[CANCER RESEARCH 42. 4999-5003, 0008-5472/82/0042-OOOOS02.00 December 1982] Kinetic Heterogeneity in Density-separated Subpopulations1 William A. Brock,2 Douglas E. Swartzendruber, Murine Fibrosarcoma and David J. Ordina Department of Experimental Radiotherapy, University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 [W. A. B., D. J. GJ, and Department of Biology, University of Colorado at Colorado Springs, Colorado Springs, Colorado 80933 [D. E. SJ ABSTRACT Murine fibrosarcoma cells can be separated into subpopu lations by centrifugation through 10 to 35% Renografin density gradients. Previous work has shown that the heavier cell pop ulations are rich in chronically hypoxic cells. In this study, each subpopulation was characterized for thymidine incorporation, thymidine transport, thymidine triphosphate pool sizes, and thymidine triphosphate specific activities. The heavier cell pop ulations have less accessibility to exogenous thymidine, and they have lower endogenous pools of thymidine triphosphate and synthesize lower levels of DNA than do the lighter cell populations. However, if the cells are removed from the tumors and labeled with [3H]thymidine in vitro, all subpopulations syn thesize DNA at similar rates. Two-parameter flow cytometry using acridine orange staining following partial acid denaturation of chromatin identified a small quiescent population in the most dense cell fraction, but the small number of these cells cannot account for the results of the biochemical studies. It appears that the hypoxic cells in the fibrosarcoma tumors are noncycling or slowly cycling, are in all phases of the cell cycle, and recover their ability to synthesize DNA when cultured under in vitro conditions. INTRODUCTION Cells isolated from a murine FSA3 can be separated into 5 distinct subpopulations by centrifugation through Renografin density gradients. These subpopulations differ in clonogenic ability, as shown by lung colony formation (7), and in their sensitivity to both low- (5) and high-linear energy transfer radiation (6). The radiation response suggests that the moredense cell populations contain a high fraction of hypoxic cells. This conclusion is supported by studies of FSA tumors irradi ated in mice made acutely hypoxic (4) and in mice treated with hypoxic cell sensitizers (8). It has been reported recently that the LI of the relatively lowdensity cells is the same as the percentage of S-phase cells in these fractions, while the LI of the denser cells is only about one-half of that predicted by the percentage of S-phase cells (9). This suggests that the S-phase cells in the more dense populations are stationary, synthesize DNA very slowly, or receive inadequate labeled precursor. All possibilities are con1 This work was supported in part by Research Grants CA-18628, CA-06294, and CA-16672, awarded by the National Cancer Institute. * To whom requests for reprints should be addressed, at Department of Experimental Radiotherapy. M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, Texas 77030. 3 The abbreviations used are: FSA, fibrosarcoma; LI, labeling index; FCM. flow cytometry; AO. acridine orange: dsDNA, double-stranded DNA; ssDNA, singlestranded DNA. Received May 18, 1982: accepted September 8, 1982. DECEMBER 1982 sistent with a cell population that is isolated from the functional vasculature of the tumor. In this study, we examined DNA metabolism in isolated FSA subpopulations in greater detail by measuring in vivo and in vitro [3H]thymidine incorporation and LI, TTP specific activities and pool sizes, [3H]thymidine uptake, and 2-parameter FCM using AO. Our purpose was to characterize further, in terms of tumor biology, the variant DNA metabolism exhibited by the more-dense cell populations. MATERIALS AND METHODS Materials. The [5-metfjy/-3H]thymidine (specific activity, 50 Ci/ mmol), [5-me</7y/-3H]TTP (specific activity, 42 Ci/mmol), and [MC]dCTP (specific activity, 452 mCi/mmol) were obtained from Amersham/ Searle Corp. (Arlington Heights, III.). All nonradioactive deoxyribonucleoside triphosphates (TTP, dCTP, dATP, and dGTP) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Escherichia coli DNA