Somatic Cell Genetics, Vol. 5, No. 6, 1979, pp. 1045-1059
Restoration of Metabolic Cooperation in Heterokaryons Between HGPRT-Deficient Mouse A9 Fibroblasts and Chick Embryo Erythrocytes N.C. Bols, 1 A.B. Kane, 2 and N.R. Ringertz Institute for Medical Cell Genetics, Medical Nobel Institute, Karolinska Institute, S-104 01 Stockholm 60, Sweden Received 19 July 1979
Abstract--Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate FH]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT § cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated FH]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.
INTRODUCTION Intercellular communication and transfer of low-molecular-weight compounds between cells in contact can be demonstrated by at least three ~Current address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. 2Current address: Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 1045
0098-0366/79/1100-1045503.00/0 9 1979PlenumPublishingCorporation
1046
Bols et al.
techniques: (1) electronic coupling, (2) transfer of fluorescent dyes (1), and (3) transfer of labeled nucleotides which is called metabolic cooperation (2). These types of intercellular communication take place at specialized membrane structures identified as gap junctions (3). These structures appear as a 2- to 4-nm gap between adjacent cells in transmission electron micrographs or as hexagonal aggregates of intramembranous particles in freeze-fracture preparations (4). Most invertebrate and vertebrate cells possess these specialized gap junctions and have the ability to participate in intercellular communication (5). Notable exceptions among vertebrate cells are erythrocytes, A9 and C1-D1 cells (sublines of mouse L cells), and some varieties of HeLa and other malignant cell lines (reviewed in 6). In order to investigate the defect in those cells which are unable to participate in metabolic cooperation, Azarnia et al. (7) analyzed heterokaryons and hybrid cells derived from fusion between cooperators and noncooperators. Heterokaryons between fibroblasts from Lesch-Nyhan patients and mouse C1-DI cells showed ionic coupling, fluorescein transfer, and gap junction formation within 24 h after fusion (7). In a similar experiment, Clements and Subak-Sharpe (8) demonstrated metabolic cooperation between BHK-chick embryo erythrocyte heterokaryons and unfused BHK cells. Azarnia et al. (7) also tested hybrids of human Lesch-Nyhan + mouse C1-D1 cells for their ability to communicate and isolated several segregants which retained the ability to communicate together with a few human chromosomes. With complete loss of human chromosomes, the hybrids could not communicate and no gap junctions could be detected by electron microscopy. Both investigations with heterokaryons suggest that the cooperating phenotype dominates. Segregation of ionic coupling and gap junction formation with retention of human chromosomes in hybrids suggests that expression of human genes enables the hybrids to participate in intercellular communication (7). We have investigated the ability of heterokaryons between two noncooperating cells, mouse A9 fibroblasts and chick embryo erythrocytes, to participate in metabolic cooperation with human Lesch-Nyhan fibroblasts (HGPRT-). Complementation of the defects in metabolic cooperation between these two noncooperating cells could occur, first, at the membrane level immediately after fusion or, second, at the genetic level with reactivation of the chick erythrocyte nucleus some days after fusion. Complementation of membrane functions in heterokaryons between defective cells has already been reported for activation of adenylate cyclase by the prostaglandin E1 or catecholamine receptor (9). Thus, it is possible that chick erythrocyte membrane components could complement mouse A9 components to allow the heterokaryon to participate in metabolic cooperation. The second possible mechanism of complementation in heterokaryons
Metabolic Cooperationin Heterokaryons
1047
between mouse A9 fibroblasts and chick embryo erythrocytes is through reactivation of the chick erythrocyte nucleus. Some days after fusion there is eventual reactivation and reexpression of chick genetic information (reviewed in 10). With the reappearance of the chick nucleolus, heterokaryons express chick surface antigens (11), chick nueleolus-specific antigens (12), and susceptibility to diphtheria toxin (13). We have discovered that with the reactivation of the chick erythrocyte nucleus four days after fusion, A9erythrocyte heterokaryons acquire the ability to participate in metabolic cooperation. At earlier times after fusion there is no complementation of the defects in metabolic cooperation in heterokaryons between mouse A9 fibroblasts and chick embryo erythrocytes. MATERIALS AND METHODS Cell Cultures. Human fibroblasts from Lesch-Nyhan patients deficient in hypoxanthine-guanine phosphoribosyltransferase ( H G P R T - ) activity were used as recipient cells in metabolic cooperation experiments. The GM29 strain was obtained from the Human Genetic Mutant Cell Repository, Camden, New Jersey; another strain (VIC) was a generous gift of Dr. B. Migeon, Johns Hopkins Hospital, Baltimore, Maryland. All cells were used between passages 22 and 35. Mouse A9 fibroblasts are derivatives of the L-cell line deficient in HGPRT activity, adenine phosphoribosyltransferase (APRT) activity (14), and are unable to undergo metabolic cooperation (3). Prior to fusion with A9 cells, chick embryo erythrocytes were obtained by collection of blood from the chorioallantoic vessels of 10-day-old embryos. The blood was centrifuged and the cells washed three times with Hanks' balanced salt solution. Primary chick embryo fibroblasts were grown from brief trypsinization and dispersion of skin from 10-day-old embryos. These cultures were used within 2-4 passages, at which time a homogeneous population of fibroblasts was present. All cultures were grown in Dulbecco's modified Eagles' medium containing 10% calf serum or fetal calf serum (for human fibroblasts) which had been inactivated at 56~ for 20 rain. Incubation of cultures was at 37~ in a humidified 95% air-5% CO2 atmosphere. Stock cultures were passaged once weekly at a split ratio of 1:2 and monitored for mycoplasma contamination according to the procedure of Chen (15). All experiments were conducted with exponentially growing fibroblast cultures. Cell Fusion. Heterokaryons between chick embryo erythrocytes and fibroblasts were obtained by fusion with UV-inactivated Sendai virus. Fibroblasts were plated overnight on Btirker cover glasses at a density of 1.3 x 103 cells/cm 2. Prior to fusion, the cultures were rinsed three times with cold (4~ Hanks' balanced salt solution and placed at 4~ for 15 min with
1048
Bols et al.
