Early Transport Changes during Erythroid Differentiation of Friend Leukemic Cells DIXIE MAGER AND ALAN BERNSTEIN Department ofMedical Biophysics, University of Toronto, and The Ontario Cancer Institute, 500Sherbourne Street, Toronto, Ontario, Canada M4X 1K9

ABSTRACT

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rbtransport was observed beginning approximately five hours after stimulation with 1.5%dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized a t close to 60%of control level. This decrease in the rate of 86Rbtransport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rbinflux. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rbinflux. The transport of two amino acid analogues, a-aminoisobutyric acid and 2-aminobicyclo [2,2,11-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast,the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes- those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process a t which a particular variant line is blocked.

Friend erythroleukemic cells are permanent cell lines which originated from the spleens of mice infected with Friend virus. These cells can be induced to differentiate in vitro along the erythroid pathway by treatment with certain chemical agents such as dimethylsulfoxide (DMSO) (Friend et al., '71). Although the overt expression of erythroid differentiation, as measured by hemoglobin synthesis and other characteristic markers, has been extensively studied (for review, see Harrison '761, very little is known about the initial and presumably controlling biochemical events which occur in Friend cells after stimulation by an inducing agent. In addition to DMSO, a variety of other organic solvents also induce hemoglobin J. CELL. PHYSIOL. (1978)94: 275-286.

synthesis in these cells (Tanaka et al., '75; Preisler and Lyman, '75; Reuben et al., '76). Friend cells can also be induced to differentiate by several purines and purine analogues (Gusella and Housman, '761, butyric acid (Leder and Leder, '75) and ouabain, a specific inhibitor of the Na+/K+ATPase (Bernstein et al., '76). The observation that such a wide variety of compounds induce Friend cell differentiation raises the question as to whether the different inducers act by distinct or common mechanisms. Because ouabain appears to induce Friend cell differentiation via its binding to the Na+/K+ ATPase (Bernstein e t al., '76), a membrane-bound enzyme involved in Received Aug. 15, '77.Accepted Oct. 4, '77.

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was measured as follows. I4C-labelledAIB, 2.0 pM and 0.1 pCi/ml of tissue culture fluid, was added to cells in normal medium plus 10% fetal calf serum in a 37" shaker bath. The incorporation was stopped by pipetting a 2-ml cell sample into 8 ml of ice cold PBS containing 10mM unlabelled AIB. The cell sample was pelleted by centrifugation and washed MATERIALS AND METHODS twice in cold PBS containing 10 mM AIB. The Cell cultures resulting pellet was resuspended in 1ml H 2 0 Suspension cultures were maintained in ex- and added to 10ml Aquasol (New England ponential growth in a medium (Stanners e t Nuclear) which was counted for 14Cin a scinal., '71) supplemented with 10% fetal calf tillation counter. serum (Reheis). The cultures were grown a t To measure the uptake of 2-amino-bicyclo 37°C in a 5%C 0 2 atmosphere. [2,2,ll-heptane carboxylic acid (BCH), 14C-laThe inducible Friend erythroleukemic cell belled BCH, a t a final concentration of 0.4 line used, 745a JG, was derived from line 745a pCi/ml and 0.09 mM, was added to cells in norisolated by Doctor C . Friend, and provided by mal medium plus 10% fetal calf serum to J. Gusella. The non-inducible variant line, which 10 mM phenylalanine had been added M18D1, was isolated from 745a by continuous immediately prior to the experiment. Phenylgrowth in suspension in 1.5%DMSO for three alanine competes with BCH for uptake and its weeks after which time the cells were cloned presence is necessary to slow the uptake of in the absence of DMSO. One clone obtained 14C-BCHso that it can be measured in a 10in this manner was M18D1. The cell line minute time interval (Christensen e t al., '69). M18DlR, a spontaneous revertant of M18D1, The cells were incubated a t 37°C in a shaker has partially regained the ability to differen- bath and the uptake was stopped by pipetting tiate in response to DMSO. The variant line a 2-ml cell sample into 8 m l of ice cold PBS 745-TG-13 is a thioguanine-resistant, spon- containing 25 mM phenylalanine. The protaneous, DMSO non-inducible clone provided cedure after this point was the same as in the 14C-AIBuptake experiments. by Doctor D. Housman. The amount of extracellular label in these 86Rbuptake experiments was again estimated by using The rate of K' ion uptake was measured parallel cultures to which l4C-inu1in was using the K' analogue 86Rb+.86Rubidiumchlo- added. This background value, which was subride, a t a final concentration of 0.5-1.0 pCi/ml tracted from each time point measured, was and 0.5-1.0 pM, was added to exponentially independent of time and was approximately growing cells in normal growth medium con- 7%of the sample cpm of the 10-minute time taining 10% fetal calf serum. The cultures point. were maintained a t 37°C in a water bath Colchicine uptake shaker. At specified time intervals, samples The uptake of 3H-colchicine was performed were taken and the incorporation of 86Rb stopped by sedimenting the cell sample, con- as described previously (See e t al., '74). Cells taining 1-2 x lo6cells, in a clinical centrifuge were harvested by centrifugation and resusat 1,500 rpm for three to four minutes. Cell pended in PBS at a concentration of 2 X lo6 pellets were washed twice in phosphate-buf- cells/ml. This cell suspension was added to fered saline (PBS) and resuspended in 10 ml tubes containing 3H-colchicine a t a final conH,O. The incorporation of 86Rb was deter- centration of 1pM and specific activity of mined by measuring Cerenkov radiation in a 1Ci/mmole, and the cells were incubated at liquid scintillation counter. The amount of 37°C in a shaker bath. At specified time interextracellular 86Rb present in each sample vals, 1-ml aliquots were taken and added to after the washing procedure, estimated using 2 mi PBS containing 20 pM colchicine. The 14C-inulin in parallel experiments, was less cells were immediately pelleted by centrifugathan 1.0%of the total 86Rbcounted. tion and the cell pellet washed twice in PBS containing 20 pM colchicine. The pellet was Amino acid uptake then resuspended in 1ml HzO which was addThe uptake of a-aminoisobutyric acid (AIB) ed to a scintillation vial containing 1 0 m l