polymerase I and nicked calf thymus DNA were purchased from Bethesda Research Laboratories (Gaithersburg, Md.). Renografin-60 is a product of Squibb and Sons (Princeton, N.J.). AO was purchased from Polysciences, Inc. (Warrington, Pa.). The balanced salt solution con tained 8 g NaCI, 0.4 g KCI. 1.0 g glucose, and 0.35 g NaHCO3 per liter water. Animals. Eight- to 10-week-old C3Hf/Kam mice were used for tumor growth. These mice were syngeneic with the FSA. Tumors. The FSA used in these experiments was induced with methylcholanthrene (11), and, after 5 generations of isotransplants, was stored frozen; thus, experiments, both present and published previously, used a comparable tumor. Experimental tumors were formed by the s.c. injection of 3 x 10s cells into the hind legs of the animals, and the tumors were used after they reached a 12-mm diameter. Single-cell suspensions were prepared by a protocol using trypsin and DNase I published previously (7). Density Gradient Centrifugation. Suspensions containing 5 x 10r tumor cells in 3.0 ml of culture medium were layered over 34-ml 10 to 35% linear gradients of Renografin-60 diluted with Ringer's solution and containing DNase I (200 units/ml) (type I; Sigma Chemical Co.) (7). The gradients were centrifuged at 13,000 x g at 4°for 30 min, and 3 ml were removed from each band of cells by aspiration through a 25-gauge needle. The cells were finally washed twice with a balanced salt solution to remove the Renografin. Thymidine Labeling. Mice bearing 12-mm tumors were given injec tions of 100 fiCi of [mef/7y/-3HJthymidine (specific activity, 53 Ci/mmol; Schwarz/Mann, Spring Valley, N. Y.) and sacrificed 1 hr later. The tumors were removed, and a single-cell suspension was prepared as described above. For in vitro labeling, tumor cells were first suspended in McCoy's modified Medium 5A with 5% calf serum at 5.0 x 107 cells/ml. Next, [3H]thymidine (6 Ci/mmol) was added for a final con centration of 0.1 /iCi/ml, and the mixture was incubated for 15 min in a shaking water bath at 37°. After incubation, the cells were cooled rapidly on ice, and 3-ml aliquots of the suspension Renografin gradients. TTP Pools and Specific Activities. density populations were determined were layered over The TTP levels in the different by the method of Solter and 4999 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. W. A. Brock et al. Handschumacher (10) with some modifications. In each experiment, FSA cells were density banded on 6 density gradients, and the corre sponding fractions were pooled. Between 8 x 107 and 2 x 10e cells from each density fraction were extracted with 2.0 ml of 0.5 M HCIO4 at 4° for 2 hr, followed by a second extraction with 1.0 ml. The combined extracts were neutralized to pH 7.4 with KOH, centrifuged to remove the KCIO4 crystals, and lyophilized. The lyophilized powder was resuspended in 0.5 ml of distilled water and centrifuged to remove undissolved crystals of KCIO4, and samples of up to 0.1 ml were used for each assay. The reaction mixture contained [MC]dCTP adjusted to contain 150 ng dCTP and 20,000 cpm, 300 ng dGTP, 300 ng dATP, and 7 /ig nicked calf thymus DNA in a total volume of 0.2 ml reaction buffer [90 mw Tris-HCI (pH 7.4): 4 mM MgCI2:1.3 mw 2-mercaptoethanol]. The reaction was started by adding 0.1 ml of E. coli DNA polymerase I (3.5 units). Standards contained between 10 and 100 ng "FTP. After 60 min, the samples were cooled on ice, 0.3 ml of cold 33% trichloroacetic acid were added to each tube, and the suspension was transferred to a GF/C filter. The filters were washed with 10 ml of cold 5% trichloroacetic acid and then dried after being washed with 5 ml of 95% cold ethanol. The radioactive content of each filter was then determined by liquid scintillation counting. To determine the specific activity of the intracellular TTP pools, cells were first labeled with high-specific-activity [5-mei/?y/-3H]thymidine and then extracted and assayed as described above. In a separate exper iment, the fraction of total [3H]TTP incorporated into the calf thymus DNA by polymerase I was determined in the presence of several different concentrations of nonradioactive TTP. Therefore, by