0.5 ml of UV-inactivated Sendai virus in 10 ml of Earles' balanced salt solution, pH 8.0. Chick embryo erythrocytes were added to the cultures at a density of 5 • 105 cells/cm 2 for an additional 15 rain at 4~ with occasional gentle agitation. After incubation at 37~ for 45 min, the medium was gently aspirated and replaced with prewarmed Dulbecco's modified Eagles' medium containing 10% calf serum. Details of the fusion procedures are described by Appels et al. (16). Where indicated, A9-chick erythrocyte heterokaryons were placed in Dulbecco's modified Eagles' medium containing 0.5% calf serum or Dulbecco's modified Eagles' medium containing 10% calf serum plus hypoxanthine, aminopterin, and thymidine (HAT medium) for four days before the assay of metabolic cooperation. Heterokaryons between A9 fibroblasts and primary chick embryo fibroblasts were obtained following fusion with 50% polyethylene glycol (w/v) in phosphate-buffered saline, pH 7.2. Freshly trypsinized stock cultures were cocultivated on Bfirker cover glasses at a density of 1.3 • 103 cells/cm 2 for 1 h at 37~ Prior to fusion, cultures were rinsed three times with phosphate-buffered saline, pH 7.2, at 20~ Fusion was carried out by exposing the cultures to the polyethylene glycol solution at 20~ for 60 sec as described by Pontecorvo (1975). After gentle washing with phosphatebuffered saline, pH 7.2, the cultures were incubated in Dulbecco's modified Eagles' medium containing 10% calf serum for 2-24 h before the assay of metabolic cooperation. Assay for Metabolic Cooperation. For these 'experiments, H G P R T human fibroblasts were used as recipient cells. They were first allowed to phagocytize polystyrene beads. After having been grown with beads for 24 h, 90% of the cells contained more than 20 beads, which were confined to the cytoplasms of the cells. They were trypsinized, washed several times to remove any noningested beads, and plated onto Biirker cover glasses that already contained the potential donors. To this mixed culture, [3H]hypoxanthine was added immediately after the addition of the recipients 1-2 h later. After 4 h in the presence of [3H]hypoxanthine (25 #Ci/ml), the cultures were fixed overnight in absolute methanol or ethanol-acetone (1:1) at 4~ extracted with 5% (w/v) trichloracetic acid at 4~ for 5 min, and covered with Kodak AR 10 stripping film. After exposure for 5-6 days, the slides were developed, rinsed in running tap water, stained with H33258 (1 #g/ml in phosphate-buffered saline, pH 7.0) for 5 min, rinsed in water, dried, and mounted in glycerol. Under these staining conditions H33258 stains only nuclei, and different animal species give distinct patterns of nuclear fluoresence (18). Autoradiograms were examined with fluorescence microscopes, either a Zeiss 4902 or a Nikon Apophot, that were equipped with phase optics. Fluorescence microscopy was used to determine the nuclei present in
Metabolic Cooperation in Heterokaryons
1049
heterokaryons and the nuclear area over which grain counts were to be made. Chick erythrocyte nuclei were identified by their small size while mouse A9 nuclei were distinguished by their brightly fluorescent chromocenters. In contrast the human and chick fibroblast nuclei were large and uniformly fluorescent. The phase optics were used to see the beads in recipients, to determine whether recipients and donors were in contact, to count the number of grains overlying nuclei, and to ascertain if nucleoli were present in the reactivating erythrocyte nuclei. Chemicals. [3H]Hypoxanthine (1.8 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, England. Unlabeled hypoxanthine and thymidine were purchased from Sigma Scientific Co., St. Louis, Missouri. Aminopterin was obtained from the Karolinska Sjukhuset Pharmacy, Stockholm. Polystyrene beads 0.982 lzm in diameter were purchased from Polysciences, Inc., Warrington, Pennsylvania. The fluorescent dye H33258 was bought from Farbw~irke Hoechstag, Frankfurt, Germany. Polyethylene glycol (MW 1500) was Obtained from Koch-Light Lab., Ltd., Colnbrook, Bucks, England. RESULTS
Acquisition of HGPRT Activity by Fusion of HGPRT- Cells with Chick Embryo Erythrocytes. Immediately after the fusion of chick embryo erythrocytes (CE) with either human (GM29) or mouse (A9) fibroblasts deficient in HGPRT activity the heterokaryons incorporated hypoxanthine. [3H]Hypoxanthine was added to CE-GM29 heterokaryons 3 h after fusion and to CE-A9 heterokaryons 1.5 h after fusion. After a 4-h labeling period, the cultures were fixed and processed for autoradiography of acid-insoluble material. Grains were found over the fibroblast nuclei in heterokaryons while very few grains were found over the nuclei of unfused fibroblasts (Fig. 1). The ability of these heterokaryons to incorporate [3H]hypoxanthine allowed their use as donors in subsequent metabolic cooperation experiments.
Metabolic Cooperation with Heterokaryons Formedfrom Cooperating and Noncooperating Cells. Cooperating and noncooperating cells were fused together and the resulting heterokaryons tested for their ability to cooperate. Heterokaryons were formed between human fibroblasts (GM29), which do cooperate (19), and chick embryo erythrocytes, which do not (Fig. 1B), and betwen chick fibroblasts, which do cooperate (data not shown) and mouse A9 cells, which do not (3). In both cases, the heterokaryons, which have HGPRT activity and therefore are donors, were tested against human fibroblasts (GM29), which were labeled with polystyrene beads. The GM29 recipients in contact with CE-GM29 heterokaryons or chick fibroblast-A9 heterokaryons demonstrated considerable nuclear labeling while those not in contact showed
1050
Bols et al.
3H-HYPOXANTHINE INCORF:'ORATION
A. GM29-ERYTHROCYTE
HETEROKARYONS
2010t.)