K' and Na' ion transport, in this study other Friend cell inducers have been examined to determine if early changes in cell membrane transport are a common step in the sequence of biochemical events culminating in the induction of hemoglobin synthesis in these cells.

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Aquasol and counted in a scintillation counter. Extracellular 3H-colchicine was again estimated using '*C-inulin and the value obtained, approximately 5% of the 20-minute point, was subtracted from each time point.

Cell volume determinations Cell volumes and numbers were determined using an electronic cell counter fitted with a pulse height analyzer as previously described (Loritz et al., '77). Benzidine staining Cells were stained for hemoglobin using liquid benzidine staining as described previously (Orkin et al., '75). Materials All radioactive isotopes were supplied by I

New England Nuclear. Ouabain octahydrate, colchicine, hypoxanthine and xanthine were obtained from Sigma Chemical Company. RESULTS

Effect of DMSO on "Rb transport A change in the rate of uptake of 86Rb,used as a measure of K' ion influx, was observed within six hours after the addition of DMSO to exponentially growing Friend cells. Figure 1 illustrates the time course of this change. The percent difference between the initial transport rate of *6Rbin control cells and the rate in DMSO-treated cells is plotted as a function of time after DMSO addition. As is evident from figure 1, the major change in 86Rbinflux began to occur four to six hours after DMSO addition. At this time the initial rate began to decrease and continued to decline until it had

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Fig. 1 Time course of the change in 86Rbuptake rate and cell volume in response to 1.5%DMSO. At each time point, 2.5 pCi ssRb was added to duplicate 5-ml aliquots of control and DMSO-treated cells. After 20 minutes, uptake was stopped by centrifugation and the cell samples processed as described in MATERIALS AND METHODS. The incorporation after 20 minutes can be taken as a measure of the initial rate of uptake since the uptake was linear for a t least 30 minutes (fig. 2). The average percent change in rate in DMSO-treated cells relative to the rate in control cells is plotted. The rate of 86Rbuptake in control cells measured throughout the experiment varied with a standard deviation of 2.6%. The average standard deviationof each percentage point, evaluated a t aparticular time, was 2.3%.Cell volumes of duplicate samples were determined using a n electronic cell counter and pulse height analyzer. In each case the peak channel, proportional to the medal volume of the cell distribution, was recorded. The average change in cell volume of the DMSO-treated cells is plotted. The inset shows, on a during the course of the experiment. The serni-logplot, the growth of control ( 0 )and DMSO-treated cells (0) percentages of benzidine-positive or hemoglobin-containing cells in the cultures, determined five days after DMSO addition, were 85% for DMSO-treated cells and less than 1%for control cells.