using double-label scintillation counting, the total amount of TTP can be determined from the MC content, and the fraction of [3H]TTP in that same unknown sample can be determined from the tritium content. FCM. Tumor cells were recovered from Renografin gradients, washed with balanced salt solution, and analyzed by 2-parameter FCM using an Ortho ICP 22 (Westwood, Mass.) interfaced with a ModComp II/25 minicomputer. The same machine settings were used for each group of samples. Two FCM measurements were made on the cellular populations of each gradient layer, and a minimum of 10" cells was analyzed per histogram. One method uses AO to detect differential sensitivity of quiescent and cycling cells to acid denaturation of the DNA (3). Aliquots of 106 cells fixed in 70% ethanol were treated with 2000 units RNase for 1 hr at 37°and then placed on ice. The DNA was partially denatured with HCI, pH 1.5, for 30 sec. Cells were then stained with AO (S^g/ml), which fluoresces green (530-nm maximum) when bound to dsDNA and red (640-nm maximum) when bound to ssDNA. Quiescent cells have been shown to have a greater fraction of their DNA susceptible to acid denaturation, and therefore, they have a relatively increased red fluorescence with a concomitant loss of green. The second method measures RNA:DNA ratios (12). Unfixed cells are stained with AO (10 fig/ml); green fluorescence is proportional to DNA content, and the red fluorescence is indicative to RNA content. The number of cells in any region of the 2-dimension plot is determined by "box-o-gram" analysis, which is a computerized method of counting the number of cells within a given region of the histogram. Autoradiography. Cell suspensions were air dried on protein-coated slides and then fixed with 70% ethanol. The slides were coated with Kodak NTB2 liquid emulsion at 46°, dried, exposed for 2 weeks, and then developed with Kodak D-19 developer. The background was 6 grain counts/cell. The percentage of labeled nuclei was obtained by counting at least 500 cells/slide. DNA Content and Specific Activity Determinations. Total DNA was measured by the diphenylamine reaction according to the method of Burton (I). After being labeled with [3H]thymidine, the radioactivity of the DNA was determined by scintillation counting of an aliquot of the acid-hydrolyzed sample and a diphenylamine determination of another aliquot. Under these conditions, only DNA and RNA are hydrolyzed, thus reducing contamination caused by redistribution of tritium label into other macromolecules. RESULTS Tumor DNA Synthesis. The DNA replication kinetics of FSA tumor cells was studied by measuring [3H]thymidine incorpo ration into DNA, TTP pool sizes, and TTP specific activities. Tumor cells were labeled by i.p. injections of [3H]thymidine, and after 1 hr, the tumors were removed and subjected to density gradient centrifugation. The results from each density band are presented in Table 1. The acid-soluble or unincor porated label was measured as an estimate of [3H]thymidine transport or diffusion into each population. The denser cells contained less label, suggesting that they were less accessible to the functional vasculature. This same conclusion is reached if total label (unincorporated and incorporated) is measured. The acid-insoluble tritium represents incorporation of label into DNA, and here again, the denser cells showed the lowest specific activity. To determine whether the reduced rate of incorporation in the denser cells was due to the inability of label to reach these cells or to a lower rate of DNA synthesis, the specific activity of the TTP pools was measured for each density population using the DNA polymerase assay. As shown in Table 1, the absolute size of the TTP pool was lowest in the denser populations, resulting in a higher tritium specific activity. Therefore, the reduced rate of incorporation in these cell types was not caused by insufficient label. Because of the high TTP specific activity, the rate of incorporation in Bands 3, 4, and 5 is an overestimate of the actual rate of DNA synthesis. After correcting for specific activities of TTP, we determined the rates of DNA synthesis in Bands 3, 