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Reactivation of chick erythrocyte nuclei in heterokaryons with rat hepatoma cells
Experimental
REACTIVATION
Cell Research 83 (1974) 47-54
OF CHICK ERYTHROCYTE
HETEROKARYONS
WITH RAT HEPATOMA
NUCLEI
IN
CELLS
C. SZPTRERl Sir William Dunn School of Pathology, Uniwrsity Oxford OXI 3RE, UK
of Oxford,
SUMMARY Chick erythrocyte nuclei were fused with rat hepatoma cells which express a number of liver functions; among other proteins, they secrete serum albumin. Like other cells (A9, HeLa), these rat hepatoma ceils induce the reactivation of the erythrocyte nuclei, which increase in size, resume the synthesis of RNA and acquire nucleoli. In hepatoma cells deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) the reactivated chick erythrocyte nuclei will induce the synthesis of this enzyme. In populations of fused cells containing 15 to 30 % of chick nuclei, the amount of induced HGPRT is more than 1 :!, of the level found in HGPRT+ rat hepatoma cells. On the other hand no secretion of chick albumin can be detected. Given the sensitivity of the method, one can conclude that if any chick albumin is secreted, it is at a rate equal to less than 0.1 ‘$6of the rate of rat albumin secretion. This result shows either that chick albumin is synthesized but not secreted by the heterokaryons; or that the reactivation of the erythrocyte nucleus is selective (only some chick genes are expressed) and that the rat hepatoma cells cannot induce these reactivated nuclei to acquire the same pattern of gene activity as themselves.
The chick erythrocyte nucleus is inert: it does not synthesize any DNA and only very low amounts of RNA, if any, and has no well formed nucleolus. However, this state of ultimate differentiation is not irreversible: when such a nucleus is introduced by cell fusion into an active cell (for example a mouse or a human tissue culture cell) it quickly increases in volume and dry mass [I, 21, resumes the synthesis of DNA and RNA [3] and, after 3-4 days, full-sized nucleoli appear [4]. At this stage, RNA is transferred from the erythrocyte nucleus to the cytoplasm of the heterokaryon [4, 51 where it is translated. i Present address: Department de Biologie MolCculaire, Universite libre de Bruxelles. Rue des Chevaux 67, B-1640 Rhode-St-Get&e, Belgium. 4-731809
To date, it has been clearly demonstrated that reactivated chick erythrocyte nuclei direct the synthesis of the following proteins: membrane antigens [4], an enzyme, the hypoxanthine guanine phosphoribosyl transferase (HGPRT, EC 2.4.2.8) [6, 71, nucleolar antigens [8, 91 and the receptor for diphtheria toxin [IO, 111. (The HGPRT catalyses the condensation of hypoxanthine or guanine with Sphosphoribosyl- 1-pyrophosphate.) These products are found in all cells (none of them is a differentiated product); and one may wonder whether the synthesis of some differentiated products can be induced in reactivated erythrocyte nuclei. In other words, is there any selectivity in the reactivation process? Are all the chick genes turned on in Expti Cell Res 83 (1974)
48
C. Szpirer
the heterokaryons, or does the chick genome or the genome of the other parent, or both, imrtose some choice? In order to obtain some information about this cluestion, I studied the reactivation of chick erythrocyte nuclei in albumin-producing cells, derived from a rat hepatoma. These hepatoma cells have been used in other fusion experiments [12, 131: it has been shown that rat albumin synthesis is not ‘extinguished’ in hybrids with mouse fibroblasts [12], and, furthermore, that in some cases mouse albumin synthesis can be initiated [13]. The main aim of the present study was to determine whether rat hepatoma cell chick erythrocyte heterokaryons produce chick albumin. L
MATERIALS
AND METHODS
Cells. Chick embryo erythrocytes were obtained from 12-day-old fertile eggs [9]. These erythrocytes synthesize very little RNA, if any; under conditions which permit very heavy labelling of tissue culture cells (autoradiography after exposure of the cells to 10 pCi/ ml of 3H-uridine for 4 h), most of the erythrocytes are not labelled. The rat hepatoma cells, Fu 5 [I21 and 967 were kindly provided by Dr B. Ephrussi and Dr M. Weiss. These cells are hyperdiploid. They are derived from the Reuber H35 hepatoma and were adapted to growth in vitro by Pitot et al. [14]. Clone 967 has been isolated from Fu5 by Dr M. Weiss bv selection in the presence of 3 pug/ml of b-azaguaninei it lacks HGPRT (see below). These hepatoma cells were grown in Eagle’s Minimum Essential Medium (MEM; Flow Laboratories) containing IO To fetal calf serum (Biocult). They secrete rat albumin [15]. Cell fusion. Before fusion, the hepatoma cells were harvested by trypsinization and subjected to a flux of 6 000 rads of gamma irradiation. This treatment suppresses mitosis and chick nuclei remain discrete long enough to develop nucleoli and to produce chick RNA which is translated in the cytoplasm of the heterokaryons [4]. The heterokaryons were made by fusing chick ervthrocvtes with the irradiated rat hepakma cells by means of UV-inactivated Sendai virus f4]. After fusion, a fraction of the cells was transferred to a Petri dish containing coverslips, the rest seeded into glass prescription bottles. The cells were maintained in MEM. The medium was changed every other day. Cytological examination. At various times, coverslips were removed and stained with May-GriinwaldExptl Cell Res 83 (1974)
