Biochemistry 1981, 20, 6244-6250
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Slapikoff, S., & Berg, P. (1967) Biochemistry 6, 3654-3658. Symons, R. H. (1974) Methods Enzymol. 29, 102-1 15. Topal, M. D., & Fresco, J. R. (1976) Nature (London) 263, 285-289. Topal, M. D., DiGuiseppi, S. R., & Sinha, N. K. (1980) J . Biol. rhem. 255, 11717-11724.
Uematsu, T., & Suhadolnik, R. (1976) J. Chromatogr. 123, 347-354. Ward, D. C., Reich, E., & Stryer, L. (1969) J . Biol. Chem. 244, 1228-1237. Wittenberg, J . , & Kornberg, A. (1953) J . Biol. Chem. 202, 43 1-444.
Subfractionation of Malignant Variants of Metastatic Murine Lymphosarcoma Cells by Countercurrent Distribution in Two-Polymer Aqueous Phases? Karen M. Miner, Harry Walter, and Garth L. Nicolson*
ABSTRACT:The low metastatic murine lymphosarcoma parental cell line RAWl17-P and a high metastatic subline (RAW1 17-H10), selected sequentially 10 times for liver colonization, were grown in suspension and subjected to countercurrent distribution (CCD) in a dextran-poly(ethy1ene glycol) aqueous phase system having an electrostatic potential difference between the phases. Both the RAWl 17-P and the RAWl 17-H10 sublines gave broad distribution curves, with the mean partition coefficient of RAWl 17-H10 cells being higher than the mean partition coefficient of RAWl 17-P cells. RAWl 17-P cells were kept in culture over a 2-month period during which time the mean partition coefficients increased. Concomitant with their in vivo drift in partition coefficients, the RAW 1 17-P cells also displayed increasing heterogeneity as evidenced by the appearance of broader and even multipeaked CCD curves. However, the mean partition coefficients always remained lower than that of the RAWl 17-H10 subline. Cells from different cavities along the extraction train following
CCD of the RAWl 17-P line were placed into tissue culture, allowed to double overnight, and assayed in vivo by injection of 5 X lo3 cells intravenously into BALB/c mice. After 20 days, the RAW117-P cells which had the higher partition coefficients (corresponding more closely to the higher mean partition coefficient of the high metastatic RAW 117-H10 subline) formed significantly more liver tumor colonies than did the RAWl 17-P cells with lower partition coefficients. Analysis of surface proteins on the CCD-subfractionated RAW 117-P cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with ‘251-labeledconcanavalin A indicated that the cell-surface glycoproteins of the more metastatic cell subpopulations had a decrease in an -70000 molecular weight component similar to the highly metastatic subline RAWl 17-H10. These results suggested that malignant cell variants exist in the parental RAWl 17-P population and that they can be separated from the majority of cells of low malignant potential by countercurrent distribution.
T m o r metastasis involves a complex series of sequential steps whereby malignant cells spread from primary to near and distant secondary sites where they arrest, invade, and proliferate to form new tumor foci (Fidler et al., 1978; Poste & Fidler, 1980; Fidler & Nicolson, 1981). This phenomenon appears to be the end result of several highly selective steps in which fewer and fewer tumor cells survive ultimately to form secondary growth (Poste & Fidler, 1980; Fidler & Nicolson, 1981). The concept suggests that the cells capable of metastasis represent a minor subpopulation of cells comprising the primary tumor (Fidler & Kripke, 1977; Kripke et al., 1978; Nicolson et al., 1978) and that these highly malignant tumor cells have unique characteristics important in determining their metastatic properties (Nicolson et al., 1980 Fidler & Nicolson, 1981).
Several animal tumor models have been developed to establish the tumor cell and host properties important in metastasis (Poste & Fidler, 1980; Fidler & Nicolson, 1981; Nicolson et al., 1978, 1980). One such model utilizes a murine lymphosarcoma tumor line called RAWl 17 that is capable of forming solid tumor nodules in liver, lungs, spleen, and lymph nodes of injected mice. We have used the parental lyumphosarcoma line RAW 117-P to select sublines with enhanced potential to colonize liver. After ten sequential selections for liver colonization, subline RAWl 17-H10 was obtained which forms 200-250 times more gross liver tumor nodules compared to line RAW 117-P within approximately 2 weeks after injection intravenously or subcutaneously and displays enhanced malignancy when assayed by time of host death (Brunson & Nicolson, 1978; Reading et al., 1980b; Nicolson et al., 1980). The RAWl 17 sublines with enhanced malignant characteristics have cell-surface alterations that correlate with their biologic properties (Reading et al., 1980b). Utilizing sequential selection procedures based upon lack of cell adherence to immobilized lectins, such as concanavalin A (Con A),’ we have also obtained RAWl 17 sublines that possess modified mal-
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From the Departments of Developmental and Cell Biology (K.M.M. and G.L.N.) and of Physiology and Biophysics (H.W. and G.L.N.), University of California, Irvine, California 9271 7, the Laboratory of Chemical Biology, Veterans Administration Medical Center, Long Beach, California 90822 (H.W.), and the Department of Tumor Biology, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 (G.L.N.). Received April 1, 1981. This investigation was supported by U S . Public Health Service National Cancer Institute Grant ROI-CA-2957 1 (G.L.N.), the Medical Research Service of the Veterans Administration (H.W.), and National Institutes of Health Postdoctoral Fellowship IF32 CA-06697 (K.M.M.). * Correspondence should be addressed to this author at the Department of Tumor Biology, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute.