4, and 5 relative to Band 2 to be 23, 22, and 8%, respectively. From the data in Table 1, it can be concluded that the denser populations, Bands 3, 4, and 5, are partially isolated from circulating [3H]thymidine and that these cells, on the average, synthesize DNA at reduced rates when compared with Band 2. In Vitro Labeling. FSA tumors were removed and labeled with [3H]thymidine in vitro before separation on density gra dients. The incorporation of [3H]thymidine into acid-insoluble Table 1 In vivo labeling of FSA tumors with [*H}thymidine and TTPpool sizes la bel (dpm/jig (dpm/*ig Cell DNA)1 (g/cu cm)1.061.081.111.141.17Acid-soluble DNA)313 band12345S-phase cells (%)1820182325Density 8.5a150 70 ± 6.6331± 7.5100 ± ±10138 6.094 ± 7.0150± .967±1 7.892± ±3.2Acid-insolublelabel ± 3.5TTP pool (ng TTP/jig DNA)50 8.842 ± 4.030 ± 5.117.6± 0.616.8 ± ± 2.2TTP specific ac tivity (dpm [3HJTTP/ng TTP)12 6.063.9 ± 0.617.2 ± 1.68.1± 1.213 ± ± 1.6 a Mean ±S.E. '' TTP pool sizes and specific activity measurements on Band 1 were performed on an insufficient quantity of material. Therefore, because of the large S.E.. Band 1 has been omitted from the discussion. 5000 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. VOL. 42 Kinetics of Density-separated material was measured, and in addition, samples were taken for autoradiography. Since high levels of [3H]thymidine incor poration take place under these conditions, we assumed that endogenous TIP pools were altered by the added label; there fore, pool size measurements were not made. Table 2 shows the results from these experiments. When FSA tumor cells were labeled in vitro, no significant difference in incorporation was observed for any of the density populations. A slight increase in Bands 3, 4, and 5 may have been due to their lower TTP pools before labeling began. The LI of each band is also presented in Table 2, and here again, no significant differences were observed. These results suggest that the same popula tions that were synthesizing very little or no DNA in vivo are able to synthesize much greater amounts of DNA under in vitro conditions. The LI results, also shown in Table 2, suggest that all density populations incorporate [3H]thymidine in vitro con sistent with the fraction of those cells that are in S phase as determined by FCM analysis. Since the different density populations contain normal cells, it is important to consider their possible contribution to the results. Since the FSA tumor cells contain 1.8 times the DNA of normal cells, the amount of DNA in each fraction contributed by normal cells can be determined by FCM. The normal cells, however, do not cycle appreciably (see below), but they do contribute a small amount of DNA to each fraction. Table 3 shows the percentage of total DNA contributed by normal cells in each fraction and the results of [3H]thymidine incorporation after adjusting the results to eliminate the influence of normal cell DNA. These results when compared with those of Table 1 demonstrate that the differences were small. Since we do not know the TTP pools of normal cells within tumors, it would be meaningless to make this adjustment for all the data. Table 3 is presented to illustrate that normal cells probably do not influence the validity of these observations. FCM. Chart 1 shows the results of 2-parameter FCM deter minations of each density population after RNase treatment and selective acid hydrolysis. After AO staining, ssDNA fluoresces red, while dsDNA fluoresces green. DNA in noncycling, quiescent cultured cells is more sensitive to acid denaturation than that in cycling cells and has increased red fluorescence with a concomitant loss of green fluorescence (3). Chart 1 shows that the fluorescent patterns of all 5 populations were quite similar. The exception was Band 5, which had a greater Table 2 In vitro labeling of FSA tumor cells with [3H¡thymidine label Cell cm)345.06 band Density (g/cu (dpm/ng DNA)4.7 (% of labeled nuclei)25 ±0.4a .08 .11 .14 .17Acid-insoluble 5.7 7.0 6.9 8.4 ±0.4 ±0.4 ±2.0 ±0.3LI 27 24 27 26 Mean ±S.E. Table 3 Results adjusted for DNA contributed by (he normal cells in FSA tumors Cell band1 in normal cells(%)3 2 34 