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stabilized a t around 12 to 14 hours, at which time the rate had decreased by 40-50%.Cell volume was also measured concurrently since an early volume decrease has been described in differentiating Friend cells (Loritz et al., '77).Figure 1 shows that 86Rbinflux began to decrease before the early volume decrease and that the magnitude of the decrease was greater. However, the shapes of the curves were in general quite similar. The growth rates of control and DMSO-treated cells were essentially the same for the first 12 hours after DMSO addition (inset fig. I).Therefore, changes in growth rate cannot explain this decrease in 86Rbtransport.

DMSO affects the ouabain-sensitive component of the "Rb influx The K+ ion influx in animal cells is comprised of both ouabain-sensitive and ouabaininsensitive components (Hoffman, '66). The major component is actively transported by the Na+/K+ATPase and is inhibited by ouabain. The ouabain-insensitive component, represented mainly by diffusion, does not require energy and is relatively minor in magnitude. To determine which component or components

of the transport of 86Rbshown in figure 1were being affected by DMSO, n6Rb uptake was measured 12 hours after DMSO addition both in the presence and absence of 1.0mM ouabain, a concentration sufficient to inhibit completely the active component of the 86Rb influx. Figure 2 illustrates the results of this experiment. In the absence of ouabain, the uptake rate in DMSO-treated cells was decreased by 42% of control level. However, in the presence of ouabain, the influx rate of 86Rb in both control and DMSO-treated cultures was the same, suggesting that it is the active transport component of Rb+ flux that is affected by DMSO treatment. From figure 2, the passive component of the Rb+ flux can be estimated as 25% of the total influx. Since this value does not appear to change within the first 12 hours after DMSO addition, the 42% decrease in total influx corresponds to a 56% decrease in the Na+/K+ ATPase-controlled portion of the Rb+ influx.

Effect of other inducers on 86Rbtransport Decreases in the rate of 86Rbuptake also occurred when other inducers of hemoglobin

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Fig. 2 Effect of ouabain on 86Rbinflux in control and DMSO-treated cells. At time zero, *"b was added to cell cultures in normal medium (circles) or medium containing 1.0 mM ouabain added five minutes before the addition of s6Rb (squares). At seven minutes intervals, duplicate samples were taken and processed as before. Closed symbols represent uptake in control cells and open symbols represent uptake in cells treated with 1.5% DMSO for 12 hours.

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synthesis were used. Figure 3 illustrates the changes in the initial rate of uptake of 86Rbas a function of time after the addition of several different inducing agents. All the inducers causedearly decreases in 86Rbuptake rates. In DMSO- and DMA-treated cells, the kinetics of the decrease in transport were very similar. In cells treated with hypoxanthine, the uptake r a t e began to decrease earlier and continued to decline in a more gradual fashion as compared to DMSO- or DMA-treated cells. Ouabain, which is known to inhibit directly K+ or Rb influx, is another inducer of Friend cell differentiation (Bernstein e t al., '76). When 0.15 mM ouabain, a concentration not significantly toxic t o ouabain-sensitive cells, was used to induce differentiation, the rate of 86Rbuptake was immediately inhibited by 3040%. This decreased transport rate remained relatively constant for at least 12 hours (fig. 3). We have also measured the effect of xanthineon 86Rbuptake. Xanthine, which is identical to hypoxanthine except for an additional hydroxyl group, does not induce hemoglobin synthesis in these cells (Gusella and Hous-

