Presence of Spectrin in Untreated Friend Erythroleukemic Cells. Its Accumulation upon Treatment of the Cells with Dimethyl Sulfoxide GIOVANNI B. ROSSI,2 PATRIZIA ADUCCI,3ROBERTO GAMBARI,4 MAURIZIO MINETTI AND PATRIZIA VERNOLE

* Laboratorio di Malattie Batteriche e Virali, Zstituto Superiore di Sanita, Rome, Ztaly and Cattedra di Microbiologia, University of Rome, Rome, 00161, Ztaly; Laboratorio d i Biologia Cellulare e Zmmunologia, Zstituto Superiore di Sanita, Rome, 00161, Ztaly and ‘Zstituto di Biologia Generale, University of Rome, Rome, 00161, Ztaly

\

ABSTRACT Friend leukemia cells (FLC) are nucleated erythroid precursors, and are markedly stimulated towards more advanced stages of differentiation by treatment with dimethyl sulfoxide (DMSO). The presence of spectrin, an erythrocyte membrane protein, has been investigated in untreated and in DMSO-treated FLC by indirect immunofluorescence and by analysis in SDSpolyacrylamide gel electrophoresis of low-ionic-strength cell extracts immunoprecipitated with a monospecific anti-spectrin serum. Spectrin is detectable in significant amounts in the “inducible” clones prior to DMSO stimulation, and accumulates 4- to 5-fold upon addition of this compound to the cultures. Spectrin accumulation occurs rather early (24 hours after cell seeding) and reaches its peak on the third day, to decline thereafter. Semiquantitative determinations of spectrin amounts present in DMSO-stimulated 745A and A o l cells on the third day after treatment were 2.4 x lo5 and 3.0 X lo5 molecules/cell, respectively. Spectrin is also detectable in very low amounts in an “uninducible” line of FLC, and is not accumulated upon DMSO treatment thereof, whereas treatment with hemin does cause a significant increase of spectrin-positive cells. These data indicate that spectrin is a convenient “early” marker for in vitro studies of erythropoiesis. Friend leukemia cells (FLC) are murine proerythroblasts committed to erythroid differentiation and rendered leukemic by infection with Friend leukemia virus (FLV). Exposure of FLC to dimethyl sulfoxide (DMSO) as well as to several structurally related and unrelated compounds caused an increased s y r thesis of heme, hemoglobin (Hb) and globin mRNA, the appearance of erythroid membrane antigens and a decrease in the expression of the major histocompatibility antigens, H-2, on the cell surface (data reviewed in Friend, ’77). Genetic studies suggest that these compounds may not have a single common mechanism. Recently, however, it has been claimed that an early change of the N a + / K + equilibrium at the plasma membrane level may represent a metabolic step common to all “inducers” (Bernstein et al., ’76; Mager and Bernstein, ’78).

~

J. CELL.

PHYSIOL. (1978)97: 293-304.

Among the membrane proteins of mammalian red blood cells spectrin is a well studied peripheral complex associated with the inner surface of the plasma membrane, and it accounts for about 20-30% of the total membrane proteins. It appears to consist primarily of two large polypeptide chains of about 210,000-250,000 molecular weight (Fairbanks et al., ’71). Although spectrin could not be detected in homogenates of several different human nonmuscle cells, antibodies directed to human uterine smooth muscle myosin showed a small but significant reaction to human erythrocyte spectrin (Painter e t al., ’75; Sheetz et al., ’76). Recent data, however, obtained with a newly Received Feb. 22, “78. Accepted May 9, ‘78. ’ This work was supported in part by grants from N.A.T.O. (No. 1152), Consiglio Nazionale delle Ricerche (CNR), Progetto Finalizzato Virus (No. 77.00304.84). Rome.

293

294

G. B. ROSSI, P. ADUCCI, R. GAMBARI, M. MINETTI, P. VERNOLE

developed radioimmunoassay demonstrate t h a t spectrin is not antigenically detectable in non-erythrocyte cells (Hiller and Weber, ’771, which makes i t a likely candidate for a membrane marker of erythropoiesis. In view of its apparent specificity for erythrocyte membrane and of the scanty information available about its presence and role in nucleated erythroid precursors, i t was of interest to investigate whether spectrin was detectable in untreated FLC, t h a t are a pure population of such nucleated precursor cells. It was also relevant to ascertain whether DMSO-stimulated accumulation of hemoglobin (Hb) would be accompanied by the accumulation of another marker for erythropoiesis. The data reported in this paper demonstrate t h a t spectrin is detectable in t h e low-ionicstrength extracts of untreated FLC. Following exposure to DMSO, increased amounts of spectrin have been measured in the differentiating cell population. Spectrin is barely detectable also in untreated cells of an “uninducible” clone of FLC. While i t does not accumulate following DMSO stimulation, it does so upon treatment with hemin, thereby providing a suitable internal control. While this work was close to being concluded, spectrin presence in untreated FLC and its accumulation in DMSO-stimulated cells has been independently reported by Eisen et al. (’77). Our data confirm and expand their observations inasmuch they (a) were obtained working with different FLC cells and clones and (b) comprise a most suitable internal control such as the “uninducible” clone of FLC. MATERIALS AND METHODS

All reagents used were of analytical grade. Concentrations of protein extracts was achieved either by pressure dialysis (Amicon Corp., Lexington, U.S.A.) or by lyophilization, with no detectable differences.