0006-296018 110420-6244$01.25/0
’
Abbreviations used: Con A, concanavalin A; CCD, countercurrent distribution; DMEM, Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s phosphate-buffered saline; FBS, fetal bovine serum; Hepes, N-(2-hydroxyethyl)piperazine-N’2-ethanesulfonicacid.
0 1981 American Chemical Society
PARTITIONING OF LYMPHOSARCOMA CELLS
ignancies (Reading et al., 1980a). However, in these and other selection procedures, several cycles of selection and tissue culture growth are required to obtain the malignant cell variants. Viable cell subpopulations can be obtained by subfractionation utilizing partitioning in two-polymer aqueous phases. Aqueous solutions of two different polymers [e.g., dextran and poly(ethy1ene glycol)] when mixed above certain concentrations give rise to two-phase systems which, when rendered isotonic, are suitable for the separation and subfractionation of cell populations by partitioning (Albertsson & Baird, 1962; Walter, 1977). By appropriate selection of both polymer and ionic composition and concentration, cell separations can be obtained based primarily on subtle differences in charge-associated or lipid-related membrane surface properties or on biospecific affinity (Walter, 1977). Phosphates are salts which have different affinities for the dextran-rich bottom and the poly(ethy1ene glycol)-rich top phases (Johansson, 1970). This phenomenon gives rise to an electrostatic potential difference between the phases with the top phase positive (Reitherman et al., 1973). When cells are added to such a system at the appropriate polymer concentration (Walter, 1977), they will partition predominantly according to subtle differences in charge-associated membrane surface properties. We have subfractionated RAW 117 cells by partitioning in two-polymer aqueous phases to demonstrate that malignant cell variants exist in the parental RAW117-P population. In addition, we have used a method predicated on cell-surface properties whereby the highly malignant cells can be rapidly separated from the majority of cells of low malignant potential. Materials and Methods Reagents. Dextran T500 (lot no. 5556) was obtained from Pharmacia Fine Chemicals, Piscataway, NJ, poly(ethy1ene glycol) 6000 [recently renamed poly(ethy1ene glycol) “8000”] from Union Carbide, heat-inactivated fetal bovine serum (FBS) from Flow Laboratories, Inglewood, CA, and gentamicin sulfate from Schering Corp., Kenilworth, NJ. Filter units (0.45 pm) were purchased from Nalgene Labware Division, Dulbecco’s modified Eagle’s medium (DMEM) and DulbecCO’S phosphate-buffered saline (DPBS) from Gibco, Grand Island, NY, and electrophoretic reagents from Bio-Rad Laboratories, Richmond, CA, or Eastman Kodak, Rochester, NY. All chemicals used were of reagent grade. Animals. Inbred 6-8-week-old BALB/c mice were obtained from Charles River Breeding Laboratories, Wilmington, MA, and quarantined for at least 3 weeks before use. Animals were maintained on chlorine-free water and normal food. Tumor Cell Lines. Lymphosarcoma parental line RAWl 17 was induced in vitro from spleen cultures of BALB/c mice infected by Abelson leukemia virus (Raschke et al., 1975) and was designated RAWl 17-P. Cells were grown as previously described (Brunson & Nicolson, 1978) in plastic petri dishes using DMEM supplemented with 15% FBS without antibiotics. Methods of sequential in vivo selection for highly malignant liver colonizing variant subline RAW l 17-H10 have been described previously (Brunson & Nicolson, 1978). Preparation of Dextran-Poly(ethy1ene glycol) Aqueous Phase Systems. Two different phase systems were prepared for CCD which take into account charge-associated membrane surface properties. They were selected as described by Walter (1977). Phase system 1 consisted of 5% (w/w) dextran, 4% (w/w) poly(ethy1ene glycol) 6000, 160 mosM sodium phosphate buffer, pH 7.4, 120 mosM NaCl, and 5% FBS; phase system 2 contained the same polymer concentrations but with 135 mosM sodium phosphate buffer, pH 7.4, 144 mosM NaC1,