B. GM29 EZ] (3M29 IN CONTACT WITH ERYTHROCYTE GM29 ALONE
10
20
310 4'0 50 60 GRA{NS/NLICLEUS
?0
>80
Fig. 1. Abscissa: grams/nucleus; ordinate: frequency. A histogram demonstrating that CEGM29 heterokaryons can incorporate [3H]hypoxanthine. Chick embryo erythrocytes (CE) were fused to HGPRT- human fibroblasts (GM29) with Sendai virus. [3H]Hypoxanthine was added to the culture (25 gCi/ml) 3 h after fusion. The culture was fixed 4 h later and processed for autoradiography. Grains were counted over human fibroblast nuclei in (A) heterokaryons (N = 100) and in (B) unfused fibroblasts (N = 100) that were either alone (hatched region) or in contact with erythrocytes (clear region).
few grains. Thus, heterokaryons between cooperating and noncooperating cells do communicate, and the intermixing of chick and mammalian membrane components does not interfere. Using this system to study metabolic cooperation in heterokaryons, we are able to ask these two questions. First, will a hybrid membrane of a heterokaryon between two noncooperating cells, chick embryo erythrocytes and mouse A9 cells, show complementation of those components required for metabolic cooperation? Second, will a heterokaryon between A9 cells and chick embryo erythrocytes acquire the ability to participate in metabolic cooperation after reactivation of the chick erthrocyte nucleus? Absence of Metabolic Cooperation in CE-A9 Heterokaryons Shortly after Fusion. Heterokaryons were created by the fusion of mouse A9 cells
Metabolic Cooperationin Heterokaryons
1051
Fig. 2. This single microscope field, which is analyzed in three ways, shows that CE-A9 heterokaryons do not undergo metabolic cooperation shortly after fusion. Heterokaryons were formed by the fusion of chick embryo erythrocytes (CE) with mouse A9 cells. HGPRT- human fibroblasts (VIC) and [3H]hypoxanthine (25 ttCi/ml) were added 1.5 h after fusion. The culture was fixed 4 h later and processed for autoradiography. In (A) (top photograph) the cell outlines are in focus and the potential recipient, which is identified by the polystyrene beads, is in contact with a number of cells (arrows). In (B) (middle illustration) the grains are in focus and none are seen to overlie the potential recipient even though the recipient is in contact with labeled cells (arrows). In (C) (bottom illustration) the nuclei are identified with fluorescence microscopy, and the radioactively labeled cells contain a small (arrows) and large nucleus and are thus CE-A9 heterokaryons.
1052
Bols et al.
with chick embryo erythrocytes (CE) and tested with HGPRT human fibroblasts for their ability to cooperate (Figs. 2 and 3). Human fibroblasts together with [3H]hypoxanthine were added to the heterokaryons 1.5 h after fusion and the cultures fixed 4 and 24 h later. The number of grains over human fibroblast nuclei was the same whether the fibroblasts were in contact with CE-A9 heterokaryons (Fig. 3A) or alone (Fig. 3B). This result is illustrated in Fig. 3 which shows the paucity of nuclear grains over human fibroblasts in contact with a CE-A9 heterokaryon. There was no metabolic cooperation between CE-A9 heterokaryons and fibroblasts whether the assay was conducted for 4 or 24 h. Thus, there appears to be no immediate
3H-HYFOXANTHINE INCORPORATION IN H G P R T - C E L L S
. IN CONTACT WITH Ag-ERYTHROCYTE HETEROKARYONS
32 2416m, 8 _ d kd (D
u. o
10
20
30 40 50 60 GRAINS/NUCLEUS
Pf Ill m
I
~
70
I
z
~8 162432-
AL NE 40-
Fig. 3. Abscissa: grains/nucleus; ordinate: frequency. A histogram demonstrating that C E - A 9 heterokaryons do not undergo metabolic cooperation shortly after formation. See Fig. 2 for a description of the experiment. The number of grains was counted over nuclei of H G P R T - human fibroblasts in (A) contact with heterokaryons ( N = 100) and (B) alone ( N = 100).
At least one present
One present One present
B
C D
Absent Absent
At least one present
At least one present
Small uniformly fluorescent nucleus
+
+
Greater than 15 grains over the cell
+
+
Less than 15 grains over the cell
Phase Optics
14.1 52.9
2.7
30.3
Frequency c (%)
A 9 - C E heterokaryons in which the CE nucleus has been at least partially reactivated A 9 - C E heterokaryons in which the CE nucleus has failed to reactivate A 9 - C E synkaryons A9 cells
interpretation
aMouse A9 cells on Biicker slides were fused to chick erythrocytes (CE) with Sendal virus and placed on H A T medium. After 4 days the ceils were given [3H]hypoxanthine (25 ~ C i / m l ) for 4 h and then fixed. After the slides had been processed for autoradiography and developed, they were stained with the fluorochrome H33258 and examined with UV illumination and with phase optics. bThis fluorescent staining pattern is characteristic of mouse nuclei. cN = 488.
At least one present
Large granular fluorescent nucleus b
A
Cell type
UV illumination
Microscopic observations
Table 1. Cell types present four days after fusion of chick erythrocytes with mouse A9 ceils ~
t,h t~
~~
mo
o
o
1054
Bols et ai.
complementation of the structures required for metabolic cooperation in the hybrid membrane of CE-A9 heterokaryons. CE-A9 Heterokaryons Cooperate after Reactivation o f CE Nucleus. When a CE-A9 fusion mixture that had been on HAT medium for 4 days was labeled with [3H]hypoxanthine, four cell types could be identified in autoradiograms. The characteristics defining these four cell types and the interpretations given to them are outlined in Table 1. The heavily labeled cells are CE-A9 heterokaryons and synkaryons in which the erythrocyte nucleus has been at least partially reactivated. They were sufficiently labeled and occurred frequently enough, in approximately 45% of the cells present, that they could be used in subsequent metabolic cooperation experiments. In contrast, when the fusion mixture was placed in medium with only 0.5% serum, the same four cell types could be identified but the frequency of heavily labeled cells was very low. This made finding cooperating pairs in subsequent metabolic cooperation experiments difficult. After 4 days on HAT medium, the HGPRT + fusion products, CE-A9 heterokaryons and synkaryons, were tested with HGPRT- human fibroblasts for their ability to cooperate (Figs. 4 and 5). By themselves the HGPRTfibroblasts incorporated very little [3H]hypoxanthine (Fig. 5B) while some of those in contact with CE-A9 heterokaryons showed considerable incorporation (Fig. 5A). Inasmuch as 15 grains per nucleus was the most observed in fibroblasts by themselves, those fibroblasts that were in contact with HGPRT + fusion products could be divided into two classes. Of 100 fibroblasts in contact with HGPRT + fusion products, 43 had more than 15 grains per nucleus and thus cooperated, while the remaining fibroblasts had less than 15 grains per nucleus and thus did not cooperate. When heterokaryons and synkaryons were considered separately, 34 of 100 heterokaryon-fibroblast pairs and 37 of 100 synkaryon-fibroblast pairs cooperated. An example of a heterokaryon-fibroblast pair cooperating is shown in Fig. 4. When the fusion mixture was maintained on low serum medium for 4 days, a few heterokaryon-fibroblast and synkaryon-fibroblast pairs were found and a number of these cooperated as well. Thus, four days after fusion, some heterokaryons and synkaryons have acquired the ability to cooperate. DISCUSSION Three conclusions may be drawn from these data. First, we have confirmed that heterokaryons between cooperating and noncooperating cells can undergo metabolic cooperation (7, 8). Second, heterokaryons that result from the fusion of chick embryo erythrocytes to mouse A9 cells do not cooperate immediately upon formation. Third, with reactivation of the chick
Metabolic Cooperation in Heterokaryons
1055
Fig. 4. This single microscope field, which is analyzed three ways, shows a 4-day-old CE-A9 heterokaryon undergoing metabolic cooperation. After the fusion of chick embryo erythrocytes (CE) with mouse A9 cells, the fusion mixture was placed on HAT medium. After 4 days the HAT medium was replaced with normal medium and HGPRT- human fibroblasts (GM29) were added to the culture along with [3H]hypoxanthine (25 #Ci/ml). Tbe culture was fixed 4 h later and processed for autoradiography. In (A) (top illustration) the cell outlines are in focus and the potential recipient, which is identified by the polystyrene beads, is in contact (arrow) with the potential donor. In (B) (middle photograph) the grains are in focus and are seen to overlie the recipient nucleus. In (C) (bottom illustration) the nuclei are identified with fluorescence microscopy, and the potential donor contains a small (arrow) and large nucleus and is thus a CE-A9 heterokaryon.