Giemsa to check the proportion of chick nuclei introduced in the hepatoma cells and to follow the formation of the nucleoli. Cells labelling and autoradiography. Covet-slips bearing heterokaryons were exposed for 3 h to 3H-hypoxanthine, generally labelled, at a spec. act. of I Ci/mM and at a concentration of I /rCi/ml (hypoxanthine-T (G); The Radiochemical Centre, Amersham). The preparations were then fixed, extracted overnight with water and then with0.3 M TCA(30min)and subjected to autoradiography (Ilford emulsion) for 4 days. Albumin synthesis. At the desired time after fusion (day 9 or day 13) the cells were incubated for 24 h in MEM depleted of amino acids and supplemented with 3 ‘I’,, of fetal calf serum with IO &i/ml of Yuniformly labelled amino acids (‘“C-protein hydrolysate; The Radiochemical Centre, Amersham). After incubation, the medium was collected and cold chick albumin was added as a carrier; it was then centrifuged, dialysed successively against many changes of phosphatebuffered saline (PBS) at uH 7.4 containing a mixture of non-radioactive ‘amino acids, PBS and finally water. After lyophilisation, the proteins were dissolved in a small volume of PBS. By this procedure, the medium was finallv concentrated at least 100-fold. This preparation will hereafter be called the lC-labelled medium. Radioimmunoelectrophoresis. The “C-labelled medium (2 ~1) was subjected to electrophoresis in a 1 o. agar gel, pH 8.6, poured on a microscopic slide. This was sometimes done - after absorption of the rat albumin (by the addition of some anti-rat albumin serum to the well before introduction of the “‘C-labelled medium). After electrouhoresis. the troughs were filled with rabbit anti-rat or anti-chick albumin serum and the immune precipitation allowed to uroceed for 20 h. The microscope slides were then soaked for 4 days in PBS and finally in water (2 h), dried, stained with amido-black, and exposed to Kodak X-ray film fcr about 1 week, to detect the labelled bands. In two cases (see table I, expt 4) the chick albumin of the ‘*C-labelled medium was further purified and concentrated in the following way. The ‘“C-labelled medium (containing a known amount of carrier chick albumin) was passed through a column of immunoadsorbent prepared with a rabbit anti-chick albumin serum coupled to cyanogen bromide-activated Sepharose [16, 171. Most of the proteins other than the chick albumin were not retained by the immunoadsorbent. The chick albumin was then eluted with 0.023 M glycine buffer at pH 2.4. The eluted fraction was dialysed against water and lyophilized, and the proteins so purified were dissolved in PBS. This concentrated preparation of chick albumin was examined by eiectrophoresis in the same way as the original ‘3C-labelled medium. The concentration of chick albumin in these preparations was also estimated by double immunodiffusion of 2-fold serial dilutions with reference solutions of known concentration. This allowed the determination of the yield of chick albumin and thus an estimate of how many times the chick albumin was concentrated by this procedure.
Reactivation
sf chick
q*throq’te
nuclei
49
Table 1. HGPRT acticity and albumin secretion in rat hepatoma cell x chick erythrocyte heterokaryons HGPRT activity ( “0) A Cr~~fuols Fu5 967
100 0.1
B Hefrrokaryom Expt I (day 9) 2 (day 9) 3 (day 13) 4 (day 9) (day 13) Mean
Not done 1.2 0.9 1.25 1.6 1.2
Secreted albumin ( 1’0) Rat
Chick
100
co.5 Not done -: 0.5 I. 0.2 -: 0.1
100 100 too
The above data were obtained from four independent fusion experiments (I, 2, 3, 4). In each case, the hepatoma cells were irradiated (6 000 rads) and then fused with chick erythrocytes by means of inactivated Sendai virus. After fusion the cells were seeded in flat prescription bottles. Nine (day 9) or thirteen (day 13) days later. the cells from some bottles were trypsinized -and their HGPRT activity determined. The values given for the HGPRT activity nresent in these different cell homogenates are not ‘corrected for the residual activity present in the recioient 967 cells (0.1 O(,).The cont;ois Fu5 (HGPRT+)‘and 967 (HGPRT-) were treated in the same way as the hepatoma cells fused with chick erythrocytes, except that no Sendai virus was added. The chick ervthrocytes added to these control samples were removed by the subsequent medium changes. Extracts from fu5 form about 1 IO nmoles of inosinic acid/h/mg protein ( 100 %), whether the cells were irradiated or not. For the detection of albumin, 9 or 13 days after fusion, the cells were incubated with ‘“C-labelled amino acids for 24 h. In expt 4, the same cells were incubated twice: first at day 9 and then at day 13. The maximum amount of chick albumin secreted by the heterokaryons (last column) was determined as explained in the text.
The sera against rat or chick albumin were prepared in rabbits using commerical preparations of albumin (from Koch-Light). These sera were highly specific: the anti-chick albumin sera were strong enough to give, after double immunodiffusion, a visible precipitin band with chick albumin at a concentration of 10 /dg/ml, but did not give any visible reaction with rat albumin at a concentration of 20 mg/ml. assay. The assay of this enzyme in cell homogenates was carried out according to Harris & Cook [6], except that the dialysis step was omitted
HGPRT
Fig. 1. A heterokaryon containing one 967 nucleus and one reactivated erythrocyte nucleus (top) with two nucleoli (from an I l-day culture).
and thymidine-5’-triphosphate (40 mM) was added to the incubation mixture, in order to avoid the degradation of inosinic acid by the nucleotidase activity present in the rat hepatoma cells. Chick and rat HGPRT activities were separated by electrophoresis on Cellogel, as described by Cook [7]. The enzyme was assayed by autoradiography according to Shin et al. [IS].
RESULTS Appearance of nucleoli in chick erythrocyte nuclei and synthesis of HGPRT The rat hepatoma cells used in this work, Fu5 and 967, fuse easily with chick erythrocytes. In these experiments, 15 to 30 9:) of the nuclei found in the cell population after fusion (i.e. heterokaryons plus rat hepatoma cells) were chick nuclei. Like other cells, the hepatoma cells induce the reactivation of chick erythrocyte nuclei; shortly after fusion the chick nuclei enlarge and after a few days full-sized nucleoli appear (see fig. I). In order to analyse more precisely the reactivation process in these rat cells, the synthesis of HGPRT was studied in 967 x chick erythrocyte heterokaryons. The 967 cells (which are resistant to 8-azaguanine) lack the enzyme HGPRT (see table 1) and are therefore unable to incorporate hypoxanExptl Cell Rex 83 (1974)
50
C. Szpirrr
Fig. 2. Autoradiographs of various cells exposed for 3 h to 3H-hypoxanthine (a) 967 cells; (b) a heterokaryon from a 3-day culture; (c) a heterokaryon from an 11-day culture; (d) Fu5 cells.