Extraction and purification of spectrin Fresh human erythrocytes were washed three times by centrifugation in saline. After each centrifugation leukocytes were removed by aspiration. Erythrocyte ghosts were prepared by the standard method of Marchesi (’74). Extraction of spectrin from ghosts was carried in 0.1 mM EDTA (pH 8.0) a t 37°C for 15minutes as described by Marchesi (’74). Ex-

traction was carried out both in the presence and in the absence of 1mM phenylmethylsulfonylfluoride with no detectable differences in the densitometric profiles. Spectrin was purified free of small molecular weight contaminants by Sepharose 4B gel chromatography in lOmM deoxycholate according to Schechter et al. (‘76). Aliquots of purified concentrated preparations, eluted with 10 mM deoxycholate, 40 mM Tris, 0.1 mM EDTA, pH 9.0, were run on sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) according to Weber and Osborne (’691,except for acrylamide and bis-acrylamide final concentrations t h a t were 5.6% a n d 0.21%, respectively. Gels were stained with Coomassie Brilliant Bleu. The electrophoretic pattern was obtained by scanning the gel, with Gilford Spectrofotometer equipped with a gel scanner apparatus, at 550 nm. Concentrations of purified spectrin were measured spectrophotometrically: E, % in 10 mM deoxycholate at 280 nm = 10.1, according to Schechter e t al. (’76).

Spectrin antiserum Rabbits weighing about 3 k g were given 4 mg of purified spectrin in 2.0 ml of deoxycholate buffer vigorously mixed with a n equal volume of complete Freund adjuvant (BBLBioquest). The mixture was inoculated first in the foot pads (0.25 ml/foot) and then in t h e thigh muscles. The animals received weekly three additional intramuscular injections of 2 mg of spectrin in 1.0 ml deoxycholate buffer not mixed with Freund adjuvant. One week after the last injection, t h e animals were bled by intracardiac puncture under anesthesia, the sera were collected and frozen at - 20°C. A serum sample obtained prior to immunization served as control. Antibodies against spectrin were detected by immunodiffusion and immunoelectrophoresis tests. The former was performed mainly according to t h e Ouchterlony’s agar double diffusion method in 0.7% agarose gels in Veronal-glycine 0.15 M NaCl pH 7.6 buffer. The diameter of wells was 7.5 mm and t h e distance between the centres of two wells was 11 mm. Immunoelectrophoresis was carried out according to the micromethod of Scheidegger (’55)in 1%agarose gels in 0.05 M Verona1 pH 8.6 buffer.

Cells The 745A (obtained from Doctor C. Friend, New York, New York), Fw (obtained from

SPECTRIN IN NUCLEATED ERYTHROID PRECURSORS

Doctor J. Paul, Glasgow, U.K.) and A”1 (obtained in our laboratory) lines and clones of FLC were used in this study. The 745A is the “standard inducible clone” for Hb production; the Fw line is not “inducible” by DMSO to Hb synthesis, whereas t h e A”1 clone, obtained according to t h e procedure outlined by Curtis and Weissman (’76) is highly inducible to Hb production by DMSO. A detailed description of t h e features of this clone will be published elsewhere (Affabris e t al., ’78). All clones were grown in Dulbecco’s modified Eagle’s medium (Eurobio, Paris) supplemented with 15% foetal calf serum. The culture medium to be used for the “induced” FLC was adjusted to 1.5%DMSO (v/v, Merck) or to M hemin (Sigma, prepared according to Ross and Sautner, ’76) before t h e cells were added. Cells were seeded a t 1-3 x 105/mland grown in nonagitated suspension cultures without any further medium change. Control and DMSOtreated cultures reached on day 5 cell densities of 2.5-3 x 106/ml and had viability (as judged by trypan bleu dye-exclusion test) greater than 95%.Periodic assays of all cell lines for mycoplasma contamination, using PPLO broth cultures and the uridine phosphorylase assay of Schneider et al. (’741, were negative.

Immunofluorescence tests

295

incubation a t 37°C for 30 minutes and repeated washings in PBS and H20, slides were dried, mounted in buffered glycerol and observed with a Leitz fluorescence microscope.