1056
Bols et al.
3H-HYPOXANTHINE INGORPORATION IN GM29 CELLS
~t
A. IN cONTACT WITH AO-ERYTHROCYTE
HETEROKARYONS AND SYNKARYONS
3O
20m I0" .J J tlJ o u. o
tO
20
n,
r / f J
w
z
30 40 50 60 (IR~NS/NUGLE~ 9
9
!
70
80
|
!
IO-
20" 30"
40'
60" 70-~"
B.
ALONE
Fig. 5. Abscissa: grains/nucleus; ordinate: frequency. A histogram demonstrating that some 4-day-old CE-GM29 heterokaryons undergo metabolic cooperation while others do not. See Fig. 4 for a description of the experiment. The number of grains was counted over nuclei of HGPRThuman fibrobIasts in (A) contact with beterokaryons (N = 100) and (B) alone (N - 100).
erythrocyte nucleus, CE-A9 heterokaryons acquire the ability to participate in metabolic cooperation. The observation that heterokaryons between cooperating and noncooperating cells do show metabolic cooperation demonstrates that there is no extinction of this function by the noncooperating cell. This observation was made previously by the demonstration of ionic coupling in heterokaryons between human Lesch-Nyhan fibroblasts and mouse C1-D1 cells (7) and metabolic cooperation by heterokaryons between BHK cells and chick erythrocytes (8). We have extended these findings to include heterokaryons between several pairs of cooperating and noncooperating cells: human LeschNyhan fibroblasts and chick erythrocytes, and primary chick fibroblasts and
Metabolic Cooperation in Heterokaryons
1057
mouse A9 cells. In the first case this assay was possible because the chick erythrocyte-fibroblast heterokaryons acquired the ability to incorporate [3H]hypoxanthine early after fusion. The ability to metabolize hypoxanthine is most likely associated with the passive transfer of the chick enzyme HGPRT during the fusion process (8). Heterokaryons were then formed between two noncooperating cells: mouse A9 fibroblasts and chick embryo erythrocytes. From the data presented, we conclude that there is no complementation of the defects in metabolic cooperation by formation of hybrid membrane in these heterokaryons. Earlier experiments have shown that chick specific surface antigens are retained in CE-A9 heterokaryons for at least the first 24 h after fusion (11). Our experiments with heterokaryons between cooperating and noncooperating cells eliminate the possibility that the fusion process renders the hybrid membrane nonfunctional for metabolic cooperation. Similarly, those experiments also establish that the introduction of chick membrane components into a mammalian membrane does not extinguish its ability to participate in metabolic cooperation. It is still possible, however, that a heterokaryon between two noncooperating cells may participate in other forms of intercellular communication, as detected by ionic coupling or intercellular fluorescein transfer. While heterokaryons between mouse A9 cells and chick erythrocytes are unable to participate in metabolic cooperation immediately after fusion, many of the heterokaryons acquired this ability 4 days later. Inasmuch as metabolic cooperation is mediated through gap junctions (3) and that with time chick genes are expressed in heterokaryons (11), the most likely explanation for this result is that the chick nucleus directs the synthesis of components required for the formation of gap junctions. Other chick specific gene products have been reported to appear some time after fusion, and their appearance has been correlated with the development of nucleoli in the erythrocyte nuclei (20). In this study the development of metabolic cooperation and nucleoli could not be directly correlated because in the autoradiograms, in which cooperation is measured, grains often interfere with the phase microscopic view of the reactivating nuclei. However, nucleoli are clearly visible in some cooperating heterokaryons. The failure of a majority of the heterokaryons to cooperate could be due to incomplete reactivation of the erythrocyte nucleus. Others have noted incomplete reactivation at 4 days after fusion (13). However, the important point is that at least one third of the heterokaryons underwent metabolic cooperation. This is likely due to the reactivation of the chick genes for gap junctions, although there are two other possible explanations, which are outlined below. First, with reactivation of the chick erythrocyte nucleus, there could be expression of the mouse genes which are responsible for metabolic coopera-
1058
Bols et al.