thine into nucleic acids: autoradiographs of these cells after exposure to tritiated hypoxanthine do not show any significant iabelling (fig. 2). However, HGPRT activity gradually appears in the 967 x chick erythrocyte heterokaryons, which then become able to incorporate hypoxanthine. This result is illustrated Exptl Cell Res 83 (1974)
by figs 2 and 3, and shows at once that the HGPRT is induced and that the reactivated nuclei are actively engaged in RNA synthesis (see, in fig. 2c, the labelling of the chick nucleus and especially of the nucleolus). Direct assays of the cell homogenates were done in order to determine how much
Reactivation of chick erythrocyte enzyme appears when the chick nuclei are fully reactivated, As shown in table 1, unfused 967 cells exhibit only 0.1 % of the activity present in the HGPRT+ cells (Fu5) from which 967 was isolated. After full reactivation, the activity detected in the fused cell population reaches a mean value of 1.2 %, of the level found in Fu5 (see table 1). The origin (chick on rat) of this new enzyme activity has been determined by an electrophoretic method [7, 181. Fig. 4 shows that the HGPRT activity of chick erythrocytes and of rat hepatoma cells (Fu5) has a different electrophoretic mobility and that the enzyme activity which appears in the 967 x chick erythrocyte heterokaryons has the mobility of the chick enzyme. If one makes the assumption that the chick and rat enzymes have similar specific activities, it appears that in the present case, a fused cell population containing 15 to 30 Y/Oof chick erythrocyte nuclei produces, after reactivation of these nuclei, an amount of chick encyme equal on the average to 1.1% of the level found in a normal cell (Fu5). This amount can be as high as 1.5 % (table 1).
Fig. 3. Abscissa: days after cell fusion; ordinate: no. of grains/967 nucleus. The development of HGPRT activity in 967 x chick erythrocyte dikaryons (incorporation of 3H-hypoxanthine into nucleic acids: autoradiography after a 3 h exposure of the cells to the tracer).
nuclei
51
Fig. 4. Electrophoretic mobilities of HGPRT, revealed by autoradiography. (a) Fu5; (b) 967-chick erythrocyte heterokaryons, from a 9-day culture; (c) chick erythrocytes.
AIbumin production In order to detect the possible release of chick albumin by the heterokaryons, it was essential to be able to distinguish between the chick albumin and the rat albumin secreted by the hepatoma cells. Advantage was taken of the fact that these two albumins have different electrophoretic mobilities, and that specific antisera against each of them can easily be obtained. Fig. 5 shows that on electrophoresis in 1 1: agar, at pH 8.6, the chick albumin has a greater mobility than rat albumin. The I%-labelled media containing carrier chick albumin from different fusion experiments were independently analysed by immunoelectrophoresis. The patterns were developed with antisera prepared against the rat and the chick albumins, and autoradiographs were made. These autoradiographs were compared with the stained slides to Exptl Cell Res 83 (1974)
52
C. Szpirer
‘ic- Anti-rat albumin 5a
+-,i Anti-chick-albumin
Sb
+- Anti-rat albumin Fig. 5. Radioimmunoelectrophoresis of lrC-labelled medium. (a) Stained slide showing the difference in electrophoretic mobility of rat and chick albumin. After electrophoresis of a mixture of the two proteins, the pattern was developed with antisera against rat albumin (top precipitin band) or chick albumin (bottom); (b) stained slide obtained after electrophoresis of “C-labelled medium reacted with an antiserum against chick albumin; (c) autoradiograph of this slide. Note that the chick precipitin band is not labelled; (d) a 200-fold dilution of ‘“C-labelled medium in a solution of rat albumin was submitted to electrophoresis and the pattern developed with an antiserum against rat albumin. The autoradiograph shows that the labelling of the precipitin band is still easily detectable.
determine whether the precipitin band formed by the carrier chick albumin was radioactive. In no case was there any evidence of labelling of the chick precipitin band. On the other hand, the rat albumin present in the 14Clabelled media was, as expected, heavily labelled. In order to estimate the sensitivity of the method, 2-fold serial dilutions of the l%labelled media were made in a solution of carrier rat albumin. These different dilutions were subjected to immunoelectrophoresis and after reaction with an anti-rat albumin antiserum autoradiographs were made. The analysis of these dilutions was done in parallel with the l-LC-labelled media. The autoradiographic arcs obtained with these different dilutions of the 14C-labelled rat albumin were then superimposed on the autoradiographs obtained with the corresponding undiluted 14C-labelled medium, in the precise position of the precipitin band formed by Exptl Cell Res 83 (1974)
the carrier chick albumin. This enabled an estimate to be made of the smallest amount of radioactive chick albumin which could have been detected. The conclusion of this analysis is that, in the present experiments, the radioactive rat albumin gives an autoradiographic are easily detectable (at the position of the carrier chick albumin band) at a 200-fold dilution (--O.S :,]) of the Wlabelled medium (fig. 5). As described in Material and Methods, two W-labelled media containing carrier chick albumin (expt 4, table 1) were passed through an immunoadsorbent prepared with an antichick albumin serum in order to concentrate the chick albumin. A 2-fold concentration was achieved in the case of the day 9 medium and a 5-fold concentration in the case of the day 13 medium. After immunoelectrophoresis and autoradiography of these concentrated preparations, the precipitin band of the chick albumin was still unlabelled. If one takes into
Reactivation of chick erythrocyte nuclei account the threshold of sensitivity of the method (0.5 0;)) one can conclude that in this experiment the maximum amount of chick albumin produced at day 9 is less than 0.25 I:) (0.5 “:, >: l/2) and a day 13 less than 0.1 “A(O.5“/:,x l/S) of the level of rat albumin produced by the heterokaryons. A comparison of the results obtained for albumin with those obtained for HGPRT is presented in table 1. It can be seen that the radioimmunoelectrophoresis method would have allowed the detection of chick albumin produced in very small amounts, compared with the HGPRT levels. The last experiments in particular (expt 4) shows that if one takes the amounts of rat enzyme and rat albumin synthesized as references (100 9;,), the amount of chick albumin produced by the heterokaryons must be less than one-tenth of the amount of HGPRT induced.