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man, '761, nor does its presence have any significant effect on the rate of M6Rb uptake (fig. 3 ) . Effect of DMSO on amino acid transport rates To determine whether the transport of other compounds was also affected within the first 12 hours after DMSO addition, the transport of two non-metabolizable amino acid analogues, a-aminoisobutyric acid (AIB) and 2amino-bicyclo [2,2,11-heptane-2-carboxylic acid (BCH), was measured. AIB is transported mainly by a carrier system dependent on the presence of a Na+ ion gradient across the membrane. If this gradient is destroyed, either directly by lowering the concentration of Na+ in the growth medium (Lever, '76; Christensen, '691, or indirectly by using ouabain to inhibit the Na+/K+ATPase (Heinz e t al., '75; Schutz and Curran, '701, the uptake rate of AIB is greatly reduced. In comparison, the transport rate of BCH, a synthetic amino acid, has been shown to be independent of a Na+ ion gradient in Ehrlich ascites tumor cells (Christensen et al., '69). Figure 4 illustrates the percentage change in the initial rate of uptake of 14C-AIBas a I

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1423F Fig. 3 Change in 86Rbuptake rate in cell line 745a JG in response to various chemical agents. The concentrations usedwere 0.15 mM ouabain (*), 10 mM N,N-dimethylacetamide(DMA) (W), 4 mM hypoxanthine (A)and 4mM xanthine (A).The procedure for obtaining each point was the same as in figure 1. The average standard deviation of each percentage point, evaluated at a particular time, was 3.7%for ouabain, 2.9%for DMA, 1.9%for hypoxanthine and 2.4%for xanthine. The percentages of benzidine-positivecells in the cultures on day 5 were 92% in DMA-treated cultures, 62%in hypoxanthine-treatedcultures, 29%in ouabain-treatedcultures and 2%in xanthine-treated cultures.

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Fig. 4 Effect of DMSO on W-AIB uptake. At each time point shown, the rate of uptake of "C-AIB in control and DMSO-treatedcells was measured as described in MATERIALS AND METHODS.The average percent change in rate in DMSO-treatedcells relative to the rate in control cells was plotted. The rate of "C-AIB uptake in control cells measured throughout the experiment varied with a standard deviation of 5.3%.The inset shows WAIB uptake in control cells ( 0 )and in cells treated with 1.5%DMSO for 12.5 hours (0).

function of time after the addition of DMSO. There was an initial small decrease in I4C-AIB uptake rate of about 10%. This was maintained for about 8 hours after which time the transport rate began to decrease sharply to 50%of control values 12.5 hours after addition of inducer. The rate of 86Rbuptake, measured concurrently, began to decrease four to five hours after DMSO addition (data not shown). To determine whether the observed decrease in the rate of AIB transport was a result of a decrease in the Na+-dependent uptake, I4C-AIB transport was measured 12 hours after DMSO addition both in the presence and absence of 0.2 mM ouabain. The initial transport rate of I4C-AIB in DMSOtreated cells was decreased by 55%relative to control level (fig. 5a). Dissipation of the Na' gradient by a one hour pre-incubation in 0.2 mM ouabain reduced the uptake rates of 14C-AIBin parallel cultures to those shown in figure 5a to the same levels in both control and DMSO-treated cell cultures (fig. 5b). This observation suggests that the decrease in AIB transport following induction was due to a decrease in the Na+-dependent uptake. The initial rate of uptake of I4C-BCHfollowing induction by DMSO was also measured in

an analogous experiment to the one just described for AIB. The results, shown in figure 6, indicate that the uptake of I4C-BCHwas also affected after a 12-hour exposure to DMSO. From the results of this and other experiments, the decrease in the initial rate of uptake of BCH varied between 25-30%of control values. A one hour pretreatment with 0.2 mM ouabain had only a small effect on I4CBCH uptake in both control and DMSOtreated cells (fig. 6b), suggesting that the uptake of BCH, as measured by our experimental procedures, was largely independent of a Na+ ion gradient in Friend cells. Colchicine uptake To determine whether the uptake of passively diffusing compounds was also affected by the action of DMSO, the uptake of 3H-colchicine was measured. Figure 7 illustrates the results of an experiment in which 3H-colchicine uptake was measured 15 hours after the addition of 1.5%DMSO. Three other experiments measuring colchicine uptake at times ranging from 8 to 24 hours after DMSO addition gave very similar results (data not shown). In all cases, the rate of uptake of colchicine in DMSO-treated cells was decreased

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a ) I4C-A1B Uptake (0.0 mM Ouabain)

1421 F Minutes Fig. 5 Effect of ouabain on 'T-AIB transport in control and DMSO-treated cells. a Uptake was measured as described previously in control cells ( 0 )and cells treated with 1.5%DMSO for 12 hours (0). b Uptake was measured in the same cell cultures after a one hour incubation in 0.2 mM ouabain.