tion. Expression of suppressed mouse genes has been noted previously by the appearance of HGPRT of mouse origin in hybrids between A9 cells and chick erythrocytes (21) or HGPRT + human cells (22), and in hybrids between mouse IR cells (L cells deficient in HGPRT) and chick fibroblasts (23). It is possible, then, that the genes for HGPRT and metabolic cooperation are linked and reexpressed concomittantly during selection of HAT medium. This is not likely because in hybrids between mouse C1-D1 cells and human Lesch-Nyhan fibroblasts the segregants, which were able to participate in intercellular communication and showed gap junctions, did not necessarily retain HGPRT or TK activity (7). The second possibility is that the ability of heterokaryons between two noncooperating cells to participate in metabolic cooperation is unrelated to nuclear activation and gene expression. Since metabolic cooperation is closely correlated with the occurrence of specialized membrane structures called gap junctions (3), it is possible that in heterokaryons between A9 fibroblasts and chick embryo erythrocytes reorganization of membrane components into gap junctions occurs late after fusion, and independently of nuclear activation. ACKNOWLEDGEMENTS This research was supported by a grant to Dr. Nils R. Ringertz from the Swedish Cancer Society. N. C. Bols was the recipient of a National Research Council of Canada postdoctoral fellowship. A. B. Kane was supported by a National Research Service Award postdoctoral fellowship CA05180 from the National Institutes of Health, U . S . A . N . C . B . would like to thank Dr. C. Peterson of the University of Waterloo for the use of her fluorescence microscope. LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Lowenstein, W. R., (1973). Fed. Proe. 32:60. Subak-Sharpe, J. H., Burk, R. R., and Pitts, J. D. (1966). Heredity 21:342. Gilula, N. B., Reeves, O. R., and Steinbach, A. (1972). Nature 235:262. Pitts, J. D. 1976. In The Developmental Biology o f Plants and Animals. (ed) Graham, C. F., and Waveing, E. F. (Blackwell, Oxford) p. 96. Epstein, M. L., and Gilula, N. B. (1977). J Cell Biol. 75:769. Weinstein, R. S., Merk, F. B., and Alroy, J. (1976). Adv Cancer Res 23:23. Azarnia, R., Larsen, W. J., and Lowenstein, W. R. (1974). Proc Natl. Acad. Sci. U.S.A. 71:880. Clements, G. B., and Subak-Sharpe, J. H. (1975). Exp Cell Res. 95:25. Schramm, M., Orly, J., Eimerl, S., and Korner, M. (1977). Nature 268:310. Ringertz, N. R., and Savage, R. E. (1976). Cell Hybrids, (Academic Press, New York), p. 87. Harris, H., Sidebottom, E., Grace, D. M., and Bramwell, M. E. (1969). J Cell Sci. 4:499. Ringertz, N. R., Carlsson, S. A., Ege, T., and Bolund, L. (1971). Proc Natl. Acad. Sci. U.S.A. 68:3228.
Metabofic Cooperation in Heterokaryons
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
1059
Dendy, P. R., and Harris, H. (1973). J Cell Sci. 12"831. Littlefield, J. W. (1964). Nature 203:1142. Chen, T. R. (1977). Exp CellRes. 104:255. Appels, R., Bolund, L., and Ringertz, N. R. (1974). JMol. Biol. 87:339. Pontecorvo, G. (1975). Somat. Cell Genet. 1:397. Hilwig, I., and Gropp, A. (1975). Exp Cell Res. 91:457. Bols, N. C., and Ringertz, N. R. (1979). Exp Cell Res. 120:15. Sidebottom, E., and Deak, I. I. (1976). Int Rev. Cytol. 44:29. Schwartz, A. G., Cook, P. R., and Harris, H. (1971). Nature New Biol. (London), 230:5. Watson, B., Gormley, I. P., Gardiner, S. E., Evans, H. J., and Harris, H. (1972). Exp Cell Res. 75:401. 23. Bakay, B., Croce, C. M., Koprowski, H., and Nyhan, W. L. (1973). Proc Natl Acad Sci U.S.A. 70:1998.
Restoration of Metabolic Cooperation in Heterokaryons Between HGPRT-Deficient Mouse A9 Fibroblasts and Chick Embryo Erythrocytes N.C. Bols, 1 A.B. Kane, 2 and N.R. Ringertz Institute for Medical Cell Genetics, Medical Nobel Institute, Karolinska Institute, S-104 01 Stockholm 60, Sweden Received 19 July 1979
Abstract--Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate FH]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT § cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated FH]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.
INTRODUCTION Intercellular communication and transfer of low-molecular-weight compounds between cells in contact can be demonstrated by at least three ~Current address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. 2Current address: Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 1045
0098-0366/79/1100-1045503.00/0 9 1979PlenumPublishingCorporation
1046
Bols et al.
techniques: (1) electronic coupling, (2) transfer of fluorescent dyes (1), and (3) transfer of labeled nucleotides which is called metabolic cooperation (2). These types of intercellular communication take place at specialized membrane structures identified as gap junctions (3). These structures appear as a 2- to 4-nm gap between adjacent cells in transmission electron micrographs or as hexagonal aggregates of intramembranous particles in freeze-fracture preparations (4). Most invertebrate and vertebrate cells possess these specialized gap junctions and have the ability to participate in intercellular communication (5). Notable exceptions among vertebrate cells are erythrocytes, A9 and C1-D1 cells (sublines of mouse L cells), and some varieties of HeLa and other malignant cell lines (reviewed in 6). In order to investigate the defect in those cells which are unable to participate in metabolic cooperation, Azarnia et al. (7) analyzed heterokaryons and hybrid cells derived from fusion between cooperators and noncooperators. Heterokaryons between fibroblasts from Lesch-Nyhan patients and mouse C1-DI cells showed ionic coupling, fluorescein transfer, and gap junction formation within 24 h after fusion (7). In a similar experiment, Clements and Subak-Sharpe (8) demonstrated metabolic cooperation between BHK-chick embryo erythrocyte heterokaryons and unfused BHK cells. Azarnia et al. (7) also tested hybrids of human Lesch-Nyhan + mouse C1-D1 cells for their ability to communicate and isolated several segregants which retained the ability to communicate together with a few human chromosomes. With complete loss of human chromosomes, the hybrids could not communicate and no gap junctions could be detected by electron microscopy. Both investigations with heterokaryons suggest that the cooperating phenotype dominates. Segregation of ionic coupling and gap junction formation with retention of human chromosomes in hybrids suggests that expression of human genes enables the hybrids to participate in intercellular communication (7). We have investigated the ability of heterokaryons between two noncooperating cells, mouse A9 fibroblasts and chick embryo erythrocytes, to participate in metabolic cooperation with human Lesch-Nyhan fibroblasts (HGPRT-). Complementation of the defects in metabolic cooperation between these two noncooperating cells could occur, first, at the membrane level immediately after fusion or, second, at the genetic level with reactivation of the chick erythrocyte nucleus some days after fusion. Complementation of membrane functions in heterokaryons between defective cells has already been reported for activation of adenylate cyclase by the prostaglandin E1 or catecholamine receptor (9). Thus, it is possible that chick erythrocyte membrane components could complement mouse A9 components to allow the heterokaryon to participate in metabolic cooperation. The second possible mechanism of complementation in heterokaryons
Metabolic Cooperationin Heterokaryons
1047