DlSCUSSTON The above results show that in rat hepatoma cell chick erythrocyte heterokaryons, the chick nuclei are efficiently reactivated. Nucleoli appear after a few days, RNA synthesis is induced and chick HGPRT is synthesized. However, it was not possible to detect any secretion of chick albumin. In the following discussion, it is assumed that rat albumin synthesis is maintained in the 967 x chick erythrocyte heterokaryons. Since the fused cell populations contain a large fraction of unfused hepatoma cells a clear-cut demonstration of this point could be obtained only by a single-cell method. Unfortunately the intracellular albumin of these hepatoma cells cannot be stained by immunofluorescence (see [ 151). An ‘extinction’ of rat albumin synthesis in the heterokaryons seems however to be highly unlikely. This synthesis is maintained, although at a
53
reduced rate, in hybrids between these hepatoma cells and mouse fibroblasts [13] and chick erythrocytes do not suppress synthesis of rat myosin when they are fused with rat myoblasts [ 191. The absence of chick albumin production by the 967 x chick erythrocyte heterokaryons can be explained by a failure either of the synthesis of the protein or of its release into the extracellular medium. The first explanation implies that the reactivation of the erythrocyte nucleus is not a generalized or random process, for, on this view, some chick genes are not expressed by the reactivated nuclei. This idea is supported by two other findings. Ringertz et al. [19] have studied the reactivation of chick erythrocytes in heterokaryons with rat myoblasts. They found that the reactivated nuclei do not induce synthesis of chick myosin. Moreover, Dariynkiewcz & Chelmicka-Szorc [20] have shown that in heterokaryons formed by fusing hen erythrocytes with human fibroblasts deficient in the ability to repair DNA after UVirradiation, the reactivated erythrocyte nuclei do not promote any restoration of this function. The absence of chick albumin secretion by the rat hepatoma cell x chick erythrocyte heterokaryons can also be explained by a failure at the secretion step. According to this hypotesis chick albumin would be synthesized but would not cross the hybrid cell membrane or would cross it very inefficiently. This explanation cannot be ruled out as the intracellular material cannot easily be studied. However, a failure at the secretion step does not seem to be a very likely explanation. Indeed, the present evidence indicates that, in hybrid cells, the transfer of macromolecules across the cellular membranes is not species-specific (see [8,9, 21, 221 and particularly the work of Peterson & Weiss [13], who have shown that in hybrids between mouse fibroExprl Cell Res 83 (1974)
54
C. Szpirer
blasts and rat hepatoma cells, mouse albumin can be secreted through the cell membrane). The reactivation of erythrocyte nuclei thus seems to be a selective process. But the differentiated cell fused with the chick erythrocyte apparently does not force the chick nucleus to acquire the same pattern of gene expression as itself, at least in the present experimental conditions. On the contrary, in heterokaryons made by fusing young immature red cells with mouse tissue culture cells synthesis of mouse hemoglobin is probably not induced (see [23]), and synthesis of chick hemoglobin stops 5 days after fusion. What are the possible interpretations of the apparently selective character of the reactivation process? One can suggest the following two interpretations (which are not mutually exclusive): (a) The selectivity of the reactivation process is a consequence of the state of the chick nucleus itself. One could suggest that only the chick genes which were active at some time in the life of the red cell can be expressed by the reactivated nuclei. This might be due to the fact that a gene which was active in the life of the cell might not be turned off in the same way as a gene which was never active in this cell or its precursors. (b) This selectivity is due to regulatory factors which might be present in the proteins that accumulate in the chick nucleus during its reactivation [8, 9, 21, 221. Among many possibilities, one could speculate that these hypothetical regulatory factors have a speciesspecific action in the case of albumin synthesis (but not in the case of other proteins, like HGPRT and membrane or nucleolar antigens) or that their selective action is due to gene dosage effects between the two genomes of the heterokaryons. The latter assumption is suggested by the recent results of Peterson & Weiss [I 31 who have shown that mouse albumin synthesis can be induced Expti Cell Res 83 (1974)
when mouse fibroblasts are crossed with hypertetraploid rat hepatoma cells but not with hyperdiploid cells. It would be interesting to fuse hypertetraploid hepatoma cells with chick erythrocytes to see whether chick albumin might not be produced in the heterokaryons. I am greatly indebted to ProfessorR. Harris, thanks to whom this work was carried out. for his suonort and for thoughtful reading the manuscript. I*‘also thank Dr P. R. Cook for helpful advice and stimulating discussions, and Professor B. Ephrussi and Dr M. Weiss for kindly providing the rat hepatoma cell lines. This work was carried out during the tenure of a fellowship from the “European SGence Exchange Programme”. C. S. is Charge de Recherches of the Fonds National de la Recherche Scientifique.
REFERENCES 1. Harris, H, J cell sci 2 (1967) 23.
2. Bolund, L, Dariynkiewicz, Z & Ringertz, N R, Exptl cell res 56 (1969) 406. 3. Harris, H, Nature 206 (1965) 583. 4. Harris, H, Sidebottom, E, Grace, D M & Bramwell. M E. J cell sci 4 (1969) 499. 5. Sidebottom, E & Harris, H, J cellsci 5 (1969) 351. 6. Harris, H &Cook, P, J cell sci 5 (1969) 121. Cook, P R, J cell sci 7 (1970) 1. i: Ege, T, Carlsson, S-A & Ringertz, N R, Exptl cell res 69 (1971) 472. 9. Ringertz, N R, Carlsson, S-A, Ege, T & Bolund, L, Proc natl acad sci US 68 (1971) 3228. IO. Dendy, P. R & Harris, H, J cell sci 12 (1973) 831. 11. Deak, I, Sidebottom, E & Harris, H, J cell sci 11 (1972) 379. 12. Schneider, J A & Weiss, M C, Proc natl acad sci US 68 (1971) 127. 13. Peterson, J A & Weiss, M C, Proc natl acad sci US 69 (1972) 571. 14. Pitot, J. C, Peraint, C, Morse, PA & Potter, V R, Nat] cancer inst monogr 13 (1964) 229. 15. Ohanian, S H, Taubman, S B & Thorbecke, G J, J natl cancer inst 43 (1969) 397. 16. Van der Loo, W. In preparation. 17. Porath, J, Axtn, R & frnback, S, Nature 215 (1967) 1491. 18. Shin, S, Khan, P M & Cook, P R, Biochemical genetics 5 (1971) 91. 19. Ringertz, N R, Carlsson, S-A & Savange, R E, Adv in biosci 8 (1972) 219. 20. Dariynkiewicz, Z & Chemilcka-Szorc, E, Exptl cell res 74 (1972) 131. 21. Dariynkiewicz, Z, Exptl cell res 67 (1971) 477. 22. Carlsson, S-A, Moore, G P M & Ringertz, N R, Exptl cell res 76 (1973) 234. 23. Harris, H, Cell fusion. Oxford University Press, Oxford (1970). Received March 20, 1973 Revised version received August 3, 1973
REACTIVATION
Cell Research 83 (1974) 47-54
OF CHICK ERYTHROCYTE
HETEROKARYONS
WITH RAT HEPATOMA
NUCLEI
IN
CELLS
C. SZPTRERl Sir William Dunn School of Pathology, Uniwrsity Oxford OXI 3RE, UK
of Oxford,
SUMMARY Chick erythrocyte nuclei were fused with rat hepatoma cells which express a number of liver functions; among other proteins, they secrete serum albumin. Like other cells (A9, HeLa), these rat hepatoma ceils induce the reactivation of the erythrocyte nuclei, which increase in size, resume the synthesis of RNA and acquire nucleoli. In hepatoma cells deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) the reactivated chick erythrocyte nuclei will induce the synthesis of this enzyme. In populations of fused cells containing 15 to 30 % of chick nuclei, the amount of induced HGPRT is more than 1 :!, of the level found in HGPRT+ rat hepatoma cells. On the other hand no secretion of chick albumin can be detected. Given the sensitivity of the method, one can conclude that if any chick albumin is secreted, it is at a rate equal to less than 0.1 ‘$6of the rate of rat albumin secretion. This result shows either that chick albumin is synthesized but not secreted by the heterokaryons; or that the reactivation of the erythrocyte nucleus is selective (only some chick genes are expressed) and that the rat hepatoma cells cannot induce these reactivated nuclei to acquire the same pattern of gene activity as themselves.