Low-ionic-strengthextraction of Friend leukemia cells Preparation of a low-ionic-strengthextract was carried out essentially according t o Marchesi (’74) except for an additional centrifugation a t 4°C for ten minutes a t 600 x g , carried out shortly after lysing the cells in order to discard the nuclei. Samples of the lowionic-strength extracts were run on SDSpolyacrylamide gels as described above. Once again, in the experimental conditions employed, densitometric profiles of low-ionicstrength extracts obtained either in the presence or in the absence of 1 mM phenylmethylsulfonylfluoride did not detectably differ.

Immunoprecipitation of spectrin and of low-ionic-strength extracts of FLC Purified spectrin and FLC extracts were incubated with spectrin antiserum in vast antibody excess (as judged by immunodiffusion tests) for 24 hours a t 4°C. Samples were centrifuged for two hours a t 4°C a t 700 X g . The immunopellets were resuspended in 0.1 M sodium phosphate pH 7.0, 1%SDS and 1% beta-mercaptoethanol buffer. Supernatants were lyophilized and resuspended in the same buffer.

Washed samples of 1.5-2 X lo5 cells, suspended in 0.3 ml medium supplemented with 15%foetal calf serum, were centrifuged a t 300 RESULTS rev/min for five minutes on to microscopy Analysis ofpurified spectrin and rabbit slides in a Cytocentrifuge (Shandon, London), antiserum against spectrin fixed in 3%formaldehyde in cold isotonic phosPurification of erythrocyte spectrin is comphate-buffered solution (PBS) pH 7.2, for 20 minutes. Additional optional fixation in plicated by self-association and by interacchilled acetone for ten minutes did allow to tions with other erythrocyte membrane prostore slides a t - 20” in the dark so that immu- teins such as actin (Schechter et al., ’76). Gel nofluorescence tests could be performed later. chromatography in deoxycholate may free Replicate samples fixed in formaldehyde only, erythrocyte spectrin from contaminating needed to be processed and tested within one membrane lipids and from other membrane week, but exhibited a more brilliant immuno- proteins (Schechter et al., ’76). SDS-gel electrophoresis showed that spectrin purified fluorescence. All samples were washed in PBS, dried, according to this procedure consisted only of covered by a drop of antiserum and incubated the two characteristic polypeptides (fig. l a ) , at 37°C for 30 minutes in a humidified atmo- even under conditions of gel overloading. The rabbit serum prepared versus purified sphere. After repeated washings with the same buffered solution, they were again dried, spectrin was reacted against both the purified covered by a drop of fluorescein-conjugated protein and the crude 0.1 mM pH 8.0 EDTAgoat antiserum (diluted one-eighth) against soluble extract in an Ouchterlony’s double difrabbit gamma-globulins (Cappel Laboratories, fusion test. In both instances only one line of Downingtown, Pennsylvania). After a further precipitation was visible with an “identity”

296

G. B. ROSSI, P. ADUCCI, R. GAM BARI, M. MINETTI, P. VERNOLE

.oo

1

E,

a

Lo 0

I n

a-

eE 0.50 2

a

I

1

2

3

L

5

6

Gel length (cm) Fig. 1 Densitometric profiles obtained by scanning SDS-polyacrylamide gels with a Gilford linear transport a t 550 nm. Proteins were stained with Coomassie Brilliant Blue. Molecular weight values were determined from a standard curve according to t h e procedures of Weber and Osborne ('69), using t h e following standard proteins: (1) thyroglobulin (167,5001, (2) bovine serum albumin (68,000), (3) catalase (60,0001 and ovalbumin (43,000). a Purified spectrin, 30 pg. b Purified spectrin immunoprecipitated with vast excess of antispectrin antiserum and resuspended as in MATERIALS AND METHODS.

c Supernatant from t h e immunoprecipitation described in (b).

pattern (fig. 2a). This test demonstrates that the antiserum contained only one detectable class of antibodies even when reacted against a crude membrane extract. This was also shown by a n immunoelectrophoresis test (fig. 2b) using the same above-mentioned antigens as reactants. Aliquots of purified spectrin were immunoprecipitated with excess antiserum (see leg-