between mouse A9 fibroblasts and chick embryo erythrocytes is through reactivation of the chick erythrocyte nucleus. Some days after fusion there is eventual reactivation and reexpression of chick genetic information (reviewed in 10). With the reappearance of the chick nucleolus, heterokaryons express chick surface antigens (11), chick nueleolus-specific antigens (12), and susceptibility to diphtheria toxin (13). We have discovered that with the reactivation of the chick erythrocyte nucleus four days after fusion, A9erythrocyte heterokaryons acquire the ability to participate in metabolic cooperation. At earlier times after fusion there is no complementation of the defects in metabolic cooperation in heterokaryons between mouse A9 fibroblasts and chick embryo erythrocytes. MATERIALS AND METHODS Cell Cultures. Human fibroblasts from Lesch-Nyhan patients deficient in hypoxanthine-guanine phosphoribosyltransferase ( H G P R T - ) activity were used as recipient cells in metabolic cooperation experiments. The GM29 strain was obtained from the Human Genetic Mutant Cell Repository, Camden, New Jersey; another strain (VIC) was a generous gift of Dr. B. Migeon, Johns Hopkins Hospital, Baltimore, Maryland. All cells were used between passages 22 and 35. Mouse A9 fibroblasts are derivatives of the L-cell line deficient in HGPRT activity, adenine phosphoribosyltransferase (APRT) activity (14), and are unable to undergo metabolic cooperation (3). Prior to fusion with A9 cells, chick embryo erythrocytes were obtained by collection of blood from the chorioallantoic vessels of 10-day-old embryos. The blood was centrifuged and the cells washed three times with Hanks' balanced salt solution. Primary chick embryo fibroblasts were grown from brief trypsinization and dispersion of skin from 10-day-old embryos. These cultures were used within 2-4 passages, at which time a homogeneous population of fibroblasts was present. All cultures were grown in Dulbecco's modified Eagles' medium containing 10% calf serum or fetal calf serum (for human fibroblasts) which had been inactivated at 56~ for 20 rain. Incubation of cultures was at 37~ in a humidified 95% air-5% CO2 atmosphere. Stock cultures were passaged once weekly at a split ratio of 1:2 and monitored for mycoplasma contamination according to the procedure of Chen (15). All experiments were conducted with exponentially growing fibroblast cultures. Cell Fusion. Heterokaryons between chick embryo erythrocytes and fibroblasts were obtained by fusion with UV-inactivated Sendai virus. Fibroblasts were plated overnight on Btirker cover glasses at a density of 1.3 x 103 cells/cm 2. Prior to fusion, the cultures were rinsed three times with cold (4~ Hanks' balanced salt solution and placed at 4~ for 15 min with
1048
Bols et al.
0.5 ml of UV-inactivated Sendai virus in 10 ml of Earles' balanced salt solution, pH 8.0. Chick embryo erythrocytes were added to the cultures at a density of 5 • 105 cells/cm 2 for an additional 15 rain at 4~ with occasional gentle agitation. After incubation at 37~ for 45 min, the medium was gently aspirated and replaced with prewarmed Dulbecco's modified Eagles' medium containing 10% calf serum. Details of the fusion procedures are described by Appels et al. (16). Where indicated, A9-chick erythrocyte heterokaryons were placed in Dulbecco's modified Eagles' medium containing 0.5% calf serum or Dulbecco's modified Eagles' medium containing 10% calf serum plus hypoxanthine, aminopterin, and thymidine (HAT medium) for four days before the assay of metabolic cooperation. Heterokaryons between A9 fibroblasts and primary chick embryo fibroblasts were obtained following fusion with 50% polyethylene glycol (w/v) in phosphate-buffered saline, pH 7.2. Freshly trypsinized stock cultures were cocultivated on Bfirker cover glasses at a density of 1.3 • 103 cells/cm 2 for 1 h at 37~ Prior to fusion, cultures were rinsed three times with phosphate-buffered saline, pH 7.2, at 20~ Fusion was carried out by exposing the cultures to the polyethylene glycol solution at 20~ for 60 sec as described by Pontecorvo (1975). After gentle washing with phosphatebuffered saline, pH 7.2, the cultures were incubated in Dulbecco's modified Eagles' medium containing 10% calf serum for 2-24 h before the assay of metabolic cooperation. Assay for Metabolic Cooperation. For these 'experiments, H G P R T human fibroblasts were used as recipient cells. They were first allowed to phagocytize polystyrene beads. After having been grown with beads for 24 h, 90% of the cells contained more than 20 beads, which were confined to the cytoplasms of the cells. They were trypsinized, washed several times to remove any noningested beads, and plated onto Biirker cover glasses that already contained the potential donors. To this mixed culture, [3H]hypoxanthine was added immediately after the addition of the recipients 1-2 h later. After 4 h in the presence of [3H]hypoxanthine (25 #Ci/ml), the cultures were fixed overnight in absolute methanol or ethanol-acetone (1:1) at 4~ extracted with 5% (w/v) trichloracetic acid at 4~ for 5 min, and covered with Kodak AR 10 stripping film. After exposure for 5-6 days, the slides were developed, rinsed in running tap water, stained with H33258 (1 #g/ml in phosphate-buffered saline, pH 7.0) for 5 min, rinsed in water, dried, and mounted in glycerol. Under these staining conditions H33258 stains only nuclei, and different animal species give distinct patterns of nuclear fluoresence (18). Autoradiograms were examined with fluorescence microscopes, either a Zeiss 4902 or a Nikon Apophot, that were equipped with phase optics. Fluorescence microscopy was used to determine the nuclei present in
Metabolic Cooperation in Heterokaryons
1049
heterokaryons and the nuclear area over which grain counts were to be made. Chick erythrocyte nuclei were identified by their small size while mouse A9 nuclei were distinguished by their brightly fluorescent chromocenters. In contrast the human and chick fibroblast nuclei were large and uniformly fluorescent. The phase optics were used to see the beads in recipients, to determine whether recipients and donors were in contact, to count the number of grains overlying nuclei, and to ascertain if nucleoli were present in the reactivating erythrocyte nuclei. Chemicals. [3H]Hypoxanthine (1.8 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, England. Unlabeled hypoxanthine and thymidine were purchased from Sigma Scientific Co., St. Louis, Missouri. Aminopterin was obtained from the Karolinska Sjukhuset Pharmacy, Stockholm. Polystyrene beads 0.982 lzm in diameter were purchased from Polysciences, Inc., Warrington, Pennsylvania. The fluorescent dye H33258 was bought from Farbw~irke Hoechstag, Frankfurt, Germany. Polyethylene glycol (MW 1500) was Obtained from Koch-Light Lab., Ltd., Colnbrook, Bucks, England. RESULTS
Acquisition of HGPRT Activity by Fusion of HGPRT- Cells with Chick Embryo Erythrocytes. Immediately after the fusion of chick embryo erythrocytes (CE) with either human (GM29) or mouse (A9) fibroblasts deficient in HGPRT activity the heterokaryons incorporated hypoxanthine. [3H]Hypoxanthine was added to CE-GM29 heterokaryons 3 h after fusion and to CE-A9 heterokaryons 1.5 h after fusion. After a 4-h labeling period, the cultures were fixed and processed for autoradiography of acid-insoluble material. Grains were found over the fibroblast nuclei in heterokaryons while very few grains were found over the nuclei of unfused fibroblasts (Fig. 1). The ability of these heterokaryons to incorporate [3H]hypoxanthine allowed their use as donors in subsequent metabolic cooperation experiments.