The chick erythrocyte nucleus is inert: it does not synthesize any DNA and only very low amounts of RNA, if any, and has no well formed nucleolus. However, this state of ultimate differentiation is not irreversible: when such a nucleus is introduced by cell fusion into an active cell (for example a mouse or a human tissue culture cell) it quickly increases in volume and dry mass [I, 21, resumes the synthesis of DNA and RNA [3] and, after 3-4 days, full-sized nucleoli appear [4]. At this stage, RNA is transferred from the erythrocyte nucleus to the cytoplasm of the heterokaryon [4, 51 where it is translated. i Present address: Department de Biologie MolCculaire, Universite libre de Bruxelles. Rue des Chevaux 67, B-1640 Rhode-St-Get&e, Belgium. 4-731809
To date, it has been clearly demonstrated that reactivated chick erythrocyte nuclei direct the synthesis of the following proteins: membrane antigens [4], an enzyme, the hypoxanthine guanine phosphoribosyl transferase (HGPRT, EC 2.4.2.8) [6, 71, nucleolar antigens [8, 91 and the receptor for diphtheria toxin [IO, 111. (The HGPRT catalyses the condensation of hypoxanthine or guanine with Sphosphoribosyl- 1-pyrophosphate.) These products are found in all cells (none of them is a differentiated product); and one may wonder whether the synthesis of some differentiated products can be induced in reactivated erythrocyte nuclei. In other words, is there any selectivity in the reactivation process? Are all the chick genes turned on in Expti Cell Res 83 (1974)
48
C. Szpirer
the heterokaryons, or does the chick genome or the genome of the other parent, or both, imrtose some choice? In order to obtain some information about this cluestion, I studied the reactivation of chick erythrocyte nuclei in albumin-producing cells, derived from a rat hepatoma. These hepatoma cells have been used in other fusion experiments [12, 131: it has been shown that rat albumin synthesis is not ‘extinguished’ in hybrids with mouse fibroblasts [12], and, furthermore, that in some cases mouse albumin synthesis can be initiated [13]. The main aim of the present study was to determine whether rat hepatoma cell chick erythrocyte heterokaryons produce chick albumin. L
MATERIALS
AND METHODS
Cells. Chick embryo erythrocytes were obtained from 12-day-old fertile eggs [9]. These erythrocytes synthesize very little RNA, if any; under conditions which permit very heavy labelling of tissue culture cells (autoradiography after exposure of the cells to 10 pCi/ ml of 3H-uridine for 4 h), most of the erythrocytes are not labelled. The rat hepatoma cells, Fu 5 [I21 and 967 were kindly provided by Dr B. Ephrussi and Dr M. Weiss. These cells are hyperdiploid. They are derived from the Reuber H35 hepatoma and were adapted to growth in vitro by Pitot et al. [14]. Clone 967 has been isolated from Fu5 by Dr M. Weiss bv selection in the presence of 3 pug/ml of b-azaguaninei it lacks HGPRT (see below). These hepatoma cells were grown in Eagle’s Minimum Essential Medium (MEM; Flow Laboratories) containing IO To fetal calf serum (Biocult). They secrete rat albumin [15]. Cell fusion. Before fusion, the hepatoma cells were harvested by trypsinization and subjected to a flux of 6 000 rads of gamma irradiation. This treatment suppresses mitosis and chick nuclei remain discrete long enough to develop nucleoli and to produce chick RNA which is translated in the cytoplasm of the heterokaryons [4]. The heterokaryons were made by fusing chick ervthrocvtes with the irradiated rat hepakma cells by means of UV-inactivated Sendai virus f4]. After fusion, a fraction of the cells was transferred to a Petri dish containing coverslips, the rest seeded into glass prescription bottles. The cells were maintained in MEM. The medium was changed every other day. Cytological examination. At various times, coverslips were removed and stained with May-GriinwaldExptl Cell Res 83 (1974)
Giemsa to check the proportion of chick nuclei introduced in the hepatoma cells and to follow the formation of the nucleoli. Cells labelling and autoradiography. Covet-slips bearing heterokaryons were exposed for 3 h to 3H-hypoxanthine, generally labelled, at a spec. act. of I Ci/mM and at a concentration of I /rCi/ml (hypoxanthine-T (G); The Radiochemical Centre, Amersham). The preparations were then fixed, extracted overnight with water and then with0.3 M TCA(30min)and subjected to autoradiography (Ilford emulsion) for 4 days. Albumin synthesis. At the desired time after fusion (day 9 or day 13) the cells were incubated for 24 h in MEM depleted of amino acids and supplemented with 3 ‘I’,, of fetal calf serum with IO &i/ml of Yuniformly labelled amino acids (‘“C-protein hydrolysate; The Radiochemical Centre, Amersham). After incubation, the medium was collected and cold chick albumin was added as a carrier; it was then centrifuged, dialysed successively against many changes of phosphatebuffered saline (PBS) at uH 7.4 containing a mixture of non-radioactive ‘amino acids, PBS and finally water. After lyophilisation, the proteins were dissolved in a small volume of PBS. By this procedure, the medium was finallv concentrated at least 100-fold. This preparation will hereafter be called the lC-labelled medium. Radioimmunoelectrophoresis. The “C-labelled medium (2 ~1) was subjected to electrophoresis in a 1 o. agar gel, pH 8.6, poured on a microscopic slide. This was sometimes