Membrane dynamics of friend leukaemic cells.

Cell Differentiation, 9 ( 1 9 8 0 ) 211--218 © Elsevier/North-Holland Scientific Publishers Ltd.

211

MEMBRANE DYNAMICS OF FRIEND LEUKAEIVIIC CELLS. H. CHANGES ASSOCIATED WITH CELL DIFFERENTIATION

H. TAPIERO, A. F O U R C A D E and C. BILLARD

Ddpartement de Culture et Production de Cellules Humaines I.N.S.E.R.M. Unit~ 50, I.C.I.G., 16, Avenue P. V. Couturier 94800 Villejuif (France) Accepted April 15th, 1980

In a previous study, using fluorescence polarization (P) with diphenyl hexatriene (DPH), it was shown that growing and resting cells have different P-values. This property has now been used to investigate the membrane action of dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), two inducers of Friend leukaemic cell (FLC) differentiation. Both an inducible cell line (FLC) and a resistant variant cell line (RFLC), had the same characteristics regarding P-values. During the differentiation process in the FLC cell line, further changes in P-values were observed. In the resistant cell line, these changes in P-value were not seen. This suggests that these inducers of differentiation are acting at the cell membrane.

Friend leukaemic cells (FLCs) have been used as a model for the study of erythroid differentiation (Friend et al., 1971) since they can be induced to differentiate and synthesize hemoglobin in vitro when grown in the presence of a variety of structurally unrelated agents (Scher et al., 1973; Leder et al., 1975'.. Gusella et al., 1976; Ebert et al., 1976; Bemstein et al., 1976; Reuben et al., 1976; Ross et al., 1976). Among the agents particularly active as inducers are DMSO and HMBA, two polar-planar compounds which have a polar hydrophilic group and a planar hydrophobic pol~;ion. Their mode of action on this system is still unknown. However the properties of DMSO as a penetrant and cryoprotectant, its interaction with phospholipid vesicles increasing the phase transition temperature (Lyman et al., 1976), suggest that their major effect is on the cell membrane. Furtherraore, alteration of membrane properties during cell differentiation and malignant transformation have been described (Pollack et al., 1974). In our previous report (Fourcade et al., 1980), it has been shown that fluorescence polarization analysis of DPH labelled FLC reflects changes of cell membrane properties in different states of growth. The present study demonstrates that growth of FLC in the presence of DMSO or HMBA which induce erythroid differentiation alter these properties causing different fluorescence polarization values.

212 MATERIALS AND METHODS

Cell culture and stimulation o f globin synthesis. Cell culture of FLC were carried out as described in the previous work. Stimulation of globin synthesis in the inducible FLC was obtained by growing cells for 4--6 days in medium supplemented with 140--280 nM DMSO (Sigma) or 4 mM HMBA (kindly donated by Dr. Y. Gazit). Determination of benzidine reactive cells. Benzidine dihydrochloride (Sigma) 0.1 g was dissolved in 50 ml of dilute acetic acid (1.5 ml glacial acetic acid diluted to 50 ml with water) and stored at 40(3 in the dark. Just before use, a 0.5-ml portion of the benzidine solution was dispensed into a test tube at 4°C, and 10 ul of 30% H202 were added followed by immediate mixing. To this mixture, 0.5 ml of a culture containing from 2 × l 0 s to 2 × 106 cells was added and swirled rapidly during the addition. After two minutes incubation at 4°C, the percentage of blue (benzidine reactive) cells was determined in a Thoma hemocytometer. Approximately 200 cells were counted. As a control the percentage of benzidine reactive (B+) cells was determined from the non-induced cultures. Isolation o f non-inducible variant of FLC. A variant of FLC (derived from line 745 A) which is unable to synthesize hemoglobin in response to DMSO and HMBA was isolated in our laboratory by seeding inducible cells at 0.5 × 106 cells/ml in medium supplemented with 280 mM DMSO. These cells were transferred every 5 days for 3 months. They have now a low base level of B+ cells, ranging from 0% to 2% when grown in medium supplemented with DMSO or HMBA. Fluorescence polarization measurements. The stimulated and the unstimulated FLC or RFLC were labelled with DPH as described previously. Briefly, aliquots of 1 × 107 cells were incubated 30 min at 37°C in 3 ml of 2 ~M DPH dispersed in phosphate buffer saline. The fluorescent labelled samples were subjected to fluorescence polarization analysis with an Elscint microviscosimeter (Israel). All fluorescence measurements were carried out at 37°C. RESULTS

We have recently reported (Fourcade et al., 1980) that fluorescence polarization of DPH labelled FLC varies under different growth conditions. Maximum and minimum P-values were related to growing and resting state respectively. These P-value changes were shown to be controlled by an active process which suggest that changes occur in the plasma membrane during cell growth. Identical results were also obtained with an FLC variant which is resistant to DMSO induction (RFLC) when grown in the same conditions. In both cell lines, fetal calf serum (FCS) interfere with the P-value changes of growing cells by a non-active process without interfering with the stimuli inducing cell proliferation. The growth curves of FLC in

213

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DAYS DAYS Fig. 1. Comparison of the growth curves of FLC grown in presence of different concentrations of FCS and DMSO. Logarithmically growing FLC seeded at 0.5 x 106 cells/mi were grown in medium supplemented with (A) 5% or (B) 20% FCS and containing 140 mM ( o ~ e ) , 280 nM ( c - - e ) or no ( x ~ x ) DMSO. Viable cells were counted daily as described in Materials and Methods.

the proliferative state seeded at 0.5 × 106 cells/ml in medium containing 5% or 20% FCS and supplemented with 140 or 280 mM DMSO show (Fig. 1) that stationary phase was reached after 3--4 days. DMSO reduced the final cell yield. The benzidine positive (B+~ cells increased from 0--1% to 35--40% I00

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1234 DAYS DAYS DAYS Fig. 2. Fluorescence polarization analysis of FLC grown in presence of various concentration of FCS and DMSO. Aliquots of 1 x 10 "~ cells grown in medium supplemented with 5% or 20% FCS and containing 140 mM (o-------e), 280 mM ( ") or no (x-~×) DMSO, were DPH b~.belled and fluorescence polarization analyzed at 37°C as described in Materials and Methods. Fig. 3. Hemoglobin synthesis of DMSO induced FLC. were seeded in medium containing 280 mM DMSO. (x -~x ) or changed with fresh one without DMSO ( o - - - - - o ) of growth. FLC induction was determined Methods.

Logarithmically growing FLC The medium was unchanged after 24 h ( e ~ ® ) or 48 h as described in Materials and

214

by the fourth day in the presence of 140 mM DMSO (5% or 20% FCS), whereas in the presence of 280 mM DMSO, B+ cells increased to 45--50% and 6 5 - 7 0 % in the presence of 5% or 20% FCS respectively. The direct relationship between DMSO concentration, induction of B+ cells and inhibition of cell growth, suggest that the mechanism of induction may involve a change ill structure of a cellular component that might be incompatible with cell growth. Effects o f DMSO on fluorescence polarization o f DPH labelled FLC. In order to investigate these relationships, FLC grown under the above conditions, were labelled with DPH and fluorescence polarization analysis monitored every 24 h. The values of P (Fig. 2) varied according to amount of DMSO added to the medium. When FLC were grown in the presence of 140 mM DMSO, the P-values at resting state did not decrease as they did in non-induced FLC. Furthermore, with 280 mM DMSO which corresponds to optimal concentration for induction, P increases to the highest values we have observed. To maintain the high level of B+ cells, the continuous presence of DMSO in the culture medium is required (Fig. 3). When DMSO was removed after 24 or 48 h and replaced with fresh medium, only 2 - 4 %

260 240 P (37°C)

220 200 1 240 B

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220 200 240 C

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Fig. 4. Fluorescence polarization analysis of FLC grown in the presence of DMSO. Aliquots of 1 x 10 ~ treated cells -#ere DPH labelled each day and fluorescence polarization analyzed as described in Mateiials and Methods. A: FLC grown continuously in medium containing 280 mM DMSO. B: medium changed at 24 h or C: 48 h with fresh one without DMSO and cells were allowed to grow to 4 days.

215

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2 3 4 DAYS Fig. 5. Fluorescence polarization analysis of RFLC grown in presence of DMSO. Aliquots

of 1 x 10 ~ RFLC grown in medium supplemented with 20% FCS and containing 280 mM (e~e) or no (s-----o) DMSO were DPH labelled and fluorescence polarization analyzed as described in Materials and Methods.

a n d 25--'30% respectively o f t h e cells became B+ by t h e f o u r t h day. U n d e r these conditions, the P-values at resting state decreased to values corres p o n d i n g to t h e n o n - i n d u c e d F L C (Fig. 4). These results suggest t h a t the high P-values o f i n d u c e d F L C are n o t the c o n s e q u e n c e of cells being d i f f e r e n t i a t e d b u t r a t h e r due t o t h e m e m b r a n e a c t i o n o f DMSO. It was t h e r e f o r e o f interest t o us t o investigate the behavior of an F L C variant resistant t o DMSO i n d u c t i o n ( R F L C ) .

Effects of DMSO on fluorescence polarization o f DPH labelled RFLC. A d d i t i o n of 2 8 0 m M DMSO in t h e culture m e d i u m did n o t i n d u c e any e r y t h r o i d d i f f e r e n t i a t i o n . Cell g r o w t h was n o t i n h i b i t e d by DMSO and t h e final cell n u m b e r was even higher t h a n t h e n o n - t r e a t e d cells. In those cells, t h e presence o f DMSO (Fig. 5) did n o t prevent a decrease in P-values after

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Fig. 6. Comparison of the growth curves of FLC lind RFLC grown in medium containing 4 mM I-IMBAand different concentrations of FCS. Cells were seeded at 0.5 x 106 cells/ml in medium supplemented with 5%, 10% or 20% FCS and containing or not HMBA. Viable cells were counted daily as described in Materials and Methods. (e. e) FLC, (o~o) RFLC grown in medium containing 4 mM HMBA, (× .-× ) RFLC, grown in medium without HMBA.