Metabolic Cooperation with Heterokaryons Formedfrom Cooperating and Noncooperating Cells. Cooperating and noncooperating cells were fused together and the resulting heterokaryons tested for their ability to cooperate. Heterokaryons were formed between human fibroblasts (GM29), which do cooperate (19), and chick embryo erythrocytes, which do not (Fig. 1B), and betwen chick fibroblasts, which do cooperate (data not shown) and mouse A9 cells, which do not (3). In both cases, the heterokaryons, which have HGPRT activity and therefore are donors, were tested against human fibroblasts (GM29), which were labeled with polystyrene beads. The GM29 recipients in contact with CE-GM29 heterokaryons or chick fibroblast-A9 heterokaryons demonstrated considerable nuclear labeling while those not in contact showed
1050
Bols et al.
3H-HYPOXANTHINE INCORF:'ORATION
A. GM29-ERYTHROCYTE
HETEROKARYONS
2010t.)
B. GM29 EZ] (3M29 IN CONTACT WITH ERYTHROCYTE GM29 ALONE
10
20
310 4'0 50 60 GRA{NS/NLICLEUS
?0
>80
Fig. 1. Abscissa: grams/nucleus; ordinate: frequency. A histogram demonstrating that CEGM29 heterokaryons can incorporate [3H]hypoxanthine. Chick embryo erythrocytes (CE) were fused to HGPRT- human fibroblasts (GM29) with Sendai virus. [3H]Hypoxanthine was added to the culture (25 gCi/ml) 3 h after fusion. The culture was fixed 4 h later and processed for autoradiography. Grains were counted over human fibroblast nuclei in (A) heterokaryons (N = 100) and in (B) unfused fibroblasts (N = 100) that were either alone (hatched region) or in contact with erythrocytes (clear region).
few grains. Thus, heterokaryons between cooperating and noncooperating cells do communicate, and the intermixing of chick and mammalian membrane components does not interfere. Using this system to study metabolic cooperation in heterokaryons, we are able to ask these two questions. First, will a hybrid membrane of a heterokaryon between two noncooperating cells, chick embryo erythrocytes and mouse A9 cells, show complementation of those components required for metabolic cooperation? Second, will a heterokaryon between A9 cells and chick embryo erythrocytes acquire the ability to participate in metabolic cooperation after reactivation of the chick erthrocyte nucleus? Absence of Metabolic Cooperation in CE-A9 Heterokaryons Shortly after Fusion. Heterokaryons were created by the fusion of mouse A9 cells
Metabolic Cooperationin Heterokaryons
1051
Fig. 2. This single microscope field, which is analyzed in three ways, shows that CE-A9 heterokaryons do not undergo metabolic cooperation shortly after fusion. Heterokaryons were formed by the fusion of chick embryo erythrocytes (CE) with mouse A9 cells. HGPRT- human fibroblasts (VIC) and [3H]hypoxanthine (25 ttCi/ml) were added 1.5 h after fusion. The culture was fixed 4 h later and processed for autoradiography. In (A) (top photograph) the cell outlines are in focus and the potential recipient, which is identified by the polystyrene beads, is in contact with a number of cells (arrows). In (B) (middle illustration) the grains are in focus and none are seen to overlie the potential recipient even though the recipient is in contact with labeled cells (arrows). In (C) (bottom illustration) the nuclei are identified with fluorescence microscopy, and the radioactively labeled cells contain a small (arrows) and large nucleus and are thus CE-A9 heterokaryons.
1052
Bols et al.
with chick embryo erythrocytes (CE) and tested with HGPRT human fibroblasts for their ability to cooperate (Figs. 2 and 3). Human fibroblasts together with [3H]hypoxanthine were added to the heterokaryons 1.5 h after fusion and the cultures fixed 4 and 24 h later. The number of grains over human fibroblast nuclei was the same whether the fibroblasts were in contact with CE-A9 heterokaryons (Fig. 3A) or alone (Fig. 3B). This result is illustrated in Fig. 3 which shows the paucity of nuclear grains over human fibroblasts in contact with a CE-A9 heterokaryon. There was no metabolic cooperation between CE-A9 heterokaryons and fibroblasts whether the assay was conducted for 4 or 24 h. Thus, there appears to be no immediate
3H-HYFOXANTHINE INCORPORATION IN H G P R T - C E L L S
. IN CONTACT WITH Ag-ERYTHROCYTE HETEROKARYONS
32 2416m, 8 _ d kd (D
u. o
10
20
30 40 50 60 GRAINS/NUCLEUS
Pf Ill m
I
~
70
I
z
~8 162432-
AL NE 40-
Fig. 3. Abscissa: grains/nucleus; ordinate: frequency. A histogram demonstrating that C E - A 9 heterokaryons do not undergo metabolic cooperation shortly after formation. See Fig. 2 for a description of the experiment. The number of grains was counted over nuclei of H G P R T - human fibroblasts in (A) contact with heterokaryons ( N = 100) and (B) alone ( N = 100).