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by only 5-15%.The magnitude of this decrease was much smaller than the decreases observed for 86Rbor 14C-AIBuptake rates.

pendently isolated non-inducible variants and a spontaneous inducible revertant isolated from one of these clones. The results, shown in table 1, indicate that the non-inducible clones can be divided into two classes-those t h a t exhibit the early transport changes, characteristic of inducible clones, after growth in DMSO (TG-13) and those that show only very limited changes in transport (M18D1). The small decreases in transport which are seen in M18D1 may be due to the small proportion of the cells which are still differentiating, as measured by hemoglobin-containing cells on day 5. Significantly, the spontaneous inducible revertant M18DlR, isolated from M18D1, also demonstrated the characteristic early change in the initial rate of transport of 86Rb and W-AIB. However, there was no significant difference between M18D1 and M18DlR in the uptake rate of Y - B C H in response t o DMSO (table 1). This observation suggests that a change in BCH uptake, as measured by our experimental procedures, may not be a general early event in Friend cell differentiation. This finding is consistent with the observation t h a t ouabain, another inducer of Friend cell differentiation, does not cause any significant change in the uptake rate of 14CBCH (fig. 6).These genetic results, obtained using the variant clones, provide additional evidence that early changes in the transport of certain compounds are an integral part of the differentiation process in these cells. Furthermore, as evident from table l, the early transport changes should be useful in the characterization of non-inducible Friend cell clones.

Early membrane transport changes in variant cell clones Friend cell clones can be isolated which have lost the capacity to synthesize hemoglobin in response to one or more of the chemical inducers. Such non-inducible clones have been isolated either without direct selection or after exposure to an inducer, such as DMSO. In order to establish whether the early memDISCUSSION brane transport changes observed after The results presented in this study have growth in the chemical inducers are an integral part of the differentiation process in shown that three widely different classes of these cells, the transport rates of 86Rb, 14C- compounds-organic solvents such as DMSO AIB, and 14C-BCHwere measured in two inde- and DMA, the purine hypoxanthine, and the TABLE 1

Transport changesin variant Friend cell clones Clone

Percent henzidine positive cells

Percent change in S6Rhuptake

Percent change in ‘ C A I B uptake

Percent change in “C-BCH uptake

745aJG TG-13 M18DlR Ml8Dl

85.523.117) 1.220.4(3) 52.22 1.8(2) 11.02 2.3(3)

-42.62 1:6(7) - 35.32 2.3(2) -31.62 1.7(3) - 12.82 1.2(3)

-52.52 1.1(3) -48.2 (1) (1) -46.7 - 14.52 2.5(21

-28.1-C 2.3(4) -28.6 (11 - 17.3-C1.9(2) - 16.52 1.2(2)

The uptake rates of 86Rh,“C-AIB, and 14C-BCHwere measured 12-14 hours after the addition of 1.5%DMSO to the culture medium. The initial transport rates were secured from the first 20 minutes of uptake of 86Rh,the first 10 minutes of uptake of “C-AIB and the first 4 minutes of uptake of ‘%-BCHsince at those times the uptake in each case was still linear. Results are expressed as the percent difference in the initial rate of uptake between control and DMSO-treated cells measured at the same time. Benzidine staining was done on day 5. Entries are the mean and standard deviation of the mean with the number in parentheses indicating the number of experiments used for each determination.