VOL. 20, NO. 2 1 , 1981
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and 5% FBS. System 1 has a higher electrostatic potential difference between the phases as compared to phase system 2. The phase systems were filtered through a 0.45-pm filter (Nalge) and equilibrated at 4-5 OC in a separatory funnel, and top and bottom phases were separated. Countercurrent Distribution of RA W117 Lymphosarcoma Cells. Lymphosarcoma cells were harvested at a density of (2-3) X lo6 cells/mL, centrifuged at 400g for 15 min at 4 OC, and resuspended in DMEM supplemented with 10% FBS and 12 mM Hepes, pH 7.4. These cells were recentrifuged at 400g for 15 min at 4 “C, the supernatant solution was discarded, and the cells [(5-10) X lo7 cells/mL] were suspended in 4 mL of the top phase of the system to be used in countercurrent distribution. The thin-layer CCD apparatus (Buchler Instruments, Fort Lee, NJ) which was used (Albertsson, 1970) consisted of two circular Plixiglas plates with 120 concentric cavities and a bottom phase capacity of 0.7 mL. The bottom plate is a stator plate, and the top plate is a rotor plate. When only one cell preparation was to be subjected to countercurrent distribution, cavities 0-3 each received 0.5 mL of the bottom phase and 0.9 mL of the “load mix” (Le., cells suspended in the top phase). When the distributions of two cell preparations (e.g., RAW117-P and RAWl 17-H10) were compared, one population was loaded as above, and the other was loaded in an analogous manner but in cavities 60-63. By carrying out 50 or 59 transfers, we were able to run simultaneously countercurrent distributions on two preparations at 4-5 OC in the identical phase systems on opposite sides of the plate without overlap. All other cavities (Le., those not already loaded with the “load mix”) received 0.6 mL of bottom phase and 0.8 mL of top phase to assure a stationary interface [see Albertsson & Baird (1962) for a full discussion]. The automatic cycle consisted of shaking for 25 s and settling for 6 min followed by a transfer. After the preset number of transfers (50 or 59) was completed, cells were collected directly into sterile plastic centrifuge tubes and kept at 4-5 OC. Sterile, isotonic sodium chloride solution (0.7 mL) was added to each tube, and adjacent tubes were pooled into groups of four tubes. The pooled cell suspensions were diluted to 45 mL with Hanks’ solution containing 2% FBS. They were then centrifuged at 400g for 15 min, the supernatant solutions discarded, and the cells resuspended in 0.6 mL of DMEM supplemented with 10% FBS and 12 mM Hepes, pH 7.4. Tumor cell viability ranged from 60 to 96% as determined by the trypan blue dye exclusion test. The cells were washed again with DMEM plus 10% FBS and gentamicin sulfate (50 pg/mL) and incubated for 12 h at 37 OC. Since the pooled CCD fractions at the ends of the distribution contained fewer cells compared to the other pooled fractions, they required a 36-h incubation to obtain enough cells for injection into animals. In one of the experiments, cavities 8-19 and cavities 28-43 were pooled separately and cultured overnight. They were then subjected separately but simultaneously to a second CCD in a phase system having the same composition as that used in the original fractionation. This is a standard method to test whether cells from the left and right ends of a distribution are truly different (i.e., have different partition coefficients) or are merely distributed on a random basis (Walter et al., 1981). Electronic Cell Counting. Aliquots of the cell suspension obtained from different parts of the CCD extraction train as well as an aliquot of the original load mix were electronically counted with an Electrozone Celloscope (Particle Data, Chicago, IL) fitted with a 120-pm orifice tube and operating on the Coulter principle.