At least one present
One present One present
B
C D
Absent Absent
At least one present
At least one present
Small uniformly fluorescent nucleus
+
+
Greater than 15 grains over the cell
+
+
Less than 15 grains over the cell
Phase Optics
14.1 52.9
2.7
30.3
Frequency c (%)
A 9 - C E heterokaryons in which the CE nucleus has been at least partially reactivated A 9 - C E heterokaryons in which the CE nucleus has failed to reactivate A 9 - C E synkaryons A9 cells
interpretation
aMouse A9 cells on Biicker slides were fused to chick erythrocytes (CE) with Sendal virus and placed on H A T medium. After 4 days the ceils were given [3H]hypoxanthine (25 ~ C i / m l ) for 4 h and then fixed. After the slides had been processed for autoradiography and developed, they were stained with the fluorochrome H33258 and examined with UV illumination and with phase optics. bThis fluorescent staining pattern is characteristic of mouse nuclei. cN = 488.
At least one present
Large granular fluorescent nucleus b
A
Cell type
UV illumination
Microscopic observations
Table 1. Cell types present four days after fusion of chick erythrocytes with mouse A9 ceils ~
t,h t~
~~
mo
o
o
1054
Bols et ai.
complementation of the structures required for metabolic cooperation in the hybrid membrane of CE-A9 heterokaryons. CE-A9 Heterokaryons Cooperate after Reactivation o f CE Nucleus. When a CE-A9 fusion mixture that had been on HAT medium for 4 days was labeled with [3H]hypoxanthine, four cell types could be identified in autoradiograms. The characteristics defining these four cell types and the interpretations given to them are outlined in Table 1. The heavily labeled cells are CE-A9 heterokaryons and synkaryons in which the erythrocyte nucleus has been at least partially reactivated. They were sufficiently labeled and occurred frequently enough, in approximately 45% of the cells present, that they could be used in subsequent metabolic cooperation experiments. In contrast, when the fusion mixture was placed in medium with only 0.5% serum, the same four cell types could be identified but the frequency of heavily labeled cells was very low. This made finding cooperating pairs in subsequent metabolic cooperation experiments difficult. After 4 days on HAT medium, the HGPRT + fusion products, CE-A9 heterokaryons and synkaryons, were tested with HGPRT- human fibroblasts for their ability to cooperate (Figs. 4 and 5). By themselves the HGPRTfibroblasts incorporated very little [3H]hypoxanthine (Fig. 5B) while some of those in contact with CE-A9 heterokaryons showed considerable incorporation (Fig. 5A). Inasmuch as 15 grains per nucleus was the most observed in fibroblasts by themselves, those fibroblasts that were in contact with HGPRT + fusion products could be divided into two classes. Of 100 fibroblasts in contact with HGPRT + fusion products, 43 had more than 15 grains per nucleus and thus cooperated, while the remaining fibroblasts had less than 15 grains per nucleus and thus did not cooperate. When heterokaryons and synkaryons were considered separately, 34 of 100 heterokaryon-fibroblast pairs and 37 of 100 synkaryon-fibroblast pairs cooperated. An example of a heterokaryon-fibroblast pair cooperating is shown in Fig. 4. When the fusion mixture was maintained on low serum medium for 4 days, a few heterokaryon-fibroblast and synkaryon-fibroblast pairs were found and a number of these cooperated as well. Thus, four days after fusion, some heterokaryons and synkaryons have acquired the ability to cooperate. DISCUSSION Three conclusions may be drawn from these data. First, we have confirmed that heterokaryons between cooperating and noncooperating cells can undergo metabolic cooperation (7, 8). Second, heterokaryons that result from the fusion of chick embryo erythrocytes to mouse A9 cells do not cooperate immediately upon formation. Third, with reactivation of the chick
Metabolic Cooperation in Heterokaryons
1055
Fig. 4. This single microscope field, which is analyzed three ways, shows a 4-day-old CE-A9 heterokaryon undergoing metabolic cooperation. After the fusion of chick embryo erythrocytes (CE) with mouse A9 cells, the fusion mixture was placed on HAT medium. After 4 days the HAT medium was replaced with normal medium and HGPRT- human fibroblasts (GM29) were added to the culture along with [3H]hypoxanthine (25 #Ci/ml). Tbe culture was fixed 4 h later and processed for autoradiography. In (A) (top illustration) the cell outlines are in focus and the potential recipient, which is identified by the polystyrene beads, is in contact (arrow) with the potential donor. In (B) (middle photograph) the grains are in focus and are seen to overlie the recipient nucleus. In (C) (bottom illustration) the nuclei are identified with fluorescence microscopy, and the potential donor contains a small (arrow) and large nucleus and is thus a CE-A9 heterokaryon.
1056
Bols et al.
3H-HYPOXANTHINE INGORPORATION IN GM29 CELLS
~t
A. IN cONTACT WITH AO-ERYTHROCYTE
HETEROKARYONS AND SYNKARYONS
3O
20m I0" .J J tlJ o u. o
tO
20
n,
r / f J
w
z
30 40 50 60 (IR~NS/NUGLE~ 9
9
!
70
80
|
!
IO-
20" 30"
40'
60" 70-~"
B.
ALONE
Fig. 5. Abscissa: grains/nucleus; ordinate: frequency. A histogram demonstrating that some 4-day-old CE-GM29 heterokaryons undergo metabolic cooperation while others do not. See Fig. 4 for a description of the experiment. The number of grains was counted over nuclei of HGPRThuman fibrobIasts in (A) contact with beterokaryons (N = 100) and (B) alone (N - 100).
erythrocyte nucleus, CE-A9 heterokaryons acquire the ability to participate in metabolic cooperation. The observation that heterokaryons between cooperating and noncooperating cells do show metabolic cooperation demonstrates that there is no extinction of this function by the noncooperating cell. This observation was made previously by the demonstration of ionic coupling in heterokaryons between human Lesch-Nyhan fibroblasts and mouse C1-D1 cells (7) and metabolic cooperation by heterokaryons between BHK cells and chick erythrocytes (8). We have extended these findings to include heterokaryons between several pairs of cooperating and noncooperating cells: human LeschNyhan fibroblasts and chick erythrocytes, and primary chick fibroblasts and
Metabolic Cooperation in Heterokaryons
1057
mouse A9 cells. In the first case this assay was possible because the chick erythrocyte-fibroblast heterokaryons acquired the ability to incorporate [3H]hypoxanthine early after fusion. The ability to metabolize hypoxanthine is most likely associated with the passive transfer of the chick enzyme HGPRT during the fusion process (8). Heterokaryons were then formed between two noncooperating cells: mouse A9 fibroblasts and chick embryo erythrocytes. From the data presented, we conclude that there is no complementation of the defects in metabolic cooperation by formation of hybrid membrane in these heterokaryons. Earlier experiments have shown that chick specific surface antigens are retained in CE-A9 heterokaryