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Cell-surface glycoprotein synthesis during differentiation of chicken erythroblasts transformed by temperature-sensitive avian erythroblastosis virus
Cell Differentiation, 10 (1981) 163--171 Elsevier/North-Holland Biomedical Press
163
CELL-SURFACE G L Y C O P R O T E I N SYNTHESIS DURING D I F F E R E N T I A T I O N OF CHICKEN E R Y T H R O B L A S T S T R A N S F O R M E D BY T E M P E R A T U R E - S E N S I T I V E AVIAN E R Y T H R O B L A S T O S I S VIRUS KEITH W. SAVIN 1 and HARTMUT BEUG 2 1 Tumour Virology Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, L o n d o n WC2A 3PX, U.K.; and 2 Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg, F.R.G.
(Accepted 9 February 1981) Chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (ts34 AEV) have a greatly increased haemoglobin content (Graf, T., N. Ade and H. Beug: Nature 275, 496--501 (1978)) if allowed to grow for 3--5 days at the non-permissive temperature (41°C), instead of the permissive temperature (35°C) of the virus. Cell-surface molecular changes during this differentiation were investigated by examining the glycoproteins synthesized by a ts34-transformed erythroblast cell line. These cells synthesized a greatly increased amount of a 94,000 molecular weight erythrocyte cell-surface glycoprotein beginning 2--6 h after a shift in growth temperature from 35°C to 41°C, consistent with the proposal that such a shift releases these transformed cells from a differentiation block. differentiation
cell surface
glycoprotein
1. I n t r o d u c t i o n Avian erythroblastosis virus (AEV) is a replication-defective leukaemia virus causing acute erythroid leukaemia and slowly developing sarcomas in chickens (Graf et al., 1977). This virus specifically transforms fibroblasts and erythroblasts in vitro, and colonies of the latter can be obtained by transformation of chicken bone m a r r ow cultures (for review, see Graf and Beug, 1978). AEV and other viruses have been proposed to bring about leukaemic transf o r m a t i o n by blocking normal cellular differentiation (Graf et al., 1978a,b). The recent isolation o f a temperature-sensitive m u t a n t of AEV (designated ts34), which allows the synthesis of haemoglobin by virus-transf o r m e d erythroblasts u p o n a shift t o the nonpermissive t e m p e r a t u r e of the virus (Graf et al., 1978a), has lent f ur t her weight to this proposal. If the leukaemogenic mechanism of AEV does entail a block in normal e r yt hr oi d
erythroblasts
retrovirus
differentiation, analysis of the putative differentiation, perm i t t ed by the temperature-sensitive m u t a n t of AEV at the viral non-permissive temperature, should provide information about the biochemical activity of the AEV-transforming protein. In addition, it should establish w het her these cells are differentiating along the normal eryt hroi d developmental pathway or are taking an aberrant course. An analysis of t s 3 4 AEV-transformed erythroblasts was initiated along the lines of that applied to the Friend cell system (for review, see Harrison, 1977; Reuben et al., 1980). Although induction of Friend cell differentiation by chemical inducing agents might n o t be directly related to prevention of the function of a viral gene product, the Friend system provides good examples of the differentiation-associated changes one can expect to find when analysing t s 3 4 AEVtransformed cells. Differentiation can be p r o m o t e d in murine eryt hroi d cells transform ed by the Friend virus com pl ex by
0045-6039/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
164 inducing agents such as dimethyl sulphoxide. The differentiating Friend cells produce haemoglobin (Friend et al., 1971) and various other proteins including those associated with the plasma membrane, such as acetylcholinesterase (Conscience et al., 1977), transferrin receptors (Hu et al., 1977; Yeoh and Morgan, 1979) and spectrin (ArndtJovin et al., 1976). The loss of cell-surface H2 antigens (Arndt-Jovin et al., 1976) has also been demonstrated. We consider that the cell surface is a likely target area for the effect of a molecule designed to block differentiation. This could occur by, for example, preventing the synthesis or function of an essential hormone receptor or by disruption of specific ion channels. Analysis of the cell surface of ts AEV-transformed erythroblasts should provide information a b o u t possible differentiation and, in addition, establish a convenient marker for future monitoring of such differentiation. Preliminary cell-surface immunofluorescence experiments, which utilized specific anti-erythrocyte serum, indicated that cell-surface changes suggestive of erythroid maturation occurred on a majority of the ts AEV-transformed erythroblasts after a shift in growth temperature from the permissive (35°C) to the non-permissive temperature (41°C) of the virus. Neither an increase in the number of haemoglobincontaining cells nor cell-surface changes were detected on cells transformed by wildtype virus and shifted to 41°C. Biochemical analysis of the putative in vitro differentiation of ts AEV-transformed chicken erythroblasts was therefore initiated to define at the molecular level the cell-surface changes occurring as a result of the temperature shift. We report here that ts-transformed chicken erythroblasts synthesize an erythrocyte cell-surface giycoprotein, providing evidence that these cells are beginning to differentiate along the erythroid developmental pathway.
2. Materials and methods 2.1. Cell culture and labelling The cell line LSCC-HD3 (ts AE/EB/np) was used throughout. This line was recently derived (Beug et al., in prep.) from a clone of chicken erythroblasts transformed in vitro (Graf et al., 1978a,b) by ts34 AEV. A cell line transformed by wild-type (wt) AEV, designated LSCC-HD1 (AE/EB), was used as control. Transformed erythroblasts (1--2 × 107) were grown for 4 days at either 35°C or 41°C in RPMI 1640 medium containing 10% fetal calf serum, 2% chick serum, 10 -s M thiogiycerol and 10 mM Hepes, pH 7.4, and were labelled for 90 min at their growth temperature in methionine-free medium with 500 /~Ci [3SS]methionine or for 1 8 h in culture medium with 200 /~Ci~ [3H]giucosamine. For time-course analyses, cells were removed from the incubator at 35°C to that at 41°C for various times before labelling at 41°C. 2.2. Cell lysates Cells were lysed in buffer containing 0.5% Nonidet P40, 0.5% sodium deoxycholate, 0.05% NAN3, 50 mM NaC1, 20 mM NaF and 25 mM Tris--HC1, pH 8.1 (lysis buffer) at 4°C (2 ml/107 cells). Nuclear material was removed by centrifugation (3000 × g for 30 min) and the protease inhibitor phenylmethyl sulphonylfluoride added to a final concentration of 1 mM. 2.3. Affinity chromatography Lysates were passed over columns of Sepharose-4B~conjugated lectins at 4°C. Bound glycoproteins were eluted from Lens culinaris haemagglutinin (lentil) with 10% a-methylmannose in lysis buffer and from Ricinus communis agglutinin-120 (ricin) with 10% galactose in lysis buffer. Equal numbers of 3sS c.p.m, were ethanol-precipitated (85% EtOH, --20°C, 48 h) or immune-precipitated and examined by electrophoresis.
165
2.4. Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of sodium dodecyl sulphate (SDS) according to Laemmli (1970), using a sample buffer containing 5% SDS and 2.5% (v/v) 2-mercaptoethanol. Samples were heated (95°C, 3 min) in this buffer before electrophoresis. Bands were visualized by fluorography (Bonnet and Laskey, 1974; Laskey and Wills, 1975), using Kodak SB5 X-ray film. Molecular weight (MW) markers were from The Radiochemical Centre, Amersham, England. 2.5. I m m u n e precipitation
Unfractionated radiolabelled cell lysates or glycoproteins eluted from ricin-Sepharose were incubated with antiserum (20°C, 30 min) and immune complexes collected (20°C, 15 rain) with heat-killed, formaldehydefixed Staphylococcus aureus Cowan strain 1 (Kessler, 1975). After washing with lysis buffer containing 0.1% SDS, complexes were eluted (95°C, 3 min) from the bacteria with electrophoresis sample buffer and examined by SDS-PAGE. Immune precipitations were performed with antibody in excess over antigen.
After 4 days cells were labelled at their growth temperature with either [3SS]methionine or [3H]glucosamine and their glycoproteins isolated using lectin affinity chromatography with Lens culinaris lectin (lentil) which preferentially binds mannose (Lis and Sharon, 1973) and with Ricinus c o m m u n i s agglutinin 120 (ricin) which preferentially binds galactose (Lis and Sharon, 1973). The lectin-bound glycoproteins were eluted
e d
e
O0
N
2.6. Antiserum
N Anti-erythroid serum was raised in rabbits against ts34 AEV-transformed erythroblasts grown at 41°C, as described by Beug et al. (1979). Anti-chicken haemoglobin, raised in rabbits, was from Nordic Immunological Laboratories, Tilburg, The Netherlands.
m
3. Results
The cells used in this study were maintained in continuous culture at the permissive temperature (35°C). Prior to use in experiments, the cells were shifted to the nonpermissive temperature of the virus (41°C).
Fig. 1. SDS-PAGE of glycoprotein fractions eluted from columns of lentil-Sepharose (a, b) and ricinSepharose (c, d) using lysates of ts34 AEV-transformed cells grown at either 35°C (a, c) or 41°C (b, d). 14C molecular weight markers (MW in kilodaltons) were run in lane e. An exponential 6.5-12.5% polyacrylamide gradient gel was used.
166 and analyzed using immune precipitation with anti-erythroid serum and SDS-PAGE. Fig. 1 shows the lectin-binding glycoproteins from ts AEV-transformed cells grown at 35°C and 41°C. Although some minor changes were detected in the synthesis of lentil-binding glycoproteins as a result of the temperature shift, analysis of the ricinbinding glycoproteins showed the appearance after shift of a major band (arrowed) with an apparent molecular weight of 94,000 (Fig. 1, d). A small a m o u n t of this molecule may also be present at 35°C. Immune precipitation from detergent lysates of whole cells with an antiserum raised in rabbits against ts AEV-transformed erythroblasts grown at
41°C showed that increased synthesis of this 94,000 MW molecule (arrowed) occurs in cells transformed by the ts-mutant grown at the viral non-permissive temperature, but such an increase is not detectable under the same conditions in cells transformed by the wt-virus (Fig. 2). Analysis of immune precipitates from the ricin-binding glycoproteins, using anti-erythroid serum, revealed the greatly enhanced synthesis of a 94,000 MW glycoprotein (Fig. 3,a and b) in ts-transformed cells grown at the non-permissive temperature (41 ° C). This glycoprotein appears to be the same as that detected in the ricin eluate shown in Fig. 1. Absorption of the antiserum with intact erythrocytes removes
)2.5
~Ji6
Fig. 2. S D S - P A G E of i m m u n e precipitates from [3SS]methionine-labelled whole cell lysates. Anti-erythroid serum: a--d. Non-immune rabbit serum: e--h. Cells transformed by wt A E V and grown at 35°C (a, e) or 41°C (b, f). Cells transformed by is34 A E V and grown at 35°C (c, g) or 41°C (d, h). M W markers ( M W in kilodaltons) (lane m). A 7.5% polyacrylamide gel was used.
