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Behavior in two-phase aqueous polymer systems of L5178Y mouse leukemic cells
Printed in Sweden Copyright Q 1974 by Academic Press, Inc. A// rights OJ reproduction in any form reserved
Experimental Cell Research 88 (1974) 225-230
BEHAVIOR
IN TWO-PHASE AQUEOUS POLYMER OF L5178Y MOUSE LEUKEMIC II. The Lag and Exponential
SYSTEMS
CELLS
Phases of Growth1
D. M. GERSTEN and H. B. BOSMANN Department of Pharmacology and Toxicology, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642, USA
SUMMARY The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran - polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude fol the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.
It is evident that in an individual cell’s mitotic cycle and in a cell population’s growth cycle alterations occur in all cell-related parameters-enzyme levels, cell volume, amount of nucleic acid, etc. It is also evident that profound changes occur in cell membranes as dynamic structures, such as when, for example, in the M phase of the mitotic cycle, cells grown in monolayer tend to ‘round up’ and detach from their substratum. But, unlike measurement of cell volume, macromolecule or enzyme levels, ascertainment of 1 The first paper in this series is given as ref. [3].
changes in cell membranes is very difficult biochemically or biophysically because of the almost impossible task of isolating pure membrane fractions. Workers have had to resort to less direct methods of membrane analysis, such as lectin binding, whole-cell microelectrophoresis, and freeze fracture techniques. These techniques are generally not very sensitive, and in most instances the data obtained are rather descriptive in application. Dextran-polyethylene glycol phase partitioning of cells or cell organelles is a method rapidly gaining in use because it measures very subtle membrane alterations, Exptl Cell Res 88 (1974)
226
Gersten and Bosmann
principally in the ‘deep charge’ layer of the membranes of cells in essentially the native state. The present study uses this technique to investigate changes in surface membranes of L5178Y mouse leukemic cells in the population growth cycle. It is important to know what determines the membrane characteristics of a cell population as the cells progress through the population growth cycle, and whether or not build-up of metabolic wastes (e.g., changes in pH), exhaustion of nutrients, or actual cell-cell interactions determine the kinetics of the cell growth cycle. It has been known for some time that several growth parameters of mammalian cells in tissue culture are associated intimately with the pH of the supporting growth medium [l-7]. Most notably, general growth rate and protein synthesis have been studied [S-lo]. In more recent investigations, growthrelated cell surface phenomena have been described [IO-131. In particular, induction and reversal of ‘contact inhibition’ of cells growing in monolayer have been directly linked to pH of the external environment [lo, Ill. Growth-associated changes in cell surface properties have also included tumor cell invasiveness (metastasis and implantation) [12, 14, 151 and surface charge [3, 161. We have previously shown that L5178Y mouse leukemic cells undergo profound changes in surface properties as the population progresses from early stationary through to accelerated death phase. The progressive alteration of surface properties on a populational basis was not demonstrable by wholecell microelectrophoresis, but only by the technique of partition in aqueous polymers
[31. The polymers dextran and polyethylene glycol when mixed in buffered aqueous solution distribute unevenly into two phases which carry an uneven distribution of inExptI Cell Res 88 (1974)
organic salts. The cells position themselves in the system primarily in accordance with the relation of their surface charge to the charge distribution established by the salts [17]. Glutaraldehyde fixation experiments have indicated that charges deeper in the membrane than the plane of shear may also participate in partitioning behavior [18]. The fact that an analysis of the true properties measured by the partition ratio is not available is unfortunate, but the fact remains that the technique is very useful in pinpointing differences in comparative situations, such as those involved in the cell growth cycle. While the studies involving stationary and death phase cells indicated that a ‘maturation’ of the population with regard to the charge :space relationships of the cells took place and that this progression was driven by the changing pH of the growth medium, it was clear that other growth-related factors were also involved. That is, by pH reversal experiments, it was possible to deduce differences between the surfaces of early and late stationary cells. The present investigation describes the behavior in the aqueous polymer partition system, of normal lag phase and exponential phase cells as compared to cells following a lag phase artificially extended by dilution of the growth medium with high pH medium. MATERIALS
AND METHODS
Polymer systems The polymer system-5 9; (w/w) dextran T-500 (Pharmacia Fine Chemicals. Unosala. Sweden). 4 % polyethylene glycol (“Carbdwax-600& Union’Carbide Corp., New York) has been desciibed in detail [l-2]. The polymers were prepared in equimolar K,HPO,/KH,PO, buffer such that when mixed with NaCl and cells suspended in 0.1 M K,HP0,/KH,P04 buffer, pH 7.0, the final characteristics were: NaCI, 0.05 Mj buffer, 0.1 M with respect to phosphate; dextran, 5 % by weight; polyethylene glycol, 4 % by weight; total volume, 9.75 ml per partition. A11 partitions were performed at neutral pH. Immediately after mixing by gentle inversion, a
Partition of L5178Y cells
221
0.5 ml aliquot of the total system was sampled. The phases were allowed to separate for 1 h at 4”C, at which time the top phase was withdrawn and a 0.5 ml aliquot sampled. The cell numbers in each aliquot were determined by counting in a Coulter Counter Model ZB fitted with a 100 pm orifice and are expressed as a ratio: (cells/unit volume top phase)/ (ceils/unit volume total system).