done - after absorption of the rat albumin (by the addition of some anti-rat albumin serum to the well before introduction of the “‘C-labelled medium). After electrouhoresis. the troughs were filled with rabbit anti-rat or anti-chick albumin serum and the immune precipitation allowed to uroceed for 20 h. The microscope slides were then soaked for 4 days in PBS and finally in water (2 h), dried, stained with amido-black, and exposed to Kodak X-ray film fcr about 1 week, to detect the labelled bands. In two cases (see table I, expt 4) the chick albumin of the ‘*C-labelled medium was further purified and concentrated in the following way. The ‘“C-labelled medium (containing a known amount of carrier chick albumin) was passed through a column of immunoadsorbent prepared with a rabbit anti-chick albumin serum coupled to cyanogen bromide-activated Sepharose [16, 171. Most of the proteins other than the chick albumin were not retained by the immunoadsorbent. The chick albumin was then eluted with 0.023 M glycine buffer at pH 2.4. The eluted fraction was dialysed against water and lyophilized, and the proteins so purified were dissolved in PBS. This concentrated preparation of chick albumin was examined by eiectrophoresis in the same way as the original ‘3C-labelled medium. The concentration of chick albumin in these preparations was also estimated by double immunodiffusion of 2-fold serial dilutions with reference solutions of known concentration. This allowed the determination of the yield of chick albumin and thus an estimate of how many times the chick albumin was concentrated by this procedure.
Reactivation
sf chick
q*throq’te
nuclei
49
Table 1. HGPRT acticity and albumin secretion in rat hepatoma cell x chick erythrocyte heterokaryons HGPRT activity ( “0) A Cr~~fuols Fu5 967
100 0.1
B Hefrrokaryom Expt I (day 9) 2 (day 9) 3 (day 13) 4 (day 9) (day 13) Mean
Not done 1.2 0.9 1.25 1.6 1.2
Secreted albumin ( 1’0) Rat
Chick
100
co.5 Not done -: 0.5 I. 0.2 -: 0.1
100 100 too
The above data were obtained from four independent fusion experiments (I, 2, 3, 4). In each case, the hepatoma cells were irradiated (6 000 rads) and then fused with chick erythrocytes by means of inactivated Sendai virus. After fusion the cells were seeded in flat prescription bottles. Nine (day 9) or thirteen (day 13) days later. the cells from some bottles were trypsinized -and their HGPRT activity determined. The values given for the HGPRT activity nresent in these different cell homogenates are not ‘corrected for the residual activity present in the recioient 967 cells (0.1 O(,).The cont;ois Fu5 (HGPRT+)‘and 967 (HGPRT-) were treated in the same way as the hepatoma cells fused with chick erythrocytes, except that no Sendai virus was added. The chick ervthrocytes added to these control samples were removed by the subsequent medium changes. Extracts from fu5 form about 1 IO nmoles of inosinic acid/h/mg protein ( 100 %), whether the cells were irradiated or not. For the detection of albumin, 9 or 13 days after fusion, the cells were incubated with ‘“C-labelled amino acids for 24 h. In expt 4, the same cells were incubated twice: first at day 9 and then at day 13. The maximum amount of chick albumin secreted by the heterokaryons (last column) was determined as explained in the text.
The sera against rat or chick albumin were prepared in rabbits using commerical preparations of albumin (from Koch-Light). These sera were highly specific: the anti-chick albumin sera were strong enough to give, after double immunodiffusion, a visible precipitin band with chick albumin at a concentration of 10 /dg/ml, but did not give any visible reaction with rat albumin at a concentration of 20 mg/ml. assay. The assay of this enzyme in cell homogenates was carried out according to Harris & Cook [6], except that the dialysis step was omitted
HGPRT
Fig. 1. A heterokaryon containing one 967 nucleus and one reactivated erythrocyte nucleus (top) with two nucleoli (from an I l-day culture).
and thymidine-5’-triphosphate (40 mM) was added to the incubation mixture, in order to avoid the degradation of inosinic acid by the nucleotidase activity present in the rat hepatoma cells. Chick and rat HGPRT activities were separated by electrophoresis on Cellogel, as described by Cook [7]. The enzyme was assayed by autoradiography according to Shin et al. [IS].
RESULTS Appearance of nucleoli in chick erythrocyte nuclei and synthesis of HGPRT The rat hepatoma cells used in this work, Fu5 and 967, fuse easily with chick erythrocytes. In these experiments, 15 to 30 9:) of the nuclei found in the cell population after fusion (i.e. heterokaryons plus rat hepatoma cells) were chick nuclei. Like other cells, the hepatoma cells induce the reactivation of chick erythrocyte nuclei; shortly after fusion the chick nuclei enlarge and after a few days full-sized nucleoli appear (see fig. I). In order to analyse more precisely the reactivation process in these rat cells, the synthesis of HGPRT was studied in 967 x chick erythrocyte heterokaryons. The 967 cells (which are resistant to 8-azaguanine) lack the enzyme HGPRT (see table 1) and are therefore unable to incorporate hypoxanExptl Cell Rex 83 (1974)
50
C. Szpirrr
Fig. 2. Autoradiographs of various cells exposed for 3 h to 3H-hypoxanthine (a) 967 cells; (b) a heterokaryon from a 3-day culture; (c) a heterokaryon from an 11-day culture; (d) Fu5 cells.