167
The eluted antibodies could also precipitate this band when cells were labelled with [3H]glucosamine (Fig. 3, f). Figs. 4 and 5 show a time course study comparing the synthesis of the 94,000 MW glycoprotein (Fig. 4) to that of globin (Fig. 5) at various time intervals after the shift to 41°C. A low level of 94,000 MW glycoprotein was present before the shift to the nonpermissive temperature. At 6 h after the shift, an increase in 94,000 MW glycoprotein synthesis is obvious, and by 10 h it appears to have reached a plateau. Synthesis of globin
69
Fig. 3. SDS-PAGE of immune precipitates of glycoproteins in ts34 AEV-transformed cells grown at 35°C (a) or at 41°C (b--f). Immune precipitation was with rabbit anti-erythroid serum (a, b); nonimmune rabbit serum (c); antiserum absorbed with erythrocytes by incubation for 1 h at 20°C with an equal packed cell volume of chicken erythrocytes (d); antibodies eluted from the above erythrocytes by incubation at pH 2.5 for 10 rain at 4°C (e, f). For lane f, cells grown at 41°C were labelled with [3H]glucosamine at 41°C and ricin-binding glycoproteins extracted, immune Precipitated and electrophoresed as described. Lane g, 14C MW markers (MW in kilodaltons). A 7.5% polyacrylamide gel was used.
the antibody activity directed against the 94,000 MW glycoprotein (Fig. 3,d). Antibodies eluted by low pH from the erythrocytes used for absorption (without apparent cell damage) were re-used to precipitate the 94,000 MW band from ricin-binding glycoprotein fractions of differentiating erythroblasts grown at 41°C (Fig. 3, e and f).
Fig. 4. SDS-PAGE of immune precipitates showing a time course of 94,000 MW glycoprotein appearance. Cells transformed by ts34 AEV were grown at 41°C for the times shown (0--120 h) before labelling with [3SS]methionine. Lysates were immune precipitated with anti-erythroid serum eluted from erythrocytes (see Fig. 3), or non-immune rabbit serum (n, 120 h lysate). MW markers, lane m (MW in kilodaltons). An exponential 6.5--12.5% polyacrylamide gradient gel was used.
168
Fig. 5. SDS-PAGE of immune precipitates showing a time course of globin appearance. Cells transformed by ts34 AEV were grown at either 35°C, time zero; or at 41°C for the times shown (0--120 h) before labelling with [3SS]methionine. Lysates were immune-precipitated with rabbit anti-chicken haemoglobin (Nordic) or nonimmune rabbit serum (n, 120 h lysate). MW markers (MW in kilodaltons) lane m. A 15% polyacrylamide gel was used.
at 6 h is definitely above the zero-time level, b u t the increase is far less obvious than with the glycoprotein. Globin synthesis c ont i nue d to increase t h r o u g h o u t the analysis period.
4. Discussion In earlier studies by others, temperaturesensitive mutants of Rous sarcoma virus (RSV), th e genome of which codes for the 'sarc' transforming protein (for review, see Bishop, 1978), have been used in analyses of differentiating myogenic cells (Fiszman and Fuchs, 1975; Holtzer et al., 1975; Moss et al., 1979). In the latter system, differentia-
tion can be induced by a simple upward shift in growth temperature, and the aim has been to define the poi nt at which RSV blocks differentiation. We have adopt ed this approach using ts34 AEV, an acute leukaemia virus whose genome contains a transforming gene quite different from that in RSV (Stehelin and Graf, 1978}. The data presented here show t hat when ts AEV-transformed erythroblasts are shifted to the viral non-permissive t em perat ure, the greatly enhanced synthesis of a cellular 94,000 MW molecule rapidly ensues (Figs. 1--4}. This 94,000 MW molecule is a glycoprotein as shown by its ability to bind a galactose-specific lectin (Figs. 1 and 3) and to incorporate [3H]glucosamine and [3SS]me-
169
thionine (Fig. 3). The number of temperature shift-associated changes occurring in the total glycoprotein fraction is very limited. Of the 30 or so different glycoproteins seen in Fig. 1, only one or two are significantly affected by deactivation of the viral-transforming function (by shifting to 41°C). Only synthesis of the 94,000 MW ricin-binding glycoprotein proved to be detectable by an antiserum raised against these cells (Figs. 2--4) for use as a probe for newly appearing cell-surface molecules. The spectrum of the effects of AEV on differentiation then, with respect to cellular glycoproteins, may be quite narrow. The 94,000 MW glycoprotein is a cellsurface molecule. This is shown by three points: 1) an antiserum raised in rabbits by injecting intact ts-transformed cells, and therefore presumably directed against cellsurface antigens, precipitates the 94,000 MW glycoprotein; 2) antibodies directed against this glycoprotein can be absorbed out of the anti-erythroid serum by intact mature erythrocytes; and 3 ) a n t i b o d i e s which will precipitate the 94,000 MW glycoprotein can be eluted from the intact erythrocytes used for absorption (Fig. 3). The 94,000 MW glycoprotein is found on the surface of cells in later stages of erythroid differentiation, i.e. reticulocytes, bone-marrow erythrocytes, and peripheral blood erythrocytes, as evidenced by absorption experiments with anti-erythroid serum (Fig. 3) (Savin and Beug, H., unpubl, obs.). Synthesis of this glycoprotein subsequent to the shift to the viral non-permissive temperature is therefore indicative of further differentiation along the erythroid pathway. The temperature shift-induced increase in synthesis of this 94,000 MW glycoprotein is not a property of AEV-transformed cells. When wt AEV-transformed cells were grown at 41°C, no significant increase in synthesis of this glycoprotein was observed (Fig. 2). Only cells transformed b y the ts-mutant exhibit this property. Other data (not shown) indicate that this glycoprotein is present in freshly isolated colonies of ts34 AEV-