6246
BIOC HE M ISTR Y
In Vivo Assays. RAWl 17 cells were assayed for organ colonization (experimental metastasis) after intravenous injection of 5000 viable cells into at least ten animals per group (Brunson & Nicolson, 1978). After 14-23 days, organs were removed, and the number of tumor colonies was determined visually. Liver colonization was confirmed by staining with hematoxylin (Reading et al., 1980a). Analysis of Cell-Surface Proteins. Unlabeled cellular glycoproteins were identified by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis using '*'I-labeled Con A (Maizel, 1971; Burridge, 1976). Briefly, RAWl 17 cells were grown to a density of approximately 2 X loe cells/mL. A total of (2-4) X lo7 cells were harvested by centrifugation and washed 1 time with desalting buffer (10 mM Tris-HC1, pH 7.2, plus 0.25 M sucrose, 0.05 mM CaC12, and 10 p M phenylmethanesulfonyl fluoride). Cells pooled from CCD experiments were cultured for 36-60 h to obtain the required number of cells for electrophoresis. Cells were disrupted by 0.5% NP-40 in desalting buffer and centrifuged at 4 OC to remove nuclei. The NP-40 supernatant solutions were then completely solubilized in 2% sodium dodecyl sulfate containing 1% 2-mercaptoethanol. Aliquots containing 100 pg of protein were applied to 7.5% polyacrylamide slab gels containing sodium dodecyl sulfate and electrophoresed. After the gels were fixed, stained for protein with Coomassie brilliant blue R-250, and destained, they were brought to pH 7.4 with DPBS. The cells were then labeled with 0.3 mg of 1251-labeled Con A at a specific activity of approximately 3 X lo6cpm/mg in the presence of 2 mg/mL human hemoglobin for 2 h (Burridge, 1976). Unbound Con A was removed after extensive washing with DPBS, and autoradiograms were prepared from the dried gels. Results Countercurrent Distribution Patterns of RA W l l 7 Lymphosarcoma Cells. The CCD distribution curves of RAWl17-P (Figure 1A) and RAWl17-HlO (Figure 1B) indicated that there were cell-surface differences between these cell lines. It is clear that the mean partition coefficient of the RAWl 17-H10 subline is greater than the mean partition coefficient of the RAWl 17-P line, although there is appreciable overlap of the two distribution curves. Since our studies were undertaken in dextran-poly(ethy1ene glycol) aqueous phase systems having an electrostatic potential difference between the phases, the observed partition behavior was, presumably, surface charge associated (Walter, 1977). To examine whether cells from line RAWl 17-P with low or high partition coefficients under the distribution curve are separated on the basis of true differences in their partition coefficients, we utilized a simple and standard test. RAWl 17-P cells were subjected to CCD (Figure 2A), and cells from the left and right ends of the distribution were p l e d separately. These two subpopulations (after overnight culture) were then subjected to a second countercurrent distribution in a phase system of the same composition (Figure 2B). Since those cells obtained from the left part of the original distribution were to the left to those obtained from the right end of that distribution, true heterogeneity of cells detectable by partitioning was clearly demonstrated. This result led to the study of the biological properties of cells under different parts of the CCD distribution curve. During prolonged in vitro culture of RAWl 17-P cells, we find that the CCD distribution gradually changed. Figure 3 represents CCD distribution curves of RAW 1 17-P cells sampled at different times over a 2-month period during which time these cells were kept in culture from 1 (Figure 3A) to
MINER, WALTER, A N D NICOLSON
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FIGURE 1 : Countercurrent distribution patterns of murine lymphosarcoma cells. (A) Distribution of RAWl 17-P (see also Figure 3); (B) typical distribution of RAWl 17-H10. Data are presented as the number of cells found in the different cavities along the extraction train. The phase system contained 5% (w/w) dextran T500,4% (w/w) poly(ethy1ene glycol) 6000, 160 mosM sodium phosphate buffer, pH 7.4, 120 mosM NaC1, and 5% (w/w) FBS (heat inactivated). A total of 50 transfers were carried out at 4-5 "C with a settling time of 6 min and a shaking time of 25 s. For additional details, see the text.
7 weeks (Figure 3D). It appears that during culture the RAW 1 17-P cell population becomes increasingly heterogeneous as evidenced by the broad and multipeaked distribution curves and drifts to a higher mean partition coefficient. However, at no time is the mean partition coefficient as high that of the highly malignant RAWl 17-H10 population. Biologic Assays. The metastatic properties of RAWl 17-P cells subfractionated by CCD were compared after injection intravenously. The cavities were pooled by fours as described under Materials and Methods and given a fraction number; thus, fraction 1 contained cells from cavities 0-3 etc. The relationship between the various CCD fractions was also compared to the metastatic properties of cells from unfractionated lines RAWl 17-P and RAWl 17-Hl0. Cells with a lower partition coefficient (i.e., more to the left in the CCD profile) formed fewer liver tumor colonies compared to cells with a higher partition coefficient (Table I). Of the animals injected with cells from RAWl 17-P CCD fraction 3, only 2/10 animals had liver tumor colonies at day 23. In contrast, for animals injected with cells from RAW117-P CCD fraction 12, 10/10 animals had liver tumor colonies at day 23. A total of seven separate CCD fractionations and in vivo experiments were performed, and in all experiments, the cells with a lower partition coefficient formed fewer tumor colonies than did the cells with a higher partition coefficient. It may be significant that cells at the far right end of the CCD distribution (CCD fractions 13-15) formed fewer liver tumors than did cells from CCD fractions 11 and 12. In the experiments depicted in
VOL. 20, NO. 2 1 , 1981
PARTITIONING OF LYMPHOSARCOMA CELLS
6247
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Countercurrent distribution of RAWl 17-P lymphosarcoma cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells (after overnight culture) to a redistribution in a phase system of the same composition. (A) Distribution pattern of RAWl 17-P cells; (B) superimposed distribution patterns of cells pooled from the left (cavities 8-19, A) and right ends (cavities 28-43 A) of the distribution depicted in (A). The phase system composition was 5% (w/w) dextran, 4% (w/w) poly(ethylene glycol), 135 mosM sodium phosphate buffer, pH 7.4, 144 mosM NaC1, and 5% (w/w) FBS. This phase composition was used so that the cell population which had drifted slightly to the right (see Figure 3) would have a lower partition coefficient (i.e., be "centered" on the graph). Other conditions are as described in Figure 1. See the text for a discussion.