216 TABLE I D E G R E E OF F L U O R E S C E N C E P O L A R I Z A T I O N P A T 3 T C O F DPH L A B E L L E D FLC AND RFLC GROWN IN MEDIUM S U P P L E M E N T E D WITH D I F F E R E N T C O N C E N T R A T I O N S OF FCS AND C O N T A I N I N G 4 mM HMBA Aliquots o f 1 × 107 FLCs a n d RFLCs grown in t h e presence o f 4 mM HMBA (see Fig. 6) were DPH labelled at 37rC and P-values d e t e r m i n e d as described in Materials and Methods. Days

0 1 2 4 5

5% FCS

10% FCS

20% FCS

FLC

RFLC

FLC

RFLC

FLC

RFLC

0.212 0.218 0.229 0.233 0.239

0.208 0.207 0.223 0.231 0.230

0.212 0.206 0.224 0.231 0.236

0.208 0.200 0.211 0.226 0.211

0.212 0.200 0.211 0.224 0.239

0.208 0.189 0.199 0.217 0.200

the cells had reached the resting state. Thus it appears that the DMSO resistance of RFLC reflects membrane property changes. Effects o f HMBA on fluorescence polarization of DPH labelled FLC and RFLC. Another polar-planar compound, HMBA was also used in these studies. This compound is particularly interesting because we have found that it stimulates 85- -90% of our cells to differentiate at relatively low concentrations (4 raM) without significantly inhibiting cell growth at different serum concentrations (Fig. 6). Furthermore, the FLC variant resistant to DMSO induction is also resistant to HMBA induction. Fluorescence polarization of FLC and RFLC grown in medium containing various amount of serum and supplemented with 4 mM HMBA was analyzed (Table I). The values obtained with HMBA were similar to those already described after DMSO treatment. However in the case of RFLC, the level of the decrease in P-values varied according to the amount of serum. It is therefore concluded that membrane action of DMSO and HMBA could be of the same nature. They induce changes in membrane properties of sensitive but not of resistant cells. DISCUSSION

The addition of DMSO, HMBA and several unrelated compounds to growing FLC induce a wide variety of erythrocyte markers (Friend et al., 1971; Ross et al., 1972; Ebert et al., 1974; Kabat et al., 1975; Conscience et al., 1977) and various membrane changes including accumulation of erythrocyte membrane antigen (Sugano et al., 1973) and spectrin (AmdtJovin et al., 1976). The large number of inducers, suggests that the action might not be specific and that the specificity of expression reflects an

217 intrinsic property of the target cell. This assumption can be supported by the findings that neuroblastoma differentiation (Kimhi et al., 1 9 7 6 ) a n d terminal differentiation of human promyelotic leukaemia cells (Collins et al., 1978} are also induced by DMSO and other polar corgpounds. Erythroid differentiation of the human leukaemic K 562 cell line is induced by sodium butyrate but not by DMSO {Anderson et al., 1979}. Whether the initial site of action of the inducing compound is altering membrane function with signals subsequently relayed to the nucleus is still unknown. It is possible that structural or conformational changes of FLC membranes are induced by these compounds. It has been reported that DMSO alters reversibly the configuration of proteins (Rammler et al., 1967). It also raises the melting temperature of the acyl chains in phospholipid artificial membranes, indicating a stabilization of the bilayers probably related to a decrease in the fluidity (Lyman et al., 1976}. Studies on the early events which occur in DMSO induced FLC have shown, reduced membrane permeability for phosphate, uridine and leucine (Dube et al., 1974), increased agglutinability by lectins (Eisen et al., 1977) and decrease in the cell surface glycocalyx (Sato et al., 1979). We have used the fluorescence polarization of DPH embedded in cell membranes to obtain a direct measure of P, which has been related to the microviscosity of the membranes (Shinitzky et al., 1974; Shinitzky et al., 1976; Fuchs et al., 1975). In the previous report, we have shown that P-value changes which occur during cell growth probably reflect changes in the dynamic nature of the cell membrane. As reported here, these changes can be altered by DMSO and HMBA, two inducers of FLC differentiation. It is shown that at the end of the logarithmic phase of growth, P-values continue to increase in treated cells, whereas they decrease to minimum values in untreated cells. The continuous presence of DMSO appears to be required, since its removal at the second day will result in a decrease of P-values after a delay of 24 h despite the presence of 25--30% differentiated cells. Whether P-value changes induced by these compounds are correlated either with induction process or with the differentiation itself, has not yet been determined. The fact that they are not obtained in RFLC show that they are correlated to the induction process which favor the assumption that the membrane is the primary site of action of these compounds and the resistance of FLC is itself due to a structural modification of membranes. ACKNOWLEDGEMENTS This work was supported by contract D.G.R.S.T. No. 7872639. REFERENCES Andemon, L.C., M. Jokinen and C.G. Gahmberg: Nature 278, 364--365 (1979). Arndt-Jovin, D., W. Ostertag, H. Eisen, F. Klimek and T.M. Jovin: J. Histochem. Cyto-

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