TRANSPORT CHANGES DURING FRIEND CELL DIFFERENTIATION

cardiac glycoside ouabain-all bring about decreases in the transport of K+ ions (as measured with 86Rb)within the first eight hours of Friend cell differentiation. The decrease in t h e ouabain-sensitive component of AIB uptake, in response to DMSO, reflects a decrease in the activity of amino acid transport system A, which is directly dependent on a Na' gradient (Christensen, '69). This decrease in t h e activity of system A could result from a decrease in the Na+ gradient across the membrane or from a change in the transport system itself. Since complete inhibition of the Na' /K+ATPase by brief incubation with ouabain reduces the transport of 86Rband AIB to the same level in both control and DMSOtreated cells, the decrease in AIB transport, like the change in 86Rbtransport, can be attributed to changes in the Na+/K+ATPase. The observation t h a t the uptake of BCH was also decreased by DMSO treatment indicates t h a t the transport of a t least one compound, which is independent of a Na+ gradient, is also affected by treatment with DMSO. However, BCH uptake is not significantly affected by ouabain, another Friend cell inducer. In addition, the results using the variant clones M 1 8 D l and M18DlR indicate that while the changes in 86Rband AIB transport correlate w i t h the ability to synthesize hemoglobin, the change in BCH transport does not. Several observations suggest that the observed transport changes are not due to a generalized decrease in membrane permeability. First, the passive diffusion of K+ ions, represented by the ouabain-resistant component of the 8'Rb flux, was identical in control and DMSO-treated cells. Secondly, the uptake of colchicine, a drug which is taken up by passive diffusion (Carlsen et al., '761, was also not significantly affected by growth in DMSO. The above discussion suggests that the organic solvent inducers and hypoxanthine, as w e l l as ouabain, all bring about changes in the transport properties of the Na+/K+ ATPase. Ouabain is unique in two respects: First, unlike the organic solvent Friend cell inducers, ouabain stimulates differentiation as a result of i t s direct binding to the Na+/K+ ATPase (Bernstein et al., '76). Secondly, inhibition of t h e Na+/K+ATPase by ouabain is immediate, while the observed decrease in 86Rbuptake described in this study after growth in DMSO, DMA or hypoxanthine require about ten hours to reach the same value as brief exposure to ouabain. The mechanism by which the organic

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solvent inducers and hypoxanthine act to cause these transport changes is not evident from the present study. Alterations in the phospholipid composition of the lipid bilayer can change the activity of the Na+/K+ATPase (Kimelberg and Papahadjopoulos, '72). It has also been observed that DMSO and other cryoprotective agents which induce Friend cells cause decreases in fluidity and stabilize phospholipid membranes (Lyman et al., '76). Therefore, it is possible that the decrease in 86Rbtransport following exposure to DMSO is the result of changes in membrane phospholipids that specifically are associated with the Na+/K+ ATPase. However, it should be noted that much higher concentrations of solvents were used to observe the changes in lipid fluidity (Lyman et al., '76) than were used to induce Friend cells and to observe the transport changes described here. Furthermore, the 6- to 8-hour time lag in the observed decrease in transport suggests that these transport changes may not simply be the result of insertion of the inducers into the lipid bilayer but rather are the result of a cellular response to the presence of inducer. The transport changes described here are among the earliest events associated with Friend cell differentiation. Early changes have also been observed in the rates of RNA and protein synthesis in Friend cells exposed to DMSO, as measured by the incorporation of uridine and leucine into RNA and protein, respectively (Sherton and Kabat, '76; Mager and Bernstein, unpublished observations). In addition, an early volume decrease has been observed eight to ten hours after the induction of Friend cell differentiation (Loritz et al., '77). The volume decrease may be a reflection of a drop in intracellular K+ ion content, brought about by a decrease in K+ uptake, since it has been shown in many cell systems that one of the major functions of the Na+/K+ ATPase, through its control of intracellular Na+ and K+ ions levels, is to regulate cell volume (Tosteson, '64). However, until the actual intracellular levels of the monovalent cations have been measured during Friend cell differentiation, the relationship between the early volume shift and the intracellular levels of K+ remains unproven. Early changes in the distribution of inducible Friend cells around the cell cycle have also been recently reported (Terada et al., '77). These authors have observed a transient inhibition of initiation of S-phase 5 to 20 hours