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C A V I T Y NUMBER
FIGURE2:
Table I, cells from pooled fractions 13-1 5 formed tumors in 5 / 10 animals, whereas cells from fraction 12 formed tumors in 10/10 animals. This trend was seen in three other independent in vivo experiments. The metastatic properties of unfractionated cell lines were similar to those reported previously (Brunson & Nicolson, 1978; Reading et al., 1980a,b). The parental line formed few liver tumor colonies by day 23, whereas all the animals injected with the highly malignant RAWl 17-H10 cells had >200 liver tumor colonies and usually died before the end of the assay. Occasionally, animals injected with either RAWl 17-P or RAW117-HI0 cells develop metastases at sites other than the liver (Brunson & Nicolson, 1978). This was also observed with animals injected with cells which had been subfractionated by CCD (Table I). Analysis of Cell-Surface Proteins. Analysis of cellular glycoproteins after sodium dodecyl sulfate slab gel electrophoresis and labeling with 1251-labeledCon A indicated differences among the various RAWl 17-P CCD fractions. In these experiments, RAW 117-P cells were fractionated by CCD, pooled by fours, and cultured prior to cell-surface analysis as described under Materials and Methods. As reported previously (Brunson & Nicolson, 1978; Reading et al., 1980a,b), RAWl 17-H10 cells which have enhanced liver colonization and metastatic potentials display drastic reductions of a cell-surface component of -70000 molecular weight compared to the parental cell line (Figure 4). Cells from line RAWl 17-P which have been subfractionated by CCD have
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FIGURE 3: Countercurrent distribution patterns obtained when RAWl 17-P cells were sampled at different times during continuous culture. (A) Cells grown from a frozen sample were subjected to countercurrent distribution within a week; (B) cells as described (A) sampled after an additional week; (C) cells as described in (A) sampled 3 Weeks later; (D) cells as described in (A) sampled 6 weeks later. Conditions for countercurrent distribution are as in Figure 1 except that 59 transfers were carried out in (A), (B), and (D).
electrophoretic patterns consistent with their biological properties. As seen in Figure 4, cells in fraction 3 (cells from cavities 8-1 l), which have a lower partition coefficient, form a low number of liver tumor colonies and have an increased
6248 B I o c H E M I ST R Y
M I N E R , WALTER, A N D NICOLSON
Table I: Tumon Formed after Intravenous lniection of RAW117 Lvmohosarcoma Cells with or without Fractionation bv CCD' cells no. of liver Nmor colonies in each animal median ranm - Nmor colonies a1 other sites RAW117-P RAW1 17-P LM
RAWl17-PCCD 3 c RAWl17-PCCD 9 RAWl17-P CCD 11
RAWl17-PCCD 12 RAWl17-PCCD 13-15 RAWI17-H10
0, 0, o,o, 0, so, >200b 0 0, 10, 12, >200,b >200.6 >2OOb o,o, 0,o. 0,0, 0, 0, >200 b >200b 0, 0, 0.0, 0, 0,51, >zoo,&
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0, >200, b >200. b >200, b >200bb >200, b >200, b >200, b >200 34, >200,b >200,b >200 b >200 b >2OO,b>200,b>200,b>200,~>200b O,O, O,O,O, >200,b ,2nn - -_.,b ,2nn - -- -,b )?nnb - -- >200,b >200,b >200,b >200,b >200.b >200,b >200,b >200,b >200,b >200b
0 0
05200 0, >200
lymph nodes, 1 lymph nodes, 2; kidney, 2
0 0
0, >ZOO 0, >200
lymph nodes, I;spleen, 1 ovary, 1; kidney, 1
>200
0, >200
lymph nodes, 3;spleen. 3
>200
34, >200
spleen, 4 ; kidney, 4
0
0, >200
spleen, 2 ; ovary, 2
>zoo
>200
lymph nodes, 4
A total of SO00 viable RAW117 cells were injected intravenously in 0.2 mL of DMEM. All live animals were sacrificed a1 23 days. Animal died before assay date. Post-mortem examination indicated >200 liver tumors. E LM = load mix; CCD 3 = CCD fraction 3. CeUS with a low fraction number are those having a lower partition coefficient. Preparations of ceUs for m vivo a m y are described under Materials and Methods. The number of animals with metastases to site.