end to fig. 2). Both supernatant and immunopellet were run on SDS-PAGE. The amount of spectrin employed was all visible in the appropriate region of the gel in the case of t h e immunopellet (fig. l b ) whereas no traces of spectrin were observed in the same region in the case of the supernatant (fig. l c ) . It is apparent, however, that three peaks are visible in the spectrin region of the immunopellet (fig. l b ) as opposed to the two usual peaks observed when the same spectrin preparation was run prior to immunoprecipitation (fig. la). This finding may be due to minor degradation of spectrin taking place during the immunoprecipitation procedure. In spite of this discrepancy, this test convincingly shows t h a t spectrin precipitation by our antiserum is specific and complete. The densitometric profile of the gel regions following t h a t of spectrin is indistinguishable from t h a t of non-precipitated immunserum, run on a parallel gel. Evidence for a complete specificity of spectrin precipitation by a monospecific antiserum is also derived from the following observations recently reported in the literature: (a) antibodies against myosin cross-react weakly but specifically against spectrin, whereas antibodies directed versus spectrin do not cross-react with myosin (Sheetz et al., '761, which rules out that our serum could also precipitate myosin in our experiments; (b) although the anti-spectrin antibodies are directed exclusively to the heavier component of the dimer spectrin complex, in aqueous solutions both components are stoichiometrically precipitated by the antibodies (Sheetz et al., ' 7 6 ) . This is of course in keeping with data shown in figure 1 (panels a-c).

Detection of spectrin in FLC by immunofluorescence Cytocentrifuged sediments of FLC cultures a t various days after cell seeding with/without 1.5% DMSO were first overlayed with spectrin antiserum and then stained with a goat anti-rabbit gamma-globulins serum conjugated with fluorescein-isothyocianate in an indirect immunofluorescence reaction. As shown in figure 3a, the large majority of cells were weakly fluorescent in untreated 745A cultures. This fluorescence, even if weak, was still clearly stronger than that observed in untreated Fw cells (fig. 3b). Fluorescence had a spotty submembraneous distribution. A much more vivid fluorescence is visible in a large fraction of cells from DMSO-treated

297

SPECTRIN IN NUCLEATED ERYTHROID PRECURSORS

Fig. 2a Ouchterlony's double diffusion method for immunodiffusion, as in MATERIALS AND METHODS. A,, crude 0.1 mM EDTA pH 8.0 of erythrocyte ghosts a t a protein concentration of 2 mg/ml; A2, purified spectrin at a concentration of 1.3 mg/ml; AS,same as in A?, but a t a concentration of 0.65 mg/ml; P, preimmunization serum; S,rabbit serum from animals immunized with spectrin; S, and S, same as in S, diluted 1:2 and 1:4, respectively; X,no fluid added. b Immunoelectropboresis test according to Scheidegger's micromethod. A, and A2, as described in (a).The antiserum was run undiluted.

745A cultures harvested on day 1 (fig. 3c). Both the intensity of fluorescence and the percentage of positive cells increased in DMSOtreated 745A cells from day 1 to day 3 after cell seeding (fig. 3d). Less intensely fluorescent cells were observed on day 4 and 5. Fluorescence p a t t e r n s were even more prominent in A"1 cultures stimulated with DMSO. Also in this instance we observed an increase of intensity of fluorescence and of the percentage of positive cells from day 1to day 3 (fig. 3e), followed by a decline of the intensity of fluorescence, thereafter. In addition, differentiating A" 1 cells exhibit a more pronounced reduction of volume than do differentiating

745A cells (figs. 3e versus figs. 3c and 3d). A"1 cultures, grown in absence of DMSO, on the other hand, showed weakly fluorescent cells a t all time intervals from cell seeding. Following DMSO administration, the percent numbers of spectrin-accumulating (as measured by brightly fluorescent cells) and of Hb-containing (as measured by benzidine-positivity) cells in both inducible 745A and A"1 clones have been recorded. Data are shown in table 1. Benzidine-positive (B cells are significantly increased on day 2 or 3 whereas fluorescence-positivecells appear already on day 1 after DMSO addition. Both for 745A and A"1 cells a measurement

+

298

G. B. ROSSI, P. ADUCCI, R. GAMBARI, M. MINETTI, P. VERNOLE TABLE 1

Time-course study of the accumulation of spectrin and hemoglobin ' in DMSO-stimulated Friend Leukemia cells 745A cells Exposure time t o 1.5X7 DMSO

Brightly spectrin Dositive '

A"1 cells

Benzidineoositive I

Brightly spectrinpositive

BenzidineDositive '

%

x

x

days

%

zero

1 15

1 1

35 70 86 92

1.5 30 60 73

1 2 3 4

5

1 30 60 90 92 96

1

1 22.5 70 99 99

Expressed a s t h e percentage of Benzidine-positive cells, determined according t o the wet benzidine method IOrkin e t al.. '75). DMSO-stimulated cells were scored a s brightly fluorescent over the background of weak fluorescence observed in almost all untreated cells. I

TABLE 2

Quantification

' of spectrin by immunofluorescence in Friend Leukemra cells stimulated with DMSO

~

1

745A A" 1 Fw

Untreated cells

DMSO-stimulated cells

Time after seeding (days) 2 3 4

Time after seeding (days) 2 3 4

-

Clone

-

+-

2

*-

+

+?

2

+-

5 +

+-

1

+ ++ -

++ +++ -

+++ + +-+ +

+++ +++ ir

5

++ ++t -

++

1 The symbols. ranging from ( - 1 to I + + I . represent the arbitrary quantitation of the intensity of fluorescence made by the same individual who scored the cells in a single-blind fashion.

of t h e accumulated spectrin can be summarily derived from inmunofluorescence tests performed with increasing dilutions of t h e antiserum: on day 3, when t h e intensity of immunofluorescence was at its peak, untreated 745A and A o l cells showed specific fluorescence only with 1:2 diluted antiserum, whereas DMSO-stimulated 745A and A o l cells were fluorescent also with antiserum diluted 1:8 and 1:16, respectively. Cytocentrifuged cells of t h e Fw line were also scored for immunofluorescence with spectrin antiserum. They were consistently negative regardless as to whether DMSO had been added to t h e cultures. Only very few fluorescence-positive cells could be occasionally observed (fig. 3b). When, however, Fw cells were M hemin instead of DMSO, treated with fluorescence-positive cells were readily observed, being once again more numerous (27%) on day 3. Table 2 summarizes t h e immunofluorescence d a t a obtained from untreated and DMSO-treated FLC clones at t h e indicated days after cell seeding and DMSO treatment. Symbols, ranging (+1 to ( + 1, represent

+++

t h e arbitrary quantitation of t h e intensity of fluorescence, made by t h e same individual who scored t h e cells in a single-blind fashion. Criteria for specificity of immunofluorescence observations were as follows: (a) immunoserum had been previously and exhaustively adsorbed against 745A cells (10' viable cells incubated with 1 ml of serum for 1 hour at room temperature. This step was repeated 3 x ); (b) replicate samples of 745A cells incubated with preimmunization serum were fluorescence-negative; (c) incubation with fluorescein-conjugated anti-rabbit gammaglobulins serum in absence of spectrin antiserum did not show fluorescent cells; (d) incubation with non-conjugated fluorescein was likewise negative. Fig. 3 Formaldehyde-fixed Friend leukemia cells stained for spectrin by indirect immunofluorescence. M a g nification x 1,500. (a) untreated 745A cells, and (b) untreated Fw cells, both collected on day 4 of culture. (c) DMSO-stimulated 745A cells collected one day after cell seeding and administration of the compound. (d) same as i n ( c ) , but collected 48 hours later. (e) DMSO-stimulated A"1 cells collected three days after cell seeding and DMSO administration.

SPECTRIN IN NUCLEATED ERYTHROID PRECURSORS

Figure 3

299

300

G. B. ROSSI, P. ADUCCI, R. GAMBARI, M. MINETTI, P. VERNOLE

0.5

5

a

0.C

l0 n

ln

al

spectrin

0.3

0 C

CD

e 0,

2 0.1

0

0.5

1

0

1.5

0.5

-

spectrin

CONTROL

I

II

d

C

-

spectrin

spectrin 7

1

15

Gel length (cm)

Gel length (crn)

nll -

DMSO t

-

spectrin

Gel length (crn) Fig. 4 Densitometric profiles obtained as in figure 1. a 0.1 mM EDTA extract from 60 X lo6 untreated 745A cells. b Same as in (a); but from 60 X lo5DMSO-treated 745A cells collected on day 5 of culture. c Immunopellet obtained by precipitation of a 0.1 mM EDTA extract of 30 X lo6 untreated 745A cells with 10 p1 of rabbit serum against spectrin. d Supernatant from t h e immunoprecipitation described in (c). e As in (c) except for extracting DMSO-treated and not untreated 745A cells. f Supernatant from the immunoprecipitation described in (el.

-

PAGE analysis of spectrin in differentiating FLC After lysis, pellets of 10- to 50 x lo6 745A cells were extracted with 0.1 mM EDTA pH 8.0, as already done for red blood cells ghosts. After concentration, extracts were run on SDS-PAGE gels. Typical densitometric profiles of extracts from equal numbers of un-

treated and DMSO-stimulated 745A cells harvested on day 5 of culture are shown in figure 4 (panels a and b). Discrete amounts of two polypeptides with a molecular weight (M.W.) of 210,000-250,000 are visible in the case of untreated cells. Much higher peaks for polypeptides of the same M.W. are visible for DMSO-stimulated 745A cells (fig. 4b). (In addition to “spectrin” peaks, several other

SPECTRIN IN NUCLEATED ERYTHROID PRECURSORS

protein components with lower M.W.'s are visible in both figures 4a and 4b; they presumably represent other FLC membrane proteins and/or cytoplasm contaminants.) In view of the observed analogies in solubility and electrophoretic mobility, these data were suggestive of the presence of the two subunits of spectrin. In the extracts from DMSO-stimulated cells, these protein components are visible i n amounts roughly 4-fold higher than in the extracts from untreated cells. To prove that these polypeptides were indeed the two spectrin components, we immunoprecipitated the EDTA-extracts in conditions of vast excess of spectrin antiserum (see legend to fig. 2). Both immunopellets and supernatants were run on gels and their densitometric profiles are shown in figure 4 (panels c and d for untreated 745A cells and panels e and f for DMSO-stimulated 745A cells). All material having the indicated M.W.'s is visible in t h e two immunopellets (panels c and e) whereas no traces of the two polypeptides are observed in the supernatants (panels d and 0. Besides providing evidence for t h e presence of spectrin in extracts from 745A cells, this experiment also indicates that the amounts of antiserum employed were still in vast excess in spite of the fact that mouse, and not human, spectrin had been precipitated. Extensive cross-reactivitybetween spectrin from rodents and humans has been reported (Hiller and Weber, '77). We, therefore, conclude that polypeptides having the same M.W.'s and the same antigenic determinants of spectrin subunits are present in 4-fold larger amounts in differentiating 745A cells than in control cells. Both cell preparations were obtained on day 5 of cultures, i.e., a t the latest stage of differentiation available in this system. For quantitative purposes, areas under the peaks comprised in the 210,000-250,000 M.W. of densitometric profiles from EDTA extracts (figs. 4a,b) were compared with areas under peaks corresponding to known amounts of purified spectrin. Untreated and DMSO-stimulated 745A cells, harvested on day 5 after cell seeding, contained 0.03 pg/cell and 0.12 pg/ cell of spectrin, respectively. Assuming that spectrin is present in these cells as a dimer, these amounts correspond to 4 x lo4 and 1.8 x lo5 molecules/cell, respectively. We are aware of comparing amounts of a purified protein with amounts of the same protein present in a crude preparation obtained with ex-

301

traction procedures not shown to be exhaustive. That is to say t h a t the amounts of spectrin measured in this fashion in cell extracts may be underestimated. With this limitation, however, we wish to emphasize that the values measured are meaningful for comparative purposes. Moreover the value of molecules/cell estimated in DMSO-stimulated 745A cells is very close to the 2 x lo5 molecules/cell reported for human erythrocytes (Fairbanks et al., '71). It must also be pointed out t h a t , if one compares the 210,000-250,000 M.W.'s regions of panels c and e on one side, and of panels a and b, on the other side, it becomes apparent that three peaks are visible in the immunopellets as opposed to only two peaks in the non-immunoprecipitated EDTA extracts, as already noticed for figure lb. This discrepancy may be due to minor degradation of spectrin taking place during the time needed for the immunoprecipitation. Also for this reason all quantitative estimates of spectrin were obtained from non-immunoprecipitated EDTA extracts.

SDS-PAGE analysis of spectrin i n %on-inducible" Fw cells Pellets of 2 X lo8 Fw cells grown with/without DMSO and harvested on day 5, were lysed and extracted as previously described. Concentrated extracts were run on SDS-PAGE gels, and minute amounts of polypeptides peaking in the spectrin region were visible, without any detectable difference, between the extracts from untreated and DMSO-stimulated cells. Amounts comprised under the peaks were quantitated as previously described and found to be 0.004 pg/cells. It must be pointed out that in order to measure these low amounts of spectrin we had to overload the gels (fig. 5). Time-course studies of spectrin accumulation in untreated and DMSO-stimulated FLCclones Immunofluorescence data shown in table 2 were indicative of variable amounts of spectrin detectable as a consequence of DMSO stimulation. Table 3 shows the data obtained by SDS-PAGE analysis from the EDTA extracts of the two inducible clones (745A and A"1) a t various days after DMSO treatment. Spectrin amounts detected in untreated cells did not vary from day 1 to day 5 in both clones. After stimulation with DMSO, a 4-fold

302

E

L 0

G . B. ROSSI, P. ADUCCI, R. GAMBARI, M. MINETTI, P. VERNOLE

n

1.81 1.6

Lo u)

Q,

0

c m

-0 L

2 -0

a

I

~

L I

1

I

I

I

l

I

1 2 3 L 5 6 7 8 9 1 0

Gel length ( c m ) Fig. 5 Densitometric profile of 0.1 mM EDTA extract from 2.1 x lo8 DMSO-treated Fw cells collected on day 3. Scanning conditions as i n figure 1.

TABLE 3

Semiquantitative determination of the amounts of spectrin detected in 0.1 mM EDTA extracts of Friend Leukemia cells by SDS-PAGE '

In table 3, some 80% of the peak amount is present a t 24 hours whereas much less spectrin is observed according to t h e data of table 2. We believe that this discrepancy may be accounted for by the wide gap between t h e resolution powers of the two techniques employed. The immunofluorescent staining, on one hand, detects (by means of an arbitrary quantitation) the antigenic determinants of spectrin molecules only when their concentration per surface u n i t exceeds t h i t nf the threshold baseline of the technique. The SDSPAGE analysis, on the other hand, virtually measures the total spectrin available in t h e EDTA extracts, regardless of its structural organization in the cell and/or of t h e accessibility of its antigenic determinants. In addition, the arbitrary quantitation of spectrin presented in table 2 cannot account for the pronounced reduction of volume undergone by the differentiating cells (fig. 3, panels c-e). DISCUSSION

The evidence reported in this paper demonstrates that spectrin is present in untreated FLC. This data is of some relevance since Amounts of spectrin spectrin had not been sought for to-date in nu(pglcell) in clones Treatment cleated erythroid precursor cells a t a very ear745A AO1 Fw ly stage of differentiation. Our evidence is derived from both immunofluorescence staining None 0.03 0.04 0.004 1.5%DMSO f o r and gel-analysis of immunoprecipitated 0.1 1 day 0.13 0.15 0.004 mM EDTA-extracts of Friend cells. Untreated 2 days 0.14 0.17 0.004 FLC stained weakly and contained only 0.03 3 days 0.16 0.20 0.004 pg of spectridcell; however, confidence in t h e 4 days 0.13 0.17 0.004 5 days 0.12 0.13 0.004 significance of these findings was obtained by ' EDTA extracts of FLC (MATERIALS AND METHODS) and comparing (i) fluorescent-stained untreated known amounts of purified spectrin were run on SDS-PAGE. Areas 745A and Fw cells: although weakly, the 745A under peaks comprised between 210,000 and 250,000 daltons were cells did stain significantly stronger than did compared. Fw cells, and (ii) the amounts of spectrin measured in untreated 745A cells are small but increase of spectrin accumulation was de- significantly higher than those detected in tected already 24 hours later. Spectrin con- untreated Fw cells. Following DMSO administration, spectrin tinued to slowly accumulate until day 3 in both clones and declined thereafter. As also accumulated, percentage-wise, earlier than shown with immunofluorescence observations Hb in both inducible 745A and A"1 clones (fig. 3 and table 21, amounts of spectrin tested (table 1). Similarly measurements of detected in A"1 cells were always greater than spectrin show that a 4-fold accumulation (0.13 those found in 745A cells when both cultures and 0.15 pg/cell respectively) of the protein were exposed to DMSO. On day 3, these takes place 24 hours after DMSO administraamounts correspond to 2.4 x lo5and 3.0 X lo5 tion. This finding is not in keeping with those molecules/cell for 745A and A"1 cultures, re- reported by Eisen et al. ('77) who found t h a t a spectively. On the other hand, there is a dis- significant increase of spectrin accumulation crepancy between the SDS-PAGE data in takes place only a t 48 hours after DMSO table 3 and the immunofluorescent staining induction. However, the observed early accuresults in table 2 with regard to the amount of mulation of spectrin in DMSO-stimulated spectrin per cell a t 24 hours after induction. FLC is in good agreement with the data of

SPECTRIN IN NUCLEATED ERYTHROID PRECURSORS

Chang et al. (’76)who showed that spectrin is the protein whose synthesis is completed first, and well in advance if compared to hemoglobin, during the asynchronous course of in vivo protein production in t h e plasma membrane of red blood cells. Spectrin appears, thus, to be a convenient “early” marker for in vitro erythropoiesis as well. The observed decline of spectrin accumulation i n differentiating FLC after the third day of culture is a puzzling finding, also observed by Eisen et al. (’77).I t is certainly not what has been seen with globin that is accumulated until day 5 of culture. The mechanism for this loss of spectrin from t h e cells during the latest stages of erythroid differentiation remains to be determined. The DMSO-resistant Fw line has provided the most suitable internal control for a number of observations. (1) The extremely low amounts of spectrin detected in untreated Fw cells as compared with those found in untreated 745A and A”1 cells, may suggest t h a t Fw cells represent a population of “more early” erythroid precursors than cells of the other two clones. Gene dosage andlor phenotypic variability may represent alternative explanations. (2) Since the action of DMSO on differentiating Friend cells has been tentatively attributed to its interaction with the plasma membrane (Lyman et al., ’761,the accumulation of spectrin in DMSO-treated cells could have been interpreted as the mere result of a physico-chemical interaction of DMSO with the cell membrane (unmasking of spectrin? Increased extractability of spectrin?). This possibility is ruled out by the observed lack of spectrin accumulation in DMSO-stimulated Fw cells. (3) Moreover, the observation that spectrin is accumulated along with Hb in DMSO-stimulated differentiating 745A and A”1 cells, and is not accumulated in DMSO-stimulated nondifferentiating Fw cells, provides additional evidence for a specific role of spectrin in early erythroid differentiation as opposed to that of globin for terminal differentiation. The two “inducible” clones tested do express the erythroid markers under study in a coordinate fashion, but independent segregation of spectrin and Hb production has been reported by groups working on larger number of FLC variants (Harrison, ’77). The observation t h a t hemin-treated Fw cells do accumulate spectrin is also interest-