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after induction, suggesting that DMSO is inducing partial synchrony in differentiating Friend cell populations by prolonging or trapping cells in the G1 phase of the cell cycle. This observation is particularly relevant to the transport studies reported here because others have previously shown, using mouse lymphoblasts, that there are cell cycle-related changes in the intracellular concentrations of Na+ and K+ ions, as well as changes in the unidirectional fluxes of these cations in G1 (Jung and Rothstein, '67). Decreased transport of AIB during early G1 has also been reported in CHO and L cells (Sander and Pardee, '73). Thus, the decrease in transport activity described here may be either the cause, or a reflection, of the transient DMSO-induced synchronization in G1 described by Terada e t al. ('77). A central question raised from the present studies is whether any of the transport changes represent a necessary step in Friend cell differentiation. Several observations indicate that they do. First, such widely different compounds as DMSO, DMA, hypoxanthine and ouabain all reduce 86Rb+ion uptake to roughly the same levels in differentiating Friend cells. Secondly, xanthine, a compound which does not induce Friend cell differentiation, also does not bring about the transport changes associated with Friend cell differentiation. Thirdly, the presence of only small changes in cell membrane transport in the non-inducible variant M18D1 after growth in DMSO, and the re-appearance of the larger, characteristic changes in transport of 86Rb and I4C-AIB in t h e inducible revertant M18DlR, provides additional genetic evidence for a correlation between inducibility and the early changes in transport of 86Rband AIB described in the present study. Finally, ouabain, a compound which specifically inhibits the Na+/K+ ATPase, is also a potent inducer of hemoglobin synthesis in these cells (Bernstein e t al., '76). Studies with the non-inducible Friend cell lines indicate that these variants can be divided into two classes-those which show very limited changes in transport, as represented by M18D1, and those which show the characterispcc early transport changes, as represented by TG-13. Thus, the transport changes described in this study may be useful as phenotypic markers in classifying non-inducible variants. This classification should be

of value in the elucidation of the complex series of events which occur during Friend cell differentiation. Several laboratories have shown that continuous exposure t o inducer for approximately 24 to 48 hours is required before the cells become irreversibly "committed" to a second, inducer-independent, phase characterized by the late events in erythroid differentiation (Levy e t al., '75; McClintock and Papaconstantinou, '74; Gusella e t al., '76). While the molecular mechanisms underlying the early events in erythroid differentiation have yet to be elucidated, the experiments presented in this study suggest that these early transport changes are an integral part of Friend cell differentiation in culture. ACKNOWLEDGMENTS

The authors wish to thank Mr. Mark McCutcheon for his assistance with the cell volume determinations, and Doctors V. Ling and C. P. Stanners for their comments on the manuscript. We acknowledge discussions with Doctor Wolfram Ostertag, Gottingen, West Germany, who independently has observed early changes in the transport of uridine and 2-deoxyglucose during Friend cell differentiation. This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. Dixie Mager is a recipient of a Studentship from the Medical Research Council of Canada. LITERATURE CITED Bernstein, A., M. D. Hunt, V. Crichley and T. W. Mak 1976 Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell, 9: 375-381. Carlsen, S. A,, J. E. Till and V. Ling 1976 Modulation of membrane drug permeability in Chinese hamster ovary cells. Biochim. Biophys. Acta, 455: 900-912. Christensen, H. N. 1969 Some special kinetic problems of transport. Adv. Enzymol., 32: 1-20. Christensen, H. N., M. E. Handlogten, I. Lam, H. S. Tager and R. Zand 1969 A bicyclic amino acid to improve discriminations among transport systems. J. Biol. Chem., 244: 1510-1520. Friend, C., W. Scher, J. G. Holland and T. Sato 1971 Hemoglobin synthesis in murine virus-induced leukemic cells in uitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. (U.S.A.),68: 378-382. Gusella, J., R. Geller, B. Clarke, V. Weeks and D. Housman 1976 Commitment to erythroid differentiation by Friend erythroleukemia cells: a stochastic analysis. Cell, 9: 221-229. Gusella, J., and D. Housman 1976 Induction of erythroid differentiation in uitro by purines and purine analogues. Cell, 8: 263-269.

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Early transport changes during erythroid differentiation of Friend leukemic cells.

Early Transport Changes during Erythroid Differentiation of Friend Leukemic Cells DIXIE MAGER AND ALAN BERNSTEIN Department ofMedical Biophysics, Univ...
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