amount of the -70000 molecular weight component. whereas cells from fraction 13 (cells from cavities 48-51), which have a higher partition coefficient, form more liver tumor colonies and have a decrease in the -70000 molecular weight component. The glycoprotein components of cells fractioned by CCD were analyzed in four separate experiments and in all of these cells having a lower partition coefficient had an increased amount of the -70000 molecular weight component compared to cells with a higher partition coefficient. Discussion Partitioning of cells in dextran-poly(ethy1ene glycol) aqueous phase systems is an extremely sensitive method not only for the separation and subfractionation of cell populations but also for tracing subtle changes in surface properties that w a r as a function of normal or abnormal in vivo processes, e.&. differentiation, maturation, and aging [for a review, see Walter (1977)l. The sensitivity stems from the fact that the relationship between the partition coefficient (i.e., the quantity of cells in the top phase as a percentage of the total cells) and the properties that determine it is an exponenfial one (Walter, 1977). In order to determine tumor cell characteristics important in malignancy and metastasis, two strategies have been used, ( I ) selection sequentially in vivo (Fidler, 1973; Brunson et al., 1978; Brunson & Nicolson, 1978,1979: Schirrmacher et al., 1979; Kerbel et al., 1978; Nicolson et al., 1978; Tao et al., 1979) or in vitro (Tao & Burger, 1977; Reading et al., 198Oa; Briles & Kornfeld, 1978; Fidler et al., 1976;'Hart. 1979) to obtain variant cell lines differing in metastatic and (2) cloning in vitro unselected or selected cell lines (Fidler & Kripke, 1977; Kripkeet al., 1978; Fidler & Nicolson. 1981; Suzuki et al.. 1978: Nicolson et al.. 1978: Reading et al.. 1980b) to obtain cell clones differing in me&& potentials: Results from these experiments suggested that tumors are heterogeneous with respect to the metastatic phenotypes of individual tumor cells and that selected tumor cell lines r e p resent particular subpopulations derived from originally heterogeneous tumors. Tumor cell subpopulations should be obtained as quickly as possible so that cellular changes which can occur during the time required for selection are not superimposed on differences due to metastatic properties. Rapid separation techniques based on cell density have been used to characterize metastatic cell subpopulations. Grdina et al. (1977) found that low-density cells from a methylcholanthrene-induced fibrcsarcoma separated on linear density gradients of methyl-
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FIGURE4 Autoradiographs of "'I-labeled Con A-7.S%plyanylamide gels containing separated cell glycoproteins. Lane I. RAW1 17-P; lane 2, RAW1 17-P CCD 3 (cavities 9-12); lane 3. RAW117-PCCD I O (cavities 37-40); lane4. RAW117-PCCD 13 49-52); lane 5, R A W l 17.HI0, The prominent A
con
stliningbandsat -70000molecularweightintheRAW117-Psample (lane 1) are eouivalent to the Drominent bands in the RAW1 17-P CCD 3'(lane 2): however, these'compnents are prscnt in decleascd amounts in cells with a higher partition coefficient (lanes 3 and 4) and are almost absent in RAW1 17-H I O (lane 5 ) . Molecular weight markers are ovalbumin (M, -43000). bovine serum albumin (M, -68000). phosphorylase A (M, -94000). and @-galactosidase(M, -35000)
glucamine 3,S-bis(acetylamino)-2,4,6-triiodoben~te(Reno. grafin-60 TM) were more efficient in lung colonization assays compared to highdensity cells from the same tumor. Baniyash et al. (1981) showed that cells from the low lung colonizing potential melanoma subline B l b F l grown in vivo had a higher mean density profile in colloidal silica (Percoll TM) isopycnic density gradients compared to that of the high lung colonizing potential subline B16-FIO. By use of the original unselected
PARTITIONING OF LYMPHOSARCOMA CELLS
B16 tumor, these latter authors demonstrated broad cell separation patterns indicative of heterogeneity in cell densities. When B16 cells from various density fractions were injected intravenously into mice, the fractions containing cells of lower densities formed about 3 times more lung tumor colonies compared to cells of higher densities (Baniyash et al., 1981). In the present study, a parental cell popultion of the RAWl 17-P lymphosarcoma cell lines and a highly metastatic subline (RAW1 17-H10) selected for enhanced liver colonization were subjected to CCD in a dextran-poly(ethy1ene glycol) aqueous phase system having an electrostatic potential difference between the phases. The RAW 117 lymphosarcoma cell lines were found to be heterogeneous with respect to cell-surface properties as shown by their broad CCD curves, and the mean partition coefficient of the high metastatic RAWl 17-HI0 subline was always greater than the mean partition coefficient of the low metastatic RAW 117-P line. That the heterogeneity indicated by partitioning was based on real dissimilarities in cell-surface properties was shown when different CCD fractions were subjected to a second CCD in a phase system having the same composition and had partition coefficients corresponding to those obtained in the first fractionation. This then suggested the existence of subpopulations in the parental RAWl 17-P line having different biochemical and biological characteristics. By examination of the metastatic potentials of RAW 1 17-P cells