thine into nucleic acids: autoradiographs of these cells after exposure to tritiated hypoxanthine do not show any significant iabelling (fig. 2). However, HGPRT activity gradually appears in the 967 x chick erythrocyte heterokaryons, which then become able to incorporate hypoxanthine. This result is illustrated Exptl Cell Res 83 (1974)
by figs 2 and 3, and shows at once that the HGPRT is induced and that the reactivated nuclei are actively engaged in RNA synthesis (see, in fig. 2c, the labelling of the chick nucleus and especially of the nucleolus). Direct assays of the cell homogenates were done in order to determine how much
Reactivation of chick erythrocyte enzyme appears when the chick nuclei are fully reactivated, As shown in table 1, unfused 967 cells exhibit only 0.1 % of the activity present in the HGPRT+ cells (Fu5) from which 967 was isolated. After full reactivation, the activity detected in the fused cell population reaches a mean value of 1.2 %, of the level found in Fu5 (see table 1). The origin (chick on rat) of this new enzyme activity has been determined by an electrophoretic method [7, 181. Fig. 4 shows that the HGPRT activity of chick erythrocytes and of rat hepatoma cells (Fu5) has a different electrophoretic mobility and that the enzyme activity which appears in the 967 x chick erythrocyte heterokaryons has the mobility of the chick enzyme. If one makes the assumption that the chick and rat enzymes have similar specific activities, it appears that in the present case, a fused cell population containing 15 to 30 Y/Oof chick erythrocyte nuclei produces, after reactivation of these nuclei, an amount of chick encyme equal on the average to 1.1% of the level found in a normal cell (Fu5). This amount can be as high as 1.5 % (table 1).
Fig. 3. Abscissa: days after cell fusion; ordinate: no. of grains/967 nucleus. The development of HGPRT activity in 967 x chick erythrocyte dikaryons (incorporation of 3H-hypoxanthine into nucleic acids: autoradiography after a 3 h exposure of the cells to the tracer).
nuclei
51
Fig. 4. Electrophoretic mobilities of HGPRT, revealed by autoradiography. (a) Fu5; (b) 967-chick erythrocyte heterokaryons, from a 9-day culture; (c) chick erythrocytes.
AIbumin production In order to detect the possible release of chick albumin by the heterokaryons, it was essential to be able to distinguish between the chick albumin and the rat albumin secreted by the hepatoma cells. Advantage was taken of the fact that these two albumins have different electrophoretic mobilities, and that specific antisera against each of them can easily be obtained. Fig. 5 shows that on electrophoresis in 1 1: agar, at pH 8.6, the chick albumin has a greater mobility than rat albumin. The I%-labelled media containing carrier chick albumin from different fusion experiments were independently analysed by immunoelectrophoresis. The patterns were developed with antisera prepared against the rat and the chick albumins, and autoradiographs were made. These autoradiographs were compared with the stained slides to Exptl Cell Res 83 (1974)
52
C. Szpirer
‘ic- Anti-rat albumin 5a
+-,i Anti-chick-albumin
Sb
+- Anti-rat albumin Fig. 5. Radioimmunoelectrophoresis of lrC-labelled medium. (a) Stained slide showing the difference in electrophoretic mobility of rat and chick albumin. After electrophoresis of a mixture of the two proteins, the pattern was developed with antisera against rat albumin (top precipitin band) or chick albumin (bottom); (b) stained slide obtained after electrophoresis of “C-labelled medium reacted with an antiserum against chick albumin; (c) autoradiograph of this slide. Note that the chick precipitin band is not labelled; (d) a 200-fold dilution of ‘“C-labelled medium in a solution of rat albumin was submitted to electrophoresis and the pattern developed with an antiserum against rat albumin. The autoradiograph shows that the labelling of the precipitin band is still easily detectable.
determine whether the precipitin band formed by the carrier chick albumin was radioactive. In no case was there any evidence of labelling of the chick precipitin band. On the other hand, the rat albumin present in the 14Clabelled media was, as expected, heavily labelled. In order to estimate the sensitivity of the method, 2-fold serial dilutions of the l%labelled media were made in a solution of carrier rat albumin. These different dilutions were subjected to immunoelectrophoresis and after reaction with an anti-rat albumin antiserum autoradiographs were made. The analysis of these dilutions was done in parallel with the l-LC-labelled media. The autoradiographic arcs obtained with these different dilutions of the 14C-labelled rat albumin were then superimposed on the autoradiographs obtained with the corresponding undiluted 14C-labelled medium, in the precise position of the precipitin band formed by Exptl Cell Res 83 (1974)
the carrier chick albumin. This enabled an estimate to be made of the smallest amount of radioactive chick albumin which could have been detected. The conclusion of this analysis is that, in the present experiments, the radioactive rat albumin gives an autoradiographic are easily detectable (at the position of the carrier chick albumin band) at a 200-fold dilution (--O.S :,]) of the Wlabelled medium (fig. 5). As described in Material and Methods, two W-labelled media containing carrier chick albumin (expt 4, table 1) were passed through an immunoadsorbent prepared with an antichick albumin serum in order to concentrate the chick albumin. A 2-fold concentration was achieved in the case of the day 9 medium and a 5-fold concentration in the case of the day 13 medium. After immunoelectrophoresis and autoradiography of these concentrated preparations, the precipitin band of the chick albumin was still unlabelled. If one takes into
Reactivation of chick erythrocyte nuclei account the threshold of sensitivity of the method (0.5 0;)) one can conclude that in this experiment the maximum amount of chick albumin produced at day 9 is less than 0.25 I:) (0.5 “:, >: l/2) and a day 13 less than 0.1 “A(O.5“/:,x l/S) of the level of rat albumin produced by the heterokaryons. A comparison of the results obtained for albumin with those obtained for HGPRT is presented in table 1. It can be seen that the radioimmunoelectrophoresis method would have allowed the detection of chick albumin produced in very small amounts, compared with the HGPRT levels. The last experiments in particular (expt 4) shows that if one takes the amounts of rat enzyme and rat albumin synthesized as references (100 9;,), the amount of chick albumin produced by the heterokaryons must be less than one-tenth of the amount of HGPRT induced.