11 9 2 1In 5DNA DECEMBER 1982 vivo incorporation (dpm/ng DNA)323 tumor 372 152 153 93In vitro incorpo ration (dpm/ng tumor DNA)4.8 6.4 7.7 7.0 8.5 ruse Tumor Cells B1 B2 B3 B4 B5 i 88 DNA -~ Chart 1. Two-parameter FCM pattern of unseparated (USO and separated (81 to B5) FSA tumor cells, showing the relative sensitivity of chromatin to acid denaturation. Abscissa, relative amount of ssDNA; ordinate, relative amount of dsDNA after the cells were treated with RNase and limited acid denaturation as described in "Materials and Methods." Dots, measurement on a single cell. Arrow points to the region in which quiescent cells are usually found using this procedure. population of G, tumor cells with increased ssDNA. However, the fraction of noncycling cells in the denser populations can not be accounted for by this small "quiescent" population; we determined from cell counts taken before and after fixation that preferential loss of quiescent cells during preparation did not occur. Chart 2 shows another set of 2-parameter FCM deter minations that quantitate the DNA and RNA content of each cell. Here again, the noncycling population of the denser bands cannot be differentiated from the cycling cells using this tech nique. In conclusion, the noncycling cells of FSA tumors gen erally appear similar to the cycling population and thus far cannot be selectively identified by FCM. The results shown in Chart 2 also show that the normal cells were not cycling appreciably. S-phase normal cells accounted for 2% of the tumor population at most, as determined by "box-o-gram" analysis of Chart 2. It can be presumed, therefore, that DNA synthesis measurements reflect primarily the tumor cell popu lations. DISCUSSION Separation of FSA tumor cells into subpopulations by density centrifugation has proved to be an excellent system for study5001 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. W. A. Brock et al. the possibility that normal cells contributed significantly to the DNA synthetic fraction. It is interesting that all density subpopulations synthesize DNA at equivalent rates and have the same LI in vitro. Under these conditions, cells are well nourished and have an equal availability of labeled precursor. Recovery of the denser (i.e., radiation-resistant, hypoxic) fractions apparently is rapid when the cells are placed under good growth conditions. Two-parameter FCM analysis of cells according to the method of Darzynkiewicz ef al. (3) differentiates between quies cent and cycling cultured cells, because the chromatin from quiescent cells is more sensitive to selective acid denaturation than that from cycling cells. With a possible exception of the most dense population, the results presented here did not demonstrate the presence of a "typical" G0 quiescent popu B1 B3 »B5 Relative DNA Content —¿ Chart 2. Two-parameter FCM patterns of unseparated (USO and separated (81 to 85) FSA cells stained with AO. Abscissa, relative DNA contents; ordinate, relative RNA content of each cell. ing tumor heterogeneity. Tumors can be treated in vivo or in vitro before density separation, and then each subpopulation can be analyzed for clonogenicity, synthesis of macromolecules, cell cycle distribution, radiation and drug sensitivity, or any number of other parameters. After an i.p. injection of [3H]thymidine, the cells of heavier density accumulated label into the soluble fraction at a reduced rate compared to the lighter cells. This is consistent with the notion that these cells are in the hypoxic regions of the tumor, relatively isolated from the circulation. Band 4 cells respond to y and neutron irradiation in a manner similar to hypoxic cells (6). These same cells showed a decreased rate of incorporation of [3H]thymidine into DNA, suggesting that a large fraction of the cells was noncycling or slowly cycling compared to the lighter density cells in Band 2. Because the specific activities of the TTP pools were measured, it is correct to state that the denser populations are synthesizing less DNA. These conclu sions are consistent with the LI measurements of Sigdestad and Ordina (9) that showed a relatively low LI of dense cells 1 hr after [3H]thymidine