obtained from different parts of the CCD extraction train, this supposition was confirmed. We found that RAWl 17-P cells acquired from the right end of the CCD profile (higher partition coefficient) formed more liver tumor colonies than did cells from the left end of the distribution. These data are consistent with the presence of more highly metastatic cell subpopulations in the parental RAW 1 17-P line as was also found in cell cloning experiments (Reading et al., 1980b) and inferred from in vivo (Brunson & Nicolson, 1978) and in vitro (Reading et al., 1980a) selection experiments. When the data from 20-40 individual clones obtained from line RAW 117-P (as well as from the in vivo and in vitro selected sublines) were pooled and analyzed together, the average number and range of liver tumor colonies were similar to those of the original RAW117-P line, suggesting that the selection experiments resulted in an enrichment of certain clonal populations depending on the cellular properties determinant in the selection process (Nicolson et al., 1981). Certain characteristics of RAWl 17-P cells are not stable during prolonged culture in vitro. During the course of the present experiments, we noted that the CCD profiles of RAWl 17-P cells changed over a 2-month period, becoming more broad and multipeaked and drifting to a higher mean partition coefficient. This drift correlated with a change in the metastatic properties of line RAW117-P. In several separate experiments, we have noted that 2 months of tissue culture results in an increasingly heterogeneous and more malignant cell population when assayed in vivo, while this was not observed when the same cells were kept as tumors growing subcutaneously (unpublished experiments). Similar findings have recently been made with another metastatic tumor system based on rat 13762 mammary adenocarcinoma (A. Neri and G. L. Nicolson, submitted for publication). These results suggest that malignant cell populations can change with time. Highly malignant lymphosarcoma variant sublines and clones display cell-surface changes that correlate with their biologic properties in vivo. Reading et al. (1980b) examined a number of RAWl 17 sublines and clones and found an in-
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verse relationship between expression of RNA-tumor virus envelope glycoprotein gp70, cell-surface labeling of an -70000 molecular weight glycoprotein, and binding of '251-labeled concanavalin A. Highly metastatic RAWl 17 cell lines and clones show low levels of gp 70 and loss of a concanavalin A binding -70 OOO molecular weight cell-surface component and increased expression of an unrelated component of 135000 molecular weight. Similar results were obtained here, where separation of cell subpopulations on the basis of surface properties by CCD led to cell fractions with low metastatic potentials and high amounts of the 70 000 molecular weight component and fractions with high metastatic potentials and low amounts of the -70000 molecular weight component. Since gp70 is a major cell-surface component on RAW 117-P cells, its loss would be expected to dramatically alter surface properties such as those that are reflected by partitioning in dextran-poly(ethy1ene glycol) phase systems. In the RAWl 17 lymphosarcoma system selected sequentially in vivo, host immune mechanisms may eliminate individual lymphosarcoma cells with strong viral antigens, allowing only subpopulations of RAW 117 cells to survive that have lowered contents of gp70. This may be analogous to the findings of Mora et al. (1977), who reported that tumor-forming SV40-transformed fibrosarcoma cell lines lost SV40 surface antigens, presumably by immunological selection. We have shown that CCD in aqueous phases which fractionates cells on the basis of subtle differences in their surface properties is a useful technique for obtaining variant lymphosarcoma cell subpopulations of high and low metastatic potential without the need for lengthy selection procedures. CCD may be applicable for isolation of other cell variants which differ in their surface properties and are unstable during long-term in vitro growth.
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Acknowledgments We thank Eugene J. Krob and Mark Torrianni for expert technical assistance. References Albertsson, P. A. (1970) Sci. Tools 17, 53-57. Albertsson, P. A,, & Baird, G. D. (1962) Exp. Cell Res. 28, 296-322. Baniyash, M., Netanel, T., & Witz, I. P. (1981) Cancer Res. 41, 433-437. Briles, E. B., & Kornfeld, S . (1978) J . Natl. Cancer Inst. ( U S . ) 60, 1217-1222. Brunson, K. W., & Nicolson, G. L. (1978) J . Natl. Cancer Inst. ( U S . ) 61, 1499-1503. Brunson, K. W., & Nicolson, G. L. (1979) J . Supramol. Struct. 1 1 , 517-528. Brunson, K. W., Beattie, G., & Nicolson, G. L. (1978) Nature (London) 272, 543-545. Burridge, K. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4457-4461. Fidler, I. J . (1973) Nature (London),New Biol. 242, 148-149. Fidler, I. J., & Kripke, M. L. (1977) Science (Washington, D.C.)197, 893-895. Fidler, I. J., & Nicolson, G. L. (1981) Cancer Biol. Rev. 2, 17 1-234. Fidler, I. J., Gerstein, D. M., & Budmen, M. B. (1976) Cancer Res. 36, 3160-3165. Fidler, I. J., Gerstein, D. M., & Hart, I. R. (1978) Adv. Cancer Res. 28, 149-250. Grdina, D. J., Hittelman, W. M., White, R. A., & Meistrich, M. L. (1977) Br. J . Cancer 36, 659-669. Hart, I. R. (1979) Am. J . Pathol. 97, 587-600.