DlSCUSSTON The above results show that in rat hepatoma cell chick erythrocyte heterokaryons, the chick nuclei are efficiently reactivated. Nucleoli appear after a few days, RNA synthesis is induced and chick HGPRT is synthesized. However, it was not possible to detect any secretion of chick albumin. In the following discussion, it is assumed that rat albumin synthesis is maintained in the 967 x chick erythrocyte heterokaryons. Since the fused cell populations contain a large fraction of unfused hepatoma cells a clear-cut demonstration of this point could be obtained only by a single-cell method. Unfortunately the intracellular albumin of these hepatoma cells cannot be stained by immunofluorescence (see [ 151). An ‘extinction’ of rat albumin synthesis in the heterokaryons seems however to be highly unlikely. This synthesis is maintained, although at a
53
reduced rate, in hybrids between these hepatoma cells and mouse fibroblasts [13] and chick erythrocytes do not suppress synthesis of rat myosin when they are fused with rat myoblasts [ 191. The absence of chick albumin production by the 967 x chick erythrocyte heterokaryons can be explained by a failure either of the synthesis of the protein or of its release into the extracellular medium. The first explanation implies that the reactivation of the erythrocyte nucleus is not a generalized or random process, for, on this view, some chick genes are not expressed by the reactivated nuclei. This idea is supported by two other findings. Ringertz et al. [19] have studied the reactivation of chick erythrocytes in heterokaryons with rat myoblasts. They found that the reactivated nuclei do not induce synthesis of chick myosin. Moreover, Dariynkiewcz & Chelmicka-Szorc [20] have shown that in heterokaryons formed by fusing hen erythrocytes with human fibroblasts deficient in the ability to repair DNA after UVirradiation, the reactivated erythrocyte nuclei do not promote any restoration of this function. The absence of chick albumin secretion by the rat hepatoma cell x chick erythrocyte heterokaryons can also be explained by a failure at the secretion step. According to this hypotesis chick albumin would be synthesized but would not cross the hybrid cell membrane or would cross it very inefficiently. This explanation cannot be ruled out as the intracellular material cannot easily be studied. However, a failure at the secretion step does not seem to be a very likely explanation. Indeed, the present evidence indicates that, in hybrid cells, the transfer of macromolecules across the cellular membranes is not species-specific (see [8,9, 21, 221 and particularly the work of Peterson & Weiss [13], who have shown that in hybrids between mouse fibroExprl Cell Res 83 (1974)
54
C. Szpirer
blasts and rat hepatoma cells, mouse albumin can be secreted through the cell membrane). The reactivation of erythrocyte nuclei thus seems to be a selective process. But the differentiated cell fused with the chick erythrocyte apparently does not force the chick nucleus to acquire the same pattern of gene expression as itself, at least in the present experimental conditions. On the contrary, in heterokaryons made by fusing young immature red cells with mouse tissue culture cells synthesis of mouse hemoglobin is probably not induced (see [23]), and synthesis of chick hemoglobin stops 5 days after fusion. What are the possible interpretations of the apparently selective character of the reactivation process? One can suggest the following two interpretations (which are not mutually exclusive): (a) The selectivity of the reactivation process is a consequence of the state of the chick nucleus itself. One could suggest that only the chick genes which were active at some time in the life of the red cell can be expressed by the reactivated nuclei. This might be due to the fact that a gene which was active in the life of the cell might not be turned off in the same way as a gene which was never active in this cell or its precursors. (b) This selectivity is due to regulatory factors which might be present in the proteins that accumulate in the chick nucleus during its reactivation [8, 9, 21, 221. Among many possibilities, one could speculate that these hypothetical regulatory factors have a speciesspecific action in the case of albumin synthesis (but not in the case of other proteins, like HGPRT and membrane or nucleolar antigens) or that their selective action is due to gene dosage effects between the two genomes of the heterokaryons. The latter assumption is suggested by the recent results of Peterson & Weiss [I 31 who have shown that mouse albumin synthesis can be induced Expti Cell Res 83 (1974)
when mouse fibroblasts are crossed with hypertetraploid rat hepatoma cells but not with hyperdiploid cells. It would be interesting to fuse hypertetraploid hepatoma cells with chick erythrocytes to see whether chick albumin might not be produced in the heterokaryons. I am greatly indebted to ProfessorR. Harris, thanks to whom this work was carried out. for his suonort and for thoughtful reading the manuscript. I*‘also thank Dr P. R. Cook for helpful advice and stimulating discussions, and Professor B. Ephrussi and Dr M. Weiss for kindly providing the rat hepatoma cell lines. This work was carried out during the tenure of a fellowship from the “European SGence Exchange Programme”. C. S. is Charge de Recherches of the Fonds National de la Recherche Scientifique.
REFERENCES 1. Harris, H, J cell sci 2 (1967) 23.
2. Bolund, L, Dariynkiewicz, Z & Ringertz, N R, Exptl cell res 56 (1969) 406. 3. Harris, H, Nature 206 (1965) 583. 4. Harris, H, Sidebottom, E, Grace, D M & Bramwell. M E. J cell sci 4 (1969) 499. 5. Sidebottom, E & Harris, H, J cellsci 5 (1969) 351. 6. Harris, H &Cook, P, J cell sci 5 (1969) 121. Cook, P R, J cell sci 7 (1970) 1. i: Ege, T, Carlsson, S-A & Ringertz, N R, Exptl cell res 69 (1971) 472. 9. Ringertz, N R, Carlsson, S-A, Ege, T & Bolund, L, Proc natl acad sci US 68 (1971) 3228. IO. Dendy, P. R & Harris, H, J cell sci 12 (1973) 831. 11. Deak, I, Sidebottom, E & Harris, H, J cell sci 11 (1972) 379. 12. Schneider, J A & Weiss, M C, Proc natl acad sci US 68 (1971) 127. 13. Peterson, J A & Weiss, M C, Proc natl acad sci US 69 (1972) 571. 14. Pitot, J. C, Peraint, C, Morse, PA & Potter, V R, Nat] cancer inst monogr 13 (1964) 229. 15. Ohanian, S H, Taubman, S B & Thorbecke, G J, J natl cancer inst 43 (1969) 397. 16. Van der Loo, W. In preparation. 17. Porath, J, Axtn, R & frnback, S, Nature 215 (1967) 1491. 18. Shin, S, Khan, P M & Cook, P R, Biochemical genetics 5 (1971) 91. 19. Ringertz, N R, Carlsson, S-A & Savange, R E, Adv in biosci 8 (1972) 219. 20. Dariynkiewicz, Z & Chemilcka-Szorc, E, Exptl cell res 74 (1972) 131. 21. Dariynkiewicz, Z, Exptl cell res 67 (1971) 477. 22. Carlsson, S-A, Moore, G P M & Ringertz, N R, Exptl cell res 76 (1973) 234. 23. Harris, H, Cell fusion. Oxford University Press, Oxford (1970). Received March 20, 1973 Revised version received August 3, 1973