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ing as it indicates (i) that t h e spectrin gene is present and is functional i n the Fw variant, (ii) that the hemin and DMSO mechanisms of action on Friend cells are different (see also Ross and Sautner, ’761,and (iii) that a n exogenous supply of heme is apparently able to activate the package of genes leading to the synthesis of spectrin. One could also imply t h a t in the Fw cells DMSO is simply unable to activate the package of genes leading to t h e synthesis of heme. The surface-exposed proteins of mammalian erythrocyte membrane seem relatively immobile in the plane of the membrane, and in this respect they differ from those of nucleated mammalian cells (Loor et al., ’72;Singer, ’74; Bretscher and Raff, ’75;Nicolson, ’76).There is increasing evidence t h a t the role of the spectrin complex in erythrocytes may be that of maintaining cell shape and of restraining the lateral mobility of surface proteins and ligand-induced movements of antigens (Nicolson, ’76; Sheetz and Singer, ’77; Birchmeier et al., ’77).Nonetheless, spectrin or even immunologically cross-reacting molecules have not been identified in other cells (Painter e t al., ’75;Hiller and Weber, ’77)and this indicates that the erythrocyte membrane may be unique in its structure and organization. In t h e Friend system one is faced with two sets of observations, that apparently fall into a coherent pattern. There is a series of early events, namely accumulation of spectrin, decrease in membrane permeability and increase in membrane microviscosity (Harrison, ’77)followed by late events such as cessation of cell division, terminal differentiation (i.e., accumulation of hemoglobin) and cell death (possibly anucleation and production of erythrocytes). In this perspective, the accumulation of spectrin may be considered as a necessary step towards a more rigid state of the plasma membrane, in keeping with what has been already shown for erythrocytes (Sheetz and Singer, ”77). ACKNOWLEDGMENTS

The authors are indebted to Doctors C . Pini and S. Ioppolo for kind advice and help in carrying out immunodiffusion and immunoelectrophoresis tests. LITERATURE CITED Affabbris, E., S . Pulciani, G. B. Rossi and M. C. Capobianchi 1978 Erythroid differentiation of Friend Erythro-

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leukemic Cells: a “variant” cell line endowed with an early expression of erythroid markers. Microbiologica, l: In press. Bernstein, A,, D. M. Hunt, V. Crichley and T. W. Mak 1976 Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell, 9: 375-381. Birchmeier, W., and S. J. Singer 1977 On the mechanism of ATP-induced shape changes in human erythrocyte membranes. 11. The role of ATP. J. Cell Biol., 73: 647-659. Bretscher, M. S., and M. C. Raff 1975 Mammalian plasma membranes. Nature, 258: 43-49. Chang, H., P. J. Langer and H. F. Lodish 1976 Asynchronous synthesis of erythrocyte membrane proteins. Proc. Natl. Acad. Sci. (U.S.A.), 73: 3206-3210. Curtis, P. J., and C. Weissmann 1976 Purification of globin messenger RNA from dimethylsulfoxide-inducedFriend cells and detection of a putative globin messenger RNA precursor. J. Mol. Biol., 106: 1061-1075. Eisen, H., R. Bach and R. Emery 1977 Induction of spectrin in erythroleukemic cells transformed by Friend virus. Proc. Natl. Acad. Sci. (U.S.A.), 74: 3898-3902. Fairbanks, G., T. L. Steck and D. F. H. Wallach 1971 Electrophoretic analysis of the major polypeptides of the human e r y t h r o c y t e membrane. Biochemistry, 10: 2606-26 17. Friend, C. 1977 The phenomenon of differentiation in murine erythroleukemic cells. Harvey Lect., in press. Harrison, P. R. 1977 The biology of the Friend cell. In: Biochemistry of Cell Differentiation. Series 2. J. Paul, ed. MTP International Review of Biochemistry. H. L. Kornberg and D. D. C. Philips, eds. University Park Press, Baltimore, Vol. 15, pp. 227-267. Hiller, G., and K. Weber 1977 Spectrin is absent in various tissue culture cells. Nature, 266: 181-183. Loor, F., L. Forni and B. Pernis 1972 The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur. J. Immunol., 2: 203-212. Lyman, G. H., H. D. Preisler and D. Papahadjopoulos 1976 Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature, 262: 360-363. Mager, D., and A. Bernstein 1978 Early transport changes

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