transformed erythroblasts shifted to 41°C, and its appearance is therefore n o t solely a property of the established cell line. As can be seen in Figs. 1--4, a small a m o u n t of a 94,000 MW glycoprotein is made by cells grown at the viral permissive temperature (35°C). Clonal variation in the intensity of this band has been observed which may correlate with the number of cells already positive by benzidine staining for haemoglobin at 350C (data not shown). The cell line LSCC-HD3 (ts AE/EB/np) contained 3--7.5% benzidine-positive cells at 35°C, rising to 57% at 41°C; however, it is n o t y e t known whether the benzidine-positive cells and those synthesizing the 94,000 MW glycoprotein are identical at either temperature. The synthesis of differentiation-associated molecules by Friend cells is also n o t necessarily de novo. Friend cells also exhibit clonal variation. Uninduced Friend erythroleukaemic cells contain a low level of spectrin which increases dramatically after treatment with differentiation-inducing agents (Eisen et al., 1977). The low level of spectrin detected has been shown to have a normal distribution over the entire cell population rather than being confined to a small number of spontaneously differentiating cells (Rifkind et al., 1979). A similar situation could apply to transferrin receptors which are present at low levels in uninduced Friend cells (Hu et al., 1977; Yeoh and Morgan, 1979). Experiments in which ts AEV-transformed erythroblasts were radiolabelled at various times after the shift from permissive to nonpermissive temperature showed that the increased synthesis of both the 94,000 MW glycoprotein and the giobin begins somewhere between 2 and 6 h post-shift, although an increase in sensitivity of the technique m a y allow earlier detection. The time course of appearance of the 94,000 MW giycoprotein is similar to that found for spectrin in Friend cells (Eisen et al., 1977; Rifldnd et al., 1979) in that it can be seen to be 'switched on' within the first 12 h of differentiation. The more gradual appearance of globin (presum-
170 a b l y h a e m o g l o b i n ) parallels t h e rise in the number of b e n z i d i n e - s t a i n e d cells ( G r a f et al., 1978a). T h e early d e t e c t i o n o f globin synthesis in ts A E V - t r a n s f o r m e d cells is comparable to induction of Friend differentiation w i t h h e m i n w h e r e globin m a y a p p e a r w i t h i n 6 h o f a d d i t i o n o f this i n d u c e r (Rifkind et al., 1 9 7 9 ) t o c e r t a i n clones o f F r i e n d cells. In c o n t r a s t , w i t h o t h e r inducers such as d i m e t h y l s u l p h o x i d e or b u t y r i c acid u p t o 24 h m a y elapse b e f o r e t h e synthesis o f n e w globin is a p p a r e n t , a l t h o u g h s o m e clones have b e e n isolated w h i c h will s h o w a m a r k e d accum u l a t i o n o f globin m R N A in less t h a n 24 h (Pragnell et al., 1977). T h e s e results m a y , if p u r s u e d , yield s o m e i m p o r t a n t i n f o r m a t i o n a b o u t the p o i n t d u r i n g e r y t h r o i d d e v e l o p m e n t a t w h i c h A E V e x e r t s its influence. T h e d a t a p r e s e n t e d h e r e establish t h a t c h i c k e n e r y t h r o b l a s t s t r a n s f o r m e d b y ts34 A E V are c a p a b l e o f s y n t h e s i z i n g a 9 4 , 0 0 0 MW e r y t h r o c y t e cell-surface g l y c o p r o t e i n w h e n shifted t o t h e viral n o n - p e r m i s s i v e temperature. The previous demonstration, c o n f i r m e d here, n a m e l y t h a t cells transf o r m e d b y this t e m p e r a t u r e - s e n s i t i v e virus are able to greatly increase t h e i r h a e m o globin synthesis u p o n shift t o t h e n o n - p e r missive t e m p e r a t u r e (41°C) ( G r a f et al., 1 9 7 8 a ) , suggested t h a t A E V b l o c k s n o r m a l e r y t h r o i d d i f f e r e n t i a t i o n . T h e p r e s e n t result provides a d d i t i o n a l e v i d e n c e t h a t a shift in g r o w t h t e m p e r a t u r e f r o m t h e permissive (35°C) to t h e n o n - p e r m i s s i v e (41°C) alters gene e x p r e s s i o n in ts34 A E V - t r a n s f o r m e d erythroblasts consistent with the notion of release f r o m a b l o c k in d i f f e r e n t i a t i o n . F u r t h e r e x p e r i m e n t ~ are n o w in progress to e x a m i n e t h e b i o s y n t h e s i s of, a n d t o c h a r a c t e r ize this 9 4 , 0 0 0 MW e r y t h r o c y t e cell-surface glycoprotein.
Acknowledgements We w o u l d like to t h a n k G a b i D o d e r l e i n a n d G a y K i t c h e n e r f o r t e c h n i c a l assistance, Drs. T. de Kretser, M. H a y m a n , T. Graf, C.
D i c k s o n a n d P. G o o d f e l l o w f o r valuable discussions a n d assistance w i t h t h e m a n u script, a n d J o y c e N e w t o n a n d G i n a Y i a n g o u for typing the manuscript.
References Arndt-Jovin, D.H., W. Ostertag, H. Eisen, F. Klimek and T.M. Jovin: J. Histochem. Cytochem. 24, 332--347 (1976). Beug, H., A. von Kirchbach, G. Doderlein, J.-F. Conscience and T. Graf: Cell 18 (2), 375--390 (1979). Bishop, J.M.: Ann. Rev. Biochem. 47, 35--88 (1978). Bonner, W.M. and R.A. Laskey: Eur. J. Biochem. 46, 83--88 (1974). Conscience, J.F., R.A. Miller, J. Henry and F.H. Ruddle: Exp. Cell Res. 105,401--412 (1977). Eisen, H., R. Bach and R. Emery: Proc. Natl. Acad. Sci. U.S.A. 74, 3898--3902 (1977). Fiszman, M.Y. and P. Fuchs: Nature 254, 429-431 (1975). Friend, C., W. Scher, J.G. Holland and T. Sato: Proc. Natl. Acad. Sci. U.S.A. 68,378--382 (1971). Graf, T.. N. Ade and H. Beug: Nature 275, 496-501 (1978a). Graf, T. and H. Beug: Biochim. Biophys. Acta 516, 269--299 (1978). Graf, T., H. Beug, B. Royer-Pokora and W. MeyerGlauner: In: Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor Conference on Cell Proliferation, Vol. 5 (1978b). Graf, T., D. Fink, H. Beug and B. Royer-Pokora: Cancer Res. 37, 59--63 (1977). Harrison, P.R.: In: International Review of Biochemistry, 15, Biochemistry of Cell Differentiation II (1977). Holtzer, H., J. Biehl, G. Yeoh, R. Meganathan and A. Kaji: Proc. Natl. Acad. Sci. U.S.A. 72, 4051-4O55 (1975). Hu, H.-Y.Y., J. Gardner, P. Aisen and A.I. Skoultchi: Science 197,559--561 (1977). Kessler, S.W.: J. Immunol. 115, 1617--1624 (1975). Laemmli, U.K.: Nature (London) 227, 680--685 (1970). Laskey, R.A. and A.D. Mills: Eur. J. Biochem. 56, 335--341 (1975). Lis, H. and N. Sharon: Ann. Rev. Biochem. 42, 541--574 (1973). Moss, P.S., N. Honeycutt, T. Pawson and G.S. Martin: Exp. Cell Res. 123, 95--105 (1979). Pragnell, I.B., W. Ostertag and J. Paul: Exp. Cell Res. 108, 269--278 (1977). Reuben, R.C., R.A. Rifkind and P.A. Marks: Biochim. Biophys. Acta 605(3), 325--346 (1980).
171 Rifkind, R.A., E. Fibach, G. Maniatis, R. Gambari and P.A. Marks: In: Cellular and Molecular Regulation of Hemoglobin Switching, eds. G. Stamatoyannopoulos and A.W. Neinhuis (Gruae and
Stratton, New York) pp. 421--436 (1979). Stehelin, D. and T. Graf: Cell 1 3 , 7 4 5 - - 7 5 0 (1978). Yeoh, G.C.T. and E.H. Morgan: Cell Differ. 8, 331-343 (1979).
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CELL-SURFACE G L Y C O P R O T E I N SYNTHESIS DURING D I F F E R E N T I A T I O N OF CHICKEN E R Y T H R O B L A S T S T R A N S F O R M E D BY T E M P E R A T U R E - S E N S I T I V E AVIAN E R Y T H R O B L A S T O S I S VIRUS KEITH W. SAVIN 1 and HARTMUT BEUG 2 1 Tumour Virology Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, L o n d o n WC2A 3PX, U.K.; and 2 Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg, F.R.G.
(Accepted 9 February 1981) Chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (ts34 AEV) have a greatly increased haemoglobin content (Graf, T., N. Ade and H. Beug: Nature 275, 496--501 (1978)) if allowed to grow for 3--5 days at the non-permissive temperature (41°C), instead of the permissive temperature (35°C) of the virus. Cell-surface molecular changes during this differentiation were investigated by examining the glycoproteins synthesized by a ts34-transformed erythroblast cell line. These cells synthesized a greatly increased amount of a 94,000 molecular weight erythrocyte cell-surface glycoprotein beginning 2--6 h after a shift in growth temperature from 35°C to 41°C, consistent with the proposal that such a shift releases these transformed cells from a differentiation block. differentiation
cell surface
glycoprotein
1. I n t r o d u c t i o n Avian erythroblastosis virus (AEV) is a replication-defective leukaemia virus causing acute erythroid leukaemia and slowly developing sarcomas in chickens (Graf et al., 1977). This virus specifically transforms fibroblasts and erythroblasts in vitro, and colonies of the latter can be obtained by transformation of chicken bone m a r r ow cultures (for review, see Graf and Beug, 1978). AEV and other viruses have been proposed to bring about leukaemic transf o r m a t i o n by blocking normal cellular differentiation (Graf et al., 1978a,b). The recent isolation o f a temperature-sensitive m u t a n t of AEV (designated ts34), which allows the synthesis of haemoglobin by virus-transf o r m e d erythroblasts u p o n a shift t o the nonpermissive t e m p e r a t u r e of the virus (Graf et al., 1978a), has lent f ur t her weight to this proposal. If the leukaemogenic mechanism of AEV does entail a block in normal e r yt hr oi d
erythroblasts
retrovirus
differentiation, analysis of the putative differentiation, perm i t t ed by the temperature-sensitive m u t a n t of AEV at the viral non-permissive temperature, should provide information about the biochemical activity of the AEV-transforming protein. In addition, it should establish w het her these cells are differentiating along the normal eryt hroi d developmental pathway or are taking an aberrant course. An analysis of t s 3 4 AEV-transformed erythroblasts was initiated along the lines of that applied to the Friend cell system (for review, see Harrison, 1977; Reuben et al., 1980). Although induction of Friend cell differentiation by chemical inducing agents might n o t be directly related to prevention of the function of a viral gene product, the Friend system provides good examples of the differentiation-associated changes one can expect to find when analysing t s 3 4 AEVtransformed cells. Differentiation can be p r o m o t e d in murine eryt hroi d cells transform ed by the Friend virus com pl ex by
0045-6039/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
164 inducing agents such as dimethyl sulphoxide. The differentiating Friend cells produce haemoglobin (Friend et al., 1971) and various other proteins including those associated with the plasma membrane, such as acetylcholinesterase (Conscience et al., 1977), transferrin receptors (Hu et al., 1977; Yeoh and Morgan, 1979) and spectrin (ArndtJovin et al., 1976). The loss of cell-surface H2 antigens (Arndt-Jovin et al., 1976) has also been demonstrated. We consider that the cell surface is a likely target area for the effect of a molecule designed to block differentiation. This could occur by, for example, preventing the synthesis or function of an essential hormone receptor or by disruption of specific ion channels. Analysis of the cell surface of ts AEV-transformed erythroblasts should provide information a b o u t possible differentiation and, in addition, establish a convenient marker for future monitoring of such differentiation. Preliminary cell-surface immunofluorescence experiments, which utilized specific anti-erythrocyte serum, indicated that cell-surface changes suggestive of erythroid maturation occurred on a majority of the ts AEV-transformed erythroblasts after a shift in growth temperature from the permissive (35°C) to the non-permissive temperature (41°C) of the virus. Neither an increase in the number of haemoglobincontaining cells nor cell-surface changes were detected on cells transformed by wildtype virus and shifted to 41°C. Biochemical analysis of the putative in vitro differentiation of ts AEV-transformed chicken erythroblasts was therefore initiated to define at the molecular level the cell-surface changes occurring as a result of the temperature shift. We report here that ts-transformed chicken erythroblasts synthesize an erythrocyte cell-surface giycoprotein, providing evidence that these cells are beginning to differentiate along the erythroid developmental pathway.
2. Materials and methods 2.1. Cell culture and labelling The cell line LSCC-HD3 (ts AE/EB/np) was used throughout. This line was recently derived (Beug et al., in prep.) from a clone of chicken erythroblasts transformed in vitro (Graf et al., 1978a,b) by ts34 AEV. A cell line transformed by wild-type (wt) AEV, designated LSCC-HD1 (AE/EB), was used as control. Transformed erythroblasts (1--2 × 107) were grown for 4 days at either 35°C or 41°C in RPMI 1640 medium containing 10% fetal calf serum, 2% chick serum, 10 -s M thiogiycerol and 10 mM Hepes, pH 7.4, and were labelled for 90 min at their growth temperature in methionine-free medium with 500 /~Ci [3SS]methionine or for 1 8 h in culture medium with 200 /~Ci~ [3H]giucosamine. For time-course analyses, cells were removed from the incubator at 35°C to that at 41°C for various times before labelling at 41°C. 2.2. Cell lysates Cells were lysed in buffer containing 0.5% Nonidet P40, 0.5% sodium deoxycholate, 0.05% NAN3, 50 mM NaC1, 20 mM NaF and 25 mM Tris--HC1, pH 8.1 (lysis buffer) at 4°C (2 ml/107 cells). Nuclear material was removed by centrifugation (3000 × g for 30 min) and the protease inhibitor phenylmethyl sulphonylfluoride added to a final concentration of 1 mM. 2.3. Affinity chromatography Lysates were passed over columns of Sepharose-4B~conjugated lectins at 4°C. Bound glycoproteins were eluted from Lens culinaris haemagglutinin (lentil) with 10% a-methylmannose in lysis buffer and from Ricinus communis agglutinin-120 (ricin) with 10% galactose in lysis buffer. Equal numbers of 3sS c.p.m, were ethanol-precipitated (85% EtOH, --20°C, 48 h) or immune-precipitated and examined by electrophoresis.
165
2.4. Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of sodium dodecyl sulphate (SDS) according to Laemmli (1970), using a sample buffer containing 5% SDS and 2.5% (v/v) 2-mercaptoethanol. Samples were heated (95°C, 3 min) in this buffer before electrophoresis. Bands were visualized by fluorography (Bonnet and Laskey, 1974; Laskey and Wills, 1975), using Kodak SB5 X-ray film. Molecular weight (MW) markers were from The Radiochemical Centre, Amersham, England. 2.5. I m m u n e precipitation
Unfractionated radiolabelled cell lysates or glycoproteins eluted from ricin-Sepharose were incubated with antiserum (20°C, 30 min) and immune complexes collected (20°C, 15 rain) with heat-killed, formaldehydefixed Staphylococcus aureus Cowan strain 1 (Kessler, 1975). After washing with lysis buffer containing 0.1% SDS, complexes were eluted (95°C, 3 min) from the bacteria with electrophoresis sample buffer and examined by SDS-PAGE. Immune precipitations were performed with antibody in excess over antigen.
After 4 days cells were labelled at their growth temperature with either [3SS]methionine or [3H]glucosamine and their glycoproteins isolated using lectin affinity chromatography with Lens culinaris lectin (lentil) which preferentially binds mannose (Lis and Sharon, 1973) and with Ricinus c o m m u n i s agglutinin 120 (ricin) which preferentially binds galactose (Lis and Sharon, 1973). The lectin-bound glycoproteins were eluted
e d
e
O0
N
2.6. Antiserum
N Anti-erythroid serum was raised in rabbits against ts34 AEV-transformed erythroblasts grown at 41°C, as described by Beug et al. (1979). Anti-chicken haemoglobin, raised in rabbits, was from Nordic Immunological Laboratories, Tilburg, The Netherlands.
m
3. Results
The cells used in this study were maintained in continuous culture at the permissive temperature (35°C). Prior to use in experiments, the cells were shifted to the nonpermissive temperature of the virus (41°C).
Fig. 1. SDS-PAGE of glycoprotein fractions eluted from columns of lentil-Sepharose (a, b) and ricinSepharose (c, d) using lysates of ts34 AEV-transformed cells grown at either 35°C (a, c) or 41°C (b, d). 14C molecular weight markers (MW in kilodaltons) were run in lane e. An exponential 6.5-12.5% polyacrylamide gradient gel was used.
166 and analyzed using immune precipitation with anti-erythroid serum and SDS-PAGE. Fig. 1 shows the lectin-binding glycoproteins from ts AEV-transformed cells grown at 35°C and 41°C. Although some minor changes were detected in the synthesis of lentil-binding glycoproteins as a result of the temperature shift, analysis of the ricinbinding glycoproteins showed the appearance after shift of a major band (arrowed) with an apparent molecular weight of 94,000 (Fig. 1, d). A small a m o u n t of this molecule may also be present at 35°C. Immune precipitation from detergent lysates of whole cells with an antiserum raised in rabbits against ts AEV-transformed erythroblasts grown at
41°C showed that increased synthesis of this 94,000 MW molecule (arrowed) occurs in cells transformed by the ts-mutant grown at the viral non-permissive temperature, but such an increase is not detectable under the same conditions in cells transformed by the wt-virus (Fig. 2). Analysis of immune precipitates from the ricin-binding glycoproteins, using anti-erythroid serum, revealed the greatly enhanced synthesis of a 94,000 MW glycoprotein (Fig. 3,a and b) in ts-transformed cells grown at the non-permissive temperature (41 ° C). This glycoprotein appears to be the same as that detected in the ricin eluate shown in Fig. 1. Absorption of the antiserum with intact erythrocytes removes
)2.5
~Ji6
Fig. 2. S D S - P A G E of i m m u n e precipitates from [3SS]methionine-labelled whole cell lysates. Anti-erythroid serum: a--d. Non-immune rabbit serum: e--h. Cells transformed by wt A E V and grown at 35°C (a, e) or 41°C (b, f). Cells transformed by is34 A E V and grown at 35°C (c, g) or 41°C (d, h). M W markers ( M W in kilodaltons) (lane m). A 7.5% polyacrylamide gel was used.