injection. In addition, FCM measurements showed that tumor cells in all fractions contained the same percentage of S-phase cells, thereby ruling out the possibility that DNA synthesis differences were due to different cell cycle distributions. Finally, the 2-parameter FCM measurements de termined that the normal cell population in each density band accounted for 2% or less of the S-phase cells. This rules out 5002 lation. In fact, it has been estimated that the hypoxic cell fraction in this tumor is 50% (8), and the appearance of these cells has not been differentiated from the well-oxygenated, growing cells by FCM. Another 2-parameter method that mea sures DNA:RNA ratios also failed to identify the radiation-re sistant, hypoxic Band 4 fraction of FSA tumor cells. It is possible that poor growth conditions in the hypoxic regions simply slowed or stopped the tumor cells in their cycle and did not provide conditions that would allow them to be identified as quiescent by FCM analysis. It is important to note, in addition, that these data show that the hypoxic, noncycling cells in this tumor can stop progression in all phases of the cell cycle, not just GL The technique of acid denaturation followed by AO staining is very sensitive to cell concentration, quality of fixation, and rehydration. Small shifts in fluorescence may be artifactual, not representing a real biological difference between 2 subpopu lations. We cannot therefore be sure that the higher density cells do not show a generalized shift in fluorescence that would indicate a noncycling population. In conclusion, the FSA tumor system described here contains a significant hypoxic cell fraction of noncycling or slowly cy cling cells as determined by studies of DNA metabolism. These cells can be isolated as a dense cell fraction on density gra dients of Renografin. FCM measurements do not demonstrate the significant G0 or quiescent cell fraction that has been described in cultured cell systems (3). When placed in culture, the hypoxic cells immediately begin to synthesize DNA at rates similar to the well-oxygenated cells, presumably because of improved nutritional conditions. This method of in vivo versus in vitro labeling may prove useful for identifying the hypoxic cell fraction of other tumors. REFERENCES 1. Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucleic acid. Biochem istry, 62: 315-323, 1956. 2. Cleaver, J. E., and Holford. R. M. Investigation into the incorporation of (3H) thymidine into DNA of L strain cells and the formation of a pool of phosphorylated derivatives during pulse labeling. Biochem. Biophys. Acta, 703. 654-671, 1965. 3. 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Mason, K., and Withers, H. R. Separation of cells from a fibrosarcoma in Renografin density gradients. J. Nati. Cancer Inst., 52. 253-257, 1974. 8. Grdina, D. J., Sigdestad, C. P.. and Jovonovich, J. A. The effect of misonidazole in situ on the radiation response of selected tumor subpopulations. Cancer Clin. Trials, 3. 149-154. 1980. 9. Sigdestad, C. P., and Grdina, 0. J. Density centrifugaron of murine fibro- DECEMBER 1982 Tumor Cells sarcoma cells following in situ labelling with tritiated thymidine. Cell Tissue Kinet., 14: 589-600, 1981. 10 Solter, A. W., and Handschumacher, R. E. A rapid and quantitative deter mination of deoxyribonucleoside triphosphate based on the enzymatic syn thesis of DMA. Biochem. Biophys. Acta, ) 74: 585-590. 1969. 11 Suit, H. 0.. and Suchato, C. Hyperbaric oxygen and radiotherapy of a fibrosarcoma and of a squamous-cell carcinoma of C ,H mice. Radiology, 89. 713-719, 1967. 12. Tráganos, F., Darzynkiewicz, Z., Sharpless, T., and Melamed. M. R. Simul taneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J. Histochem. Cytochem., 25: 46-56, 1977. 5003 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. Kinetic Heterogeneity in Density-separated Murine Fibrosarcoma Subpopulations William A. Brock, Douglas E. Swartzendruber and David J. Grdina Cancer Res 1982;42:4999-5003. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/12/4999 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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