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Reading, C. L., Belloni, P. N., & Nicolson, G. L. (1980a) JNCI, J . Natl. Cancer Inst. 64, 1241-1249. Reading, C. L., Brunson, K. W., Torrianni, M., & Nicolson, G. L. (1980b) Proc. Natl. Acad. Sci. U 3 . A . 77,5943-5947. Reitherman, R., Flanagan, S . D., & Barondes, S. H. (1973) Biochim. Biophys. Acta 297, 193-202. Schirrmacher, V., Shantz, G., Clauer, K., Komitowski, D., Zimmermann, H.-P., & Lohmann-Matthes, M.-L. (1979) Int. J . Cancer 23, 233-244. Suzuki, N., Withers, H. R., & Koehler, M. W. (1978) Cancer Res. 38, 3349-3351. Tao, T.-W., & Burger, M. M. (1977) Nature (London) 270, 437-43 8, Tao, T.-W., Matter, A., Vogel, K., & Burger, M. M. (1979) Int. J . Cancer 23, 854-857. Walter, H. (1977) in Methods in Cell Separation (Catsimpoolas, N., Ed.) Vol. 1, pp 307-354, Plenum Press, New York. Walter, H., Krob, E. J., & Ascher, G. S. (1981) Biochim. Biophys. Acta 641, 202-215.
Nuclear Magnetic Resonance Relaxation in Nucleic Acid Fragments: Models for Internal Motion+ Giovanni Lipari and Attila Szabo*
ABSTRACT: A variety of models incorporating internal motion, which can be used to extract information from nuclear magnetic resonance relaxation studies of deoxyribonucleic acid fragments, are formulated. Illustrative analyses of some recent multinuclear relaxation data are presented. Special emphasis
is placed on determining whether the information extracted is unique. It is shown that the data are consistent with several physical pictures of the internal motion. However, all the models we have considered imply the existence of large-amplitude internal motions on the nanosecond time scale.
N u c l e a r magnetic resonance (NMR)' is an important technique for probing molecular motions. Recently, a large number of 'H, 31P,and/or 13C NMR relaxation studies of DNA have appeared in the literature (Bolton & James, 1979, 1980a,b; Early & Kearns, 1979; Hogan & Jardetzky, 1979, 1980; Klevan et al., 1979; Shindo, 1980). There appears to be agreement among several groups that large-amplitude internal motions on the nanosecond time scale are present in DNA. This conclusion was extracted from the data by using highly idealized models of the internal motion (e.g., free internal rotation about an axis or two-site jumps within a plane containing the long axis of the helix). We have set out to establish whether these conclusions hold within the framework of somewhat more sophisticated models of the internal motion and whether the currently available data lead to a unique physical picture of the internal motions. In the course of this work, we found a mathematical error in the interesting work of Hogan & Jardetzky (1979, 1980) which is corrected here. Following Hogan & Jardetzky (1979, 1980), we assume that the overall reorientation of a relatively short DNA fragment
is the same as that of a freely diffusing cylinder. Motions relative to a reference frame rigidly attached to the cylinder are considered to be internal motions. Using electric dichroism, it has been shown (Hogan et al., 1978) that DNA fragments with 100-250 base pairs behave like rods to a good approximation. However, there is evidence from computer simulations (Olson, 1980) that fragments at the upper end of the above range can deviate significantly from a rodlike shape. We do not consider such bending motions although their possible influence is implicitly mimicked by our models of internal motion. In this paper, we formulate a variety of models for internal motion. First, we consider twisting or torsional motions (using both square-well and harmonic potentials) in which the polar angle between the relevant interaction vector and the long axis of the helix remains fixed. Second, we consider wobbling motions in which the interaction vector diffuses in a cone about a director which forms a fixed angle with the long axis of the helix. Finally, we consider jump models and explicitly formulate the most general form of the two-state jump model in which the interaction vector jumps between two arbitrary and energetically inequivalent positions. We explicitly state the assumptions under which our exact treatment degenerates into
+ From the Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205. Received April 13, 1981. G.L. is supported in part by US. Public Health Service Grant H L 21483 awarded to F. R. N . Gurd and in part by a fellowship from the Foundation Stiftelsen Blanceflor Boncompagni-Ludovsisi, fijdd Bildt.
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Abbreviations used: NMR, nuclear magnetic resonance; DNA, deoxyribonucleic acid; NOE, nuclear Overhauser effect.
This article not subject to U S . Copyright. Published 1981 by the American Chemical Society