167
The eluted antibodies could also precipitate this band when cells were labelled with [3H]glucosamine (Fig. 3, f). Figs. 4 and 5 show a time course study comparing the synthesis of the 94,000 MW glycoprotein (Fig. 4) to that of globin (Fig. 5) at various time intervals after the shift to 41°C. A low level of 94,000 MW glycoprotein was present before the shift to the nonpermissive temperature. At 6 h after the shift, an increase in 94,000 MW glycoprotein synthesis is obvious, and by 10 h it appears to have reached a plateau. Synthesis of globin
69
Fig. 3. SDS-PAGE of immune precipitates of glycoproteins in ts34 AEV-transformed cells grown at 35°C (a) or at 41°C (b--f). Immune precipitation was with rabbit anti-erythroid serum (a, b); nonimmune rabbit serum (c); antiserum absorbed with erythrocytes by incubation for 1 h at 20°C with an equal packed cell volume of chicken erythrocytes (d); antibodies eluted from the above erythrocytes by incubation at pH 2.5 for 10 rain at 4°C (e, f). For lane f, cells grown at 41°C were labelled with [3H]glucosamine at 41°C and ricin-binding glycoproteins extracted, immune Precipitated and electrophoresed as described. Lane g, 14C MW markers (MW in kilodaltons). A 7.5% polyacrylamide gel was used.
the antibody activity directed against the 94,000 MW glycoprotein (Fig. 3,d). Antibodies eluted by low pH from the erythrocytes used for absorption (without apparent cell damage) were re-used to precipitate the 94,000 MW band from ricin-binding glycoprotein fractions of differentiating erythroblasts grown at 41°C (Fig. 3, e and f).
Fig. 4. SDS-PAGE of immune precipitates showing a time course of 94,000 MW glycoprotein appearance. Cells transformed by ts34 AEV were grown at 41°C for the times shown (0--120 h) before labelling with [3SS]methionine. Lysates were immune precipitated with anti-erythroid serum eluted from erythrocytes (see Fig. 3), or non-immune rabbit serum (n, 120 h lysate). MW markers, lane m (MW in kilodaltons). An exponential 6.5--12.5% polyacrylamide gradient gel was used.
168
Fig. 5. SDS-PAGE of immune precipitates showing a time course of globin appearance. Cells transformed by ts34 AEV were grown at either 35°C, time zero; or at 41°C for the times shown (0--120 h) before labelling with [3SS]methionine. Lysates were immune-precipitated with rabbit anti-chicken haemoglobin (Nordic) or nonimmune rabbit serum (n, 120 h lysate). MW markers (MW in kilodaltons) lane m. A 15% polyacrylamide gel was used.
at 6 h is definitely above the zero-time level, b u t the increase is far less obvious than with the glycoprotein. Globin synthesis c ont i nue d to increase t h r o u g h o u t the analysis period.
4. Discussion In earlier studies by others, temperaturesensitive mutants of Rous sarcoma virus (RSV), th e genome of which codes for the 'sarc' transforming protein (for review, see Bishop, 1978), have been used in analyses of differentiating myogenic cells (Fiszman and Fuchs, 1975; Holtzer et al., 1975; Moss et al., 1979). In the latter system, differentia-
tion can be induced by a simple upward shift in growth temperature, and the aim has been to define the poi nt at which RSV blocks differentiation. We have adopt ed this approach using ts34 AEV, an acute leukaemia virus whose genome contains a transforming gene quite different from that in RSV (Stehelin and Graf, 1978}. The data presented here show t hat when ts AEV-transformed erythroblasts are shifted to the viral non-permissive t em perat ure, the greatly enhanced synthesis of a cellular 94,000 MW molecule rapidly ensues (Figs. 1--4}. This 94,000 MW molecule is a glycoprotein as shown by its ability to bind a galactose-specific lectin (Figs. 1 and 3) and to incorporate [3H]glucosamine and [3SS]me-
169
thionine (Fig. 3). The number of temperature shift-associated changes occurring in the total glycoprotein fraction is very limited. Of the 30 or so different glycoproteins seen in Fig. 1, only one or two are significantly affected by deactivation of the viral-transforming function (by shifting to 41°C). Only synthesis of the 94,000 MW ricin-binding glycoprotein proved to be detectable by an antiserum raised against these cells (Figs. 2--4) for use as a probe for newly appearing cell-surface molecules. The spectrum of the effects of AEV on differentiation then, with respect to cellular glycoproteins, may be quite narrow. The 94,000 MW glycoprotein is a cellsurface molecule. This is shown by three points: 1) an antiserum raised in rabbits by injecting intact ts-transformed cells, and therefore presumably directed against cellsurface antigens, precipitates the 94,000 MW glycoprotein; 2) antibodies directed against this glycoprotein can be absorbed out of the anti-erythroid serum by intact mature erythrocytes; and 3 ) a n t i b o d i e s which will precipitate the 94,000 MW glycoprotein can be eluted from the intact erythrocytes used for absorption (Fig. 3). The 94,000 MW glycoprotein is found on the surface of cells in later stages of erythroid differentiation, i.e. reticulocytes, bone-marrow erythrocytes, and peripheral blood erythrocytes, as evidenced by absorption experiments with anti-erythroid serum (Fig. 3) (Savin and Beug, H., unpubl, obs.). Synthesis of this glycoprotein subsequent to the shift to the viral non-permissive temperature is therefore indicative of further differentiation along the erythroid pathway. The temperature shift-induced increase in synthesis of this 94,000 MW glycoprotein is not a property of AEV-transformed cells. When wt AEV-transformed cells were grown at 41°C, no significant increase in synthesis of this glycoprotein was observed (Fig. 2). Only cells transformed b y the ts-mutant exhibit this property. Other data (not shown) indicate that this glycoprotein is present in freshly isolated colonies of ts34 AEV-
transformed erythroblasts shifted to 41°C, and its appearance is therefore n o t solely a property of the established cell line. As can be seen in Figs. 1--4, a small a m o u n t of a 94,000 MW glycoprotein is made by cells grown at the viral permissive temperature (35°C). Clonal variation in the intensity of this band has been observed which may correlate with the number of cells already positive by benzidine staining for haemoglobin at 350C (data not shown). The cell line LSCC-HD3 (ts AE/EB/np) contained 3--7.5% benzidine-positive cells at 35°C, rising to 57% at 41°C; however, it is n o t y e t known whether the benzidine-positive cells and those synthesizing the 94,000 MW glycoprotein are identical at either temperature. The synthesis of differentiation-associated molecules by Friend cells is also n o t necessarily de novo. Friend cells also exhibit clonal variation. Uninduced Friend erythroleukaemic cells contain a low level of spectrin which increases dramatically after treatment with differentiation-inducing agents (Eisen et al., 1977). The low level of spectrin detected has been shown to have a normal distribution over the entire cell population rather than being confined to a small number of spontaneously differentiating cells (Rifkind et al., 1979). A similar situation could apply to transferrin receptors which are present at low levels in uninduced Friend cells (Hu et al., 1977; Yeoh and Morgan, 1979). Experiments in which ts AEV-transformed erythroblasts were radiolabelled at various times after the shift from permissive to nonpermissive temperature showed that the increased synthesis of both the 94,000 MW glycoprotein and the giobin begins somewhere between 2 and 6 h post-shift, although an increase in sensitivity of the technique m a y allow earlier detection. The time course of appearance of the 94,000 MW giycoprotein is similar to that found for spectrin in Friend cells (Eisen et al., 1977; Rifldnd et al., 1979) in that it can be seen to be 'switched on' within the first 12 h of differentiation. The more gradual appearance of globin (presum-
170 a b l y h a e m o g l o b i n ) parallels t h e rise in the number of b e n z i d i n e - s t a i n e d cells ( G r a f et al., 1978a). T h e early d e t e c t i o n o f globin synthesis in ts A E V - t r a n s f o r m e d cells is comparable to induction of Friend differentiation w i t h h e m i n w h e r e globin m a y a p p e a r w i t h i n 6 h o f a d d i t i o n o f this i n d u c e r (Rifkind et al., 1 9 7 9 ) t o c e r t a i n clones o f F r i e n d cells. In c o n t r a s t , w i t h o t h e r inducers such as d i m e t h y l s u l p h o x i d e or b u t y r i c acid u p t o 24 h m a y elapse b e f o r e t h e synthesis o f n e w globin is a p p a r e n t , a l t h o u g h s o m e clones have b e e n isolated w h i c h will s h o w a m a r k e d accum u l a t i o n o f globin m R N A in less t h a n 24 h (Pragnell et al., 1977). T h e s e results m a y , if p u r s u e d , yield s o m e i m p o r t a n t i n f o r m a t i o n a b o u t the p o i n t d u r i n g e r y t h r o i d d e v e l o p m e n t a t w h i c h A E V e x e r t s its influence. T h e d a t a p r e s e n t e d h e r e establish t h a t c h i c k e n e r y t h r o b l a s t s t r a n s f o r m e d b y ts34 A E V are c a p a b l e o f s y n t h e s i z i n g a 9 4 , 0 0 0 MW e r y t h r o c y t e cell-surface g l y c o p r o t e i n w h e n shifted t o t h e viral n o n - p e r m i s s i v e temperature. The previous demonstration, c o n f i r m e d here, n a m e l y t h a t cells transf o r m e d b y this t e m p e r a t u r e - s e n s i t i v e virus are able to greatly increase t h e i r h a e m o globin synthesis u p o n shift t o t h e n o n - p e r missive t e m p e r a t u r e (41°C) ( G r a f et al., 1 9 7 8 a ) , suggested t h a t A E V b l o c k s n o r m a l e r y t h r o i d d i f f e r e n t i a t i o n . T h e p r e s e n t result provides a d d i t i o n a l e v i d e n c e t h a t a shift in g r o w t h t e m p e r a t u r e f r o m t h e permissive (35°C) to t h e n o n - p e r m i s s i v e (41°C) alters gene e x p r e s s i o n in ts34 A E V - t r a n s f o r m e d erythroblasts consistent with the notion of release f r o m a b l o c k in d i f f e r e n t i a t i o n . F u r t h e r e x p e r i m e n t ~ are n o w in progress to e x a m i n e t h e b i o s y n t h e s i s of, a n d t o c h a r a c t e r ize this 9 4 , 0 0 0 MW e r y t h r o c y t e cell-surface glycoprotein.
Acknowledgements We w o u l d like to t h a n k G a b i D o d e r l e i n a n d G a y K i t c h e n e r f o r t e c h n i c a l assistance, Drs. T. de Kretser, M. H a y m a n , T. Graf, C.
D i c k s o n a n d P. G o o d f e l l o w f o r valuable discussions a n d assistance w i t h t h e m a n u script, a n d J o y c e N e w t o n a n d G i n a Y i a n g o u for typing the manuscript.
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