Culture conditions L5178Y mouse leukemic cells were grown in Fischer’s medium (GIBCo, Grand Island, N.Y.) [4] containing 10% horse serum (Flow Laboratories, Rockville, Md) at 37°C in batch culture. The medium in the dry form was reconstituted with double glass-distilled water. Penicillin (500 units/ml) and streptomycin (5 ma/ml) were added before membrane filter sterilization. hells were utilized in early stationary phase for ‘backculture’ into lag phase by dilution into fresh medium. The dilution was 1 : 1I- as 100 ml of stationary phase culture to 1 000 ml of fresh growth medium of appropriate pH. Cells were transferred with constant stirring in order to ensure the transfer of a uniform cell suspension to each of the 2 I culture flasks. The flasks were incubated at 37°C without further agitation until use at the stated intervals posttransfer. At anorouriate intervals. the contents of the growth flasks were’ harvested by’slow speed centrifugation (400 g. 10 min) and resusoended in oH 7.0 K,HPO,/ KH&, buff& which ias 0.1 ML with respect to phosphate. Since cell concentration has a demonstrable effect on partitioning behavior [3], the cell concentration was adjusted to 3-7 x IO6 per partition by appropriate resuspension in phosphate buffer. One ml of this suspension was added to each of 10 partition assay viais containing polymer and NaCl solution as described above. Each point, therefore. represents the average of four obse;vati&s of each of 10 independent partitions. Aliquots of the remaining suspension were used for determination of electrophoretic mobility as necessary.
Electrophoretic mobility An aliquot of the remaining suspension (above) was withdrawn, centrifuged, and resuspended in 7 ml saline-sorbitol (0.0145 M NaCI, 4.5 % sorbitol, 0.6 mM NaHCO,) after the method of Bosmann et al. [5] for observation of electrophoretic mobility. Measurements were made at 25°C in a horizontal cylindrical chamber fitted with reversible blacked platinum electrodes. Each value was determined as the mean +S.E.M. of 20 observations with a reversal of polarity between each observation and the next. The alignment of the apparatus (Rank Bros., Bottisham, UK) was checked against washed human erythrocytes from healthy male donors of the phenotype A Rh-1. by the method of Heard & Seaman [6].
RESULTS The results of a normal back-culture are shown in fig. 1. The partition ratio of the
Fig. I. Normal growth. Abscissa: time post transfer (hours); or&are: (left) partition ratio, calculated as described in Materials and Methods ( l ); (right) log,,, cells/ml (0). Partition ratio before transfer, 0.52. pH of conditioned medium. 6.7: DH of fresh medium. 7.0.
early stationary inoculum was 0.52. We have demonstrated [3] that the farther the cells are into their stationary phase, the lower their partition ratio. The value, then, is a measure of the progression of the inoculum into stationary phase. The pH of the fresh medium was 7.0; the doubling time under these conditions was 14.5 h. It may be seen that the partition ratio remains depressed for the first 26 h, then rises rapidly at a constant rate for the next 48 h during exponential growth. By 74 h post-transfer the ratio returns to 0.51 as the population has returned to the late exponential/early stationary period [3]. The partition ratio is calculated as the number of cells/unit volume in the total system before the phases separate, divided into the number of cells/unit volume of the top phase after separation. The ratio, then, is directly related to the percentage of the total cell population which is recoverable from the top phase, by a constant describing the fractional volume of the total system occupied by the top phase. Since the distribution of salts and polymers is the same for all partitions used throughout the course of Exptl Cell Res 88 (1974)
228
Gersten and Bosmann
Table 1. Time course of electrophoretic mobility Electrophoretic mobility &m/s/V/cm) Hours Normal post-transfer growth’ 0
3 6 12 24 27 36 48 50 72
-2.03 -1.90 -1.98 - 2.02 -1.93 - 2.04
Prolonged lag and exponential following prolonged lagb - 2.03 - 2.05 - 1.98 ~ 2.01 -1.92 PI.95 - 2.08
a Conditions corresponding to those of fig. 1. b Conditions corresponding to those of figs 2 and 3.
this study, the increases or decreases in the partition ratio must represent increases or decreases in the proportion of the population with sufficiently different charge to align in the top phase. Table 1 indicates that the change in partition ratio is not accompanied by an appreciable change in electrophoretic mobility. Since the length of the lag phase under the present conditions is short with respect to
Fig. 2. Abscissa: time post transfer (hours); ordinate: partition ratio (0); (right) log,, cells/ml (0). Prolonged lag phase by transfer into medium of pH 7.5-7.6.
(left)
Exptl Cell Res 88 (1974)
Fig. 3. Abscissa: time post transfer (hours); ordinate: (left) partition ratio (0); (right) log,, cells/ml (0).
Logarithmic phase following prolonged lag phase.
the entire time course, it was necessary to artificially prolong the lag phase. This was accomplished by starting with a later stationary phase inoculum than above (medium pH 6.7, partition ratio 0.34) and transfer to fresh medium of high pH (7.5-7.6) [IO]. This had the effect of prolonging the lag phase for 14 h (fig. 2). The partition ratio behaves as a prolonged depression followed by a gradual rise, again trailing the onset of exponential growth. The ensuing exponential growth is similar to that shown in fig. 1 in that the doubling time was equal to 14.5 h under these conditions. It should be noted that the partition ratio had reached 0.24 when the cell number reached 1.6 x 105/ml during normal growth whereas the ratio of 0.24 was not attained until the cell number reached 2.9 x 105/ml following prolonged lag. When the exponential phase is examined in detail (fig. 3), oscillations in partition ratio. in the late lag/early exponential area are observed which are not obvious from figs 1 or 2. In order to observe the late lag/early exponential area of the curve, it was necessary to prolong the lag phase slightly to an extent intermediate between those of figs 1
Partition of L.5178Y cells 229
, \ \
J
Fig. 4. Abscissa: time post inoculation; ordinate: cell concentration or partition ratio. -, viable cells; ----, partition ratios. Schematic representation of the complete growth curve of L5178Y cells and its relation to the partition ratio. For approximate values see figs l-3. (Since this is a schematic diagram, no numerical values for the ordinates.)
and 2. The inoculum was at a concentration of 3.4 x IO5 cells/ml and had a partition ratio of 0.46. The pH of the fresh medium was 7.5-7.6. The exponential doubling time was 14 h. Except for these oscillations, the partition ratio vs time and cell concentration relationships are similar to those of fig. 2. The partition ratio of 0.24 is again reached around 50 h post-transfer when the population has matured to approx. 1.5 to 105/ml. Again, table 1 indicates that for all conditions studied, there is no appreciable change in electrophoretic mobility measured in saline-sorbitol. DISCUSSION In fig. 4 is presented a schematic diagram of the complete cell growth cycle and the applicable partition ratio determined under the conditions described in this paper. The majority of the data of fig. 4 comes from the present work; the data for the stationary and death phases are from the previous paper [3]. The most obvious fact shown in fig. 4 is that the partition ratio changes drastically during the L5178Y cell growth cycle. Thus
in aqueous polymer partition ratios we have a technique which adequately reflects the complex changes occurring at the cell periphery during the cell growth cycle. It can now be shown that just as all other cell parameters are rapidly changing during the cell division cycle, so also are the biophysical properties of the external cell surface. It is also evident from fig. 4 that although profound alterations in aqueous polymer partition ratios are occurring, little or no assayable change is measurable by the very sensitive parameter of whole cell particle microelectrophoresis. Thus changes measured by microelectrophoresis, e.g., external net bulk charge at the shear plane of the membrane (in many instances N-acetylneuraminic acid terminal residues contribute to this), are probably not occurring extensively or, since the technique measures net “surface charge”, the changes in one direction may be very rapidly offset by equal changes in the opposite direction during the cell growth cycle. For example, as anionic groups at the shear plane are deleted or inserted (alternatively ‘masked’ or ‘unmasked’), equal amounts of cationic groups may be inserted or deleted. The results presented here show that changes occurring deeper in the plane of the membrane [18] due to insertion, deletion, fluidity etc., are measurable by the technique of aqueous polymer partitioning and such changes at this level of the cell’s external membrane do occur during the cell growth cycle. It should be remembered that the changes measured by the partitioning technique are of a populational nature and that although ‘back growth’ or medium pH alterations may make it possible to recreate certain partition ratios these factors may not be those which ‘drive’ or create the partition ratio or, more appropriately, the surface conditions which dictate the partition ratio. It would Exptl Cell Res 88 (1974)
230
Gersten and Bosmann
be of great interest to determine what role medium and cellular pH play in these phenomena and, more important, how these pH changes are generated and controlled with what must be remarkable precision. It is surprising that the partition ratios of the L5178Y cells are essentially identical at the lag phase and the death phase, when the cells are certainly in different states of metabolic activity and viability, and when the external membranes of the cell are probably greatly different. Thus the technique of aqueous polymer partitioning does not serve to differentiate these properties of the cell plasma membrane which one would expect (perhaps wrongly) to be very different. Experiments are underway to use more refined techniques to study these differences. This work was supported in part by Grants CA 13220 and GM-00032 from the USPHS. Dr Bosmann is a Research Career Development Awardee of US NIGMS. We thank K. R. Case for technical assistance.
2. Brooks, D E, Seaman, G V F & Walter, H, Nature new biol 234 (1971) 61. 3. Gersten, D M & Bosmann, H B, Exptl cell res 87 (1974) 73. 4. Bosmann, H B & Bernacki, R J, Exptl cell res 61 (1970) 379. 5. Bosmann, H B, Myers, M W, De Hond, D, Ball, R & Case, K R, J cell biol 55 (1972) 147. 6. Heard, D H & Seaman, G V F, J gen physiol 43 (1960) 635. 7. Mackenzie, C G, Mackenzie, J B & Beck, P. J biophys biochem cytol9 (1961) 141. 8. Eagle, H, Science 174 (1971) 500. 9. - Ibid 130 (1959) 432. 10. Ceccarini, C & Eagle, H, Proc natl acad sci US 68 (1971) 229. Il. - Nature new biol 233 (1971) 271. 12. Bosmann, H B, Bieber, G F, Brown, A E, Case, K R, Gersten, D M, Kimmerer, T W & Lione, A, Nature 246 (1973) 487. 13. Thrash, C R & Cunningham, D D, Nature new biol 242 (1973) 399. 14. Fidler, I J, Nature new biol 242 (1973) 148. 15. -J natl cancer inst (US) 50 (1973) 1307. 16. Walter, H, Eriksson, G, Taube, 6 & Albertsson, P-A, Exptl cell res 77 (1973) 361. 17. Seaman, G V F & Walter, H, Fed proc 30 (1971) 1182a. 18. Walter, H, Tung, R, Jackson, L J & Seaman, G V F. Biochem biophys res commun 48 (1972)
REFERENCES Received April 24, 1974 1. Albertsson, P-A, Partition of cell particles and macromolecules, 2nd edn, p. 190. Wiley-lnterscience, New York (1972).
Exptl Cell Res 88 (1974)
Experimental Cell Research 88 (1974) 225-230
BEHAVIOR
IN TWO-PHASE AQUEOUS POLYMER OF L5178Y MOUSE LEUKEMIC II. The Lag and Exponential
SYSTEMS
CELLS
Phases of Growth1
D. M. GERSTEN and H. B. BOSMANN Department of Pharmacology and Toxicology, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642, USA
SUMMARY The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran - polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude fol the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.
It is evident that in an individual cell’s mitotic cycle and in a cell population’s growth cycle alterations occur in all cell-related parameters-enzyme levels, cell volume, amount of nucleic acid, etc. It is also evident that profound changes occur in cell membranes as dynamic structures, such as when, for example, in the M phase of the mitotic cycle, cells grown in monolayer tend to ‘round up’ and detach from their substratum. But, unlike measurement of cell volume, macromolecule or enzyme levels, ascertainment of 1 The first paper in this series is given as ref. [3].
changes in cell membranes is very difficult biochemically or biophysically because of the almost impossible task of isolating pure membrane fractions. Workers have had to resort to less direct methods of membrane analysis, such as lectin binding, whole-cell microelectrophoresis, and freeze fracture techniques. These techniques are generally not very sensitive, and in most instances the data obtained are rather descriptive in application. Dextran-polyethylene glycol phase partitioning of cells or cell organelles is a method rapidly gaining in use because it measures very subtle membrane alterations, Exptl Cell Res 88 (1974)
226
Gersten and Bosmann
principally in the ‘deep charge’ layer of the membranes of cells in essentially the native state. The present study uses this technique to investigate changes in surface membranes of L5178Y mouse leukemic cells in the population growth cycle. It is important to know what determines the membrane characteristics of a cell population as the cells progress through the population growth cycle, and whether or not build-up of metabolic wastes (e.g., changes in pH), exhaustion of nutrients, or actual cell-cell interactions determine the kinetics of the cell growth cycle. It has been known for some time that several growth parameters of mammalian cells in tissue culture are associated intimately with the pH of the supporting growth medium [l-7]. Most notably, general growth rate and protein synthesis have been studied [S-lo]. In more recent investigations, growthrelated cell surface phenomena have been described [IO-131. In particular, induction and reversal of ‘contact inhibition’ of cells growing in monolayer have been directly linked to pH of the external environment [lo, Ill. Growth-associated changes in cell surface properties have also included tumor cell invasiveness (metastasis and implantation) [12, 14, 151 and surface charge [3, 161. We have previously shown that L5178Y mouse leukemic cells undergo profound changes in surface properties as the population progresses from early stationary through to accelerated death phase. The progressive alteration of surface properties on a populational basis was not demonstrable by wholecell microelectrophoresis, but only by the technique of partition in aqueous polymers
[31. The polymers dextran and polyethylene glycol when mixed in buffered aqueous solution distribute unevenly into two phases which carry an uneven distribution of inExptI Cell Res 88 (1974)
organic salts. The cells position themselves in the system primarily in accordance with the relation of their surface charge to the charge distribution established by the salts [17]. Glutaraldehyde fixation experiments have indicated that charges deeper in the membrane than the plane of shear may also participate in partitioning behavior [18]. The fact that an analysis of the true properties measured by the partition ratio is not available is unfortunate, but the fact remains that the technique is very useful in pinpointing differences in comparative situations, such as those involved in the cell growth cycle. While the studies involving stationary and death phase cells indicated that a ‘maturation’ of the population with regard to the charge :space relationships of the cells took place and that this progression was driven by the changing pH of the growth medium, it was clear that other growth-related factors were also involved. That is, by pH reversal experiments, it was possible to deduce differences between the surfaces of early and late stationary cells. The present investigation describes the behavior in the aqueous polymer partition system, of normal lag phase and exponential phase cells as compared to cells following a lag phase artificially extended by dilution of the growth medium with high pH medium. MATERIALS
AND METHODS
Polymer systems The polymer system-5 9; (w/w) dextran T-500 (Pharmacia Fine Chemicals. Unosala. Sweden). 4 % polyethylene glycol (“Carbdwax-600& Union’Carbide Corp., New York) has been desciibed in detail [l-2]. The polymers were prepared in equimolar K,HPO,/KH,PO, buffer such that when mixed with NaCl and cells suspended in 0.1 M K,HP0,/KH,P04 buffer, pH 7.0, the final characteristics were: NaCI, 0.05 Mj buffer, 0.1 M with respect to phosphate; dextran, 5 % by weight; polyethylene glycol, 4 % by weight; total volume, 9.75 ml per partition. A11 partitions were performed at neutral pH. Immediately after mixing by gentle inversion, a
Partition of L5178Y cells
221
0.5 ml aliquot of the total system was sampled. The phases were allowed to separate for 1 h at 4”C, at which time the top phase was withdrawn and a 0.5 ml aliquot sampled. The cell numbers in each aliquot were determined by counting in a Coulter Counter Model ZB fitted with a 100 pm orifice and are expressed as a ratio: (cells/unit volume top phase)/ (ceils/unit volume total system).
Culture conditions L5178Y mouse leukemic cells were grown in Fischer’s medium (GIBCo, Grand Island, N.Y.) [4] containing 10% horse serum (Flow Laboratories, Rockville, Md) at 37°C in batch culture. The medium in the dry form was reconstituted with double glass-distilled water. Penicillin (500 units/ml) and streptomycin (5 ma/ml) were added before membrane filter sterilization. hells were utilized in early stationary phase for ‘backculture’ into lag phase by dilution into fresh medium. The dilution was 1 : 1I- as 100 ml of stationary phase culture to 1 000 ml of fresh growth medium of appropriate pH. Cells were transferred with constant stirring in order to ensure the transfer of a uniform cell suspension to each of the 2 I culture flasks. The flasks were incubated at 37°C without further agitation until use at the stated intervals posttransfer. At anorouriate intervals. the contents of the growth flasks were’ harvested by’slow speed centrifugation (400 g. 10 min) and resusoended in oH 7.0 K,HPO,/ KH&, buff& which ias 0.1 ML with respect to phosphate. Since cell concentration has a demonstrable effect on partitioning behavior [3], the cell concentration was adjusted to 3-7 x IO6 per partition by appropriate resuspension in phosphate buffer. One ml of this suspension was added to each of 10 partition assay viais containing polymer and NaCl solution as described above. Each point, therefore. represents the average of four obse;vati&s of each of 10 independent partitions. Aliquots of the remaining suspension were used for determination of electrophoretic mobility as necessary.
Electrophoretic mobility An aliquot of the remaining suspension (above) was withdrawn, centrifuged, and resuspended in 7 ml saline-sorbitol (0.0145 M NaCI, 4.5 % sorbitol, 0.6 mM NaHCO,) after the method of Bosmann et al. [5] for observation of electrophoretic mobility. Measurements were made at 25°C in a horizontal cylindrical chamber fitted with reversible blacked platinum electrodes. Each value was determined as the mean +S.E.M. of 20 observations with a reversal of polarity between each observation and the next. The alignment of the apparatus (Rank Bros., Bottisham, UK) was checked against washed human erythrocytes from healthy male donors of the phenotype A Rh-1. by the method of Heard & Seaman [6].
RESULTS The results of a normal back-culture are shown in fig. 1. The partition ratio of the
Fig. I. Normal growth. Abscissa: time post transfer (hours); or&are: (left) partition ratio, calculated as described in Materials and Methods ( l ); (right) log,,, cells/ml (0). Partition ratio before transfer, 0.52. pH of conditioned medium. 6.7: DH of fresh medium. 7.0.
early stationary inoculum was 0.52. We have demonstrated [3] that the farther the cells are into their stationary phase, the lower their partition ratio. The value, then, is a measure of the progression of the inoculum into stationary phase. The pH of the fresh medium was 7.0; the doubling time under these conditions was 14.5 h. It may be seen that the partition ratio remains depressed for the first 26 h, then rises rapidly at a constant rate for the next 48 h during exponential growth. By 74 h post-transfer the ratio returns to 0.51 as the population has returned to the late exponential/early stationary period [3]. The partition ratio is calculated as the number of cells/unit volume in the total system before the phases separate, divided into the number of cells/unit volume of the top phase after separation. The ratio, then, is directly related to the percentage of the total cell population which is recoverable from the top phase, by a constant describing the fractional volume of the total system occupied by the top phase. Since the distribution of salts and polymers is the same for all partitions used throughout the course of Exptl Cell Res 88 (1974)
228
Gersten and Bosmann
Table 1. Time course of electrophoretic mobility Electrophoretic mobility &m/s/V/cm) Hours Normal post-transfer growth’ 0
3 6 12 24 27 36 48 50 72
-2.03 -1.90 -1.98 - 2.02 -1.93 - 2.04
Prolonged lag and exponential following prolonged lagb - 2.03 - 2.05 - 1.98 ~ 2.01 -1.92 PI.95 - 2.08
a Conditions corresponding to those of fig. 1. b Conditions corresponding to those of figs 2 and 3.
this study, the increases or decreases in the partition ratio must represent increases or decreases in the proportion of the population with sufficiently different charge to align in the top phase. Table 1 indicates that the change in partition ratio is not accompanied by an appreciable change in electrophoretic mobility. Since the length of the lag phase under the present conditions is short with respect to
Fig. 2. Abscissa: time post transfer (hours); ordinate: partition ratio (0); (right) log,, cells/ml (0). Prolonged lag phase by transfer into medium of pH 7.5-7.6.
(left)
Exptl Cell Res 88 (1974)
Fig. 3. Abscissa: time post transfer (hours); ordinate: (left) partition ratio (0); (right) log,, cells/ml (0).
Logarithmic phase following prolonged lag phase.
the entire time course, it was necessary to artificially prolong the lag phase. This was accomplished by starting with a later stationary phase inoculum than above (medium pH 6.7, partition ratio 0.34) and transfer to fresh medium of high pH (7.5-7.6) [IO]. This had the effect of prolonging the lag phase for 14 h (fig. 2). The partition ratio behaves as a prolonged depression followed by a gradual rise, again trailing the onset of exponential growth. The ensuing exponential growth is similar to that shown in fig. 1 in that the doubling time was equal to 14.5 h under these conditions. It should be noted that the partition ratio had reached 0.24 when the cell number reached 1.6 x 105/ml during normal growth whereas the ratio of 0.24 was not attained until the cell number reached 2.9 x 105/ml following prolonged lag. When the exponential phase is examined in detail (fig. 3), oscillations in partition ratio. in the late lag/early exponential area are observed which are not obvious from figs 1 or 2. In order to observe the late lag/early exponential area of the curve, it was necessary to prolong the lag phase slightly to an extent intermediate between those of figs 1
Partition of L.5178Y cells 229
, \ \
J
Fig. 4. Abscissa: time post inoculation; ordinate: cell concentration or partition ratio. -, viable cells; ----, partition ratios. Schematic representation of the complete growth curve of L5178Y cells and its relation to the partition ratio. For approximate values see figs l-3. (Since this is a schematic diagram, no numerical values for the ordinates.)
and 2. The inoculum was at a concentration of 3.4 x IO5 cells/ml and had a partition ratio of 0.46. The pH of the fresh medium was 7.5-7.6. The exponential doubling time was 14 h. Except for these oscillations, the partition ratio vs time and cell concentration relationships are similar to those of fig. 2. The partition ratio of 0.24 is again reached around 50 h post-transfer when the population has matured to approx. 1.5 to 105/ml. Again, table 1 indicates that for all conditions studied, there is no appreciable change in electrophoretic mobility measured in saline-sorbitol. DISCUSSION In fig. 4 is presented a schematic diagram of the complete cell growth cycle and the applicable partition ratio determined under the conditions described in this paper. The majority of the data of fig. 4 comes from the present work; the data for the stationary and death phases are from the previous paper [3]. The most obvious fact shown in fig. 4 is that the partition ratio changes drastically during the L5178Y cell growth cycle. Thus
in aqueous polymer partition ratios we have a technique which adequately reflects the complex changes occurring at the cell periphery during the cell growth cycle. It can now be shown that just as all other cell parameters are rapidly changing during the cell division cycle, so also are the biophysical properties of the external cell surface. It is also evident from fig. 4 that although profound alterations in aqueous polymer partition ratios are occurring, little or no assayable change is measurable by the very sensitive parameter of whole cell particle microelectrophoresis. Thus changes measured by microelectrophoresis, e.g., external net bulk charge at the shear plane of the membrane (in many instances N-acetylneuraminic acid terminal residues contribute to this), are probably not occurring extensively or, since the technique measures net “surface charge”, the changes in one direction may be very rapidly offset by equal changes in the opposite direction during the cell growth cycle. For example, as anionic groups at the shear plane are deleted or inserted (alternatively ‘masked’ or ‘unmasked’), equal amounts of cationic groups may be inserted or deleted. The results presented here show that changes occurring deeper in the plane of the membrane [18] due to insertion, deletion, fluidity etc., are measurable by the technique of aqueous polymer partitioning and such changes at this level of the cell’s external membrane do occur during the cell growth cycle. It should be remembered that the changes measured by the partitioning technique are of a populational nature and that although ‘back growth’ or medium pH alterations may make it possible to recreate certain partition ratios these factors may not be those which ‘drive’ or create the partition ratio or, more appropriately, the surface conditions which dictate the partition ratio. It would Exptl Cell Res 88 (1974)
230
Gersten and Bosmann
be of great interest to determine what role medium and cellular pH play in these phenomena and, more important, how these pH changes are generated and controlled with what must be remarkable precision. It is surprising that the partition ratios of the L5178Y cells are essentially identical at the lag phase and the death phase, when the cells are certainly in different states of metabolic activity and viability, and when the external membranes of the cell are probably greatly different. Thus the technique of aqueous polymer partitioning does not serve to differentiate these properties of the cell plasma membrane which one would expect (perhaps wrongly) to be very different. Experiments are underway to use more refined techniques to study these differences. This work was supported in part by Grants CA 13220 and GM-00032 from the USPHS. Dr Bosmann is a Research Career Development Awardee of US NIGMS. We thank K. R. Case for technical assistance.
2. Brooks, D E, Seaman, G V F & Walter, H, Nature new biol 234 (1971) 61. 3. Gersten, D M & Bosmann, H B, Exptl cell res 87 (1974) 73. 4. Bosmann, H B & Bernacki, R J, Exptl cell res 61 (1970) 379. 5. Bosmann, H B, Myers, M W, De Hond, D, Ball, R & Case, K R, J cell biol 55 (1972) 147. 6. Heard, D H & Seaman, G V F, J gen physiol 43 (1960) 635. 7. Mackenzie, C G, Mackenzie, J B & Beck, P. J biophys biochem cytol9 (1961) 141. 8. Eagle, H, Science 174 (1971) 500. 9. - Ibid 130 (1959) 432. 10. Ceccarini, C & Eagle, H, Proc natl acad sci US 68 (1971) 229. Il. - Nature new biol 233 (1971) 271. 12. Bosmann, H B, Bieber, G F, Brown, A E, Case, K R, Gersten, D M, Kimmerer, T W & Lione, A, Nature 246 (1973) 487. 13. Thrash, C R & Cunningham, D D, Nature new biol 242 (1973) 399. 14. Fidler, I J, Nature new biol 242 (1973) 148. 15. -J natl cancer inst (US) 50 (1973) 1307. 16. Walter, H, Eriksson, G, Taube, 6 & Albertsson, P-A, Exptl cell res 77 (1973) 361. 17. Seaman, G V F & Walter, H, Fed proc 30 (1971) 1182a. 18. Walter, H, Tung, R, Jackson, L J & Seaman, G V F. Biochem biophys res commun 48 (1972)
REFERENCES Received April 24, 1974 1. Albertsson, P-A, Partition of cell particles and macromolecules, 2nd edn, p. 190. Wiley-lnterscience, New York (1972).
Exptl Cell Res 88 (1974)