPA on Synchronized possible as discussed earlier (3), these data seem to indicate that the TPA addition as such mimics a pulse treatment even though the material was not removed afterwards. The obser vations described here are similar to results obtained after treatment of synchronous cultures with X-irradiation or antitumor drugs as discussed in detail elsewhere (3). ACKNOWLEDGMENTS We thank Dr. J. Reed for critically reading the manuscript. REFERENCES 1. Brunk, C. F.. Jones, K. C., and James. T. W. Assay for nanogram quantities of ONA in cellular homogenates. Anal. Biochem., 92. 497-500, 1979. JANUARY HeLa Cells 2. Kinzel, V., Kreibich, G., Hecker, E., and Suss, R. Stimulation of choline incorporation in cell cultures by phorbol derivatives and its correlation with their irritant and tumor-promoting activity. Cancer Res., 39. 2743-2750, 1979. 3. Kinzel, V.. Richards, J., and Stöhr,M. Early effects of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate on the cell cycle tra verse of asynchronous HeLa cells. Cancer Res., 41: 300-305, 1981. 4 Mueller, G. C., and Kajiwara, K. Regulatory steps in the replication of mammalian nuclei. In: Developmental and Metabolic Control Mechanisms and Neoplasia. The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, pp. 452-474. Baltimore: The Williams & Wilkins Co., 1965. 5 Mueller, G. C., and Kajiwara. K. Synchronization of cells for DNA synthesis. In: K. Habel and N. P. Salzman (eds.). Fundamental Techniques in Virology, pp. 21-27. New York: Academic Press. Inc., 1969. 6. Suss, R.. Kreibich, G., and Kinzel. V. Phorbol esters as a tool in cell research? Eur. J. Cancer, 8. 299-304, 1972. 1981 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1981 American Association for Cancer Research. 309 Responses of Synchronized HeLa Cells to the Tumor-promoting Phorbol Ester 12- O-Tetradecanoylphorbol-13-acetate as Evaluated by Flow Cytometry Volker Kinzel, James Richards and Michael Stöhr Cancer Res 1981;41:306-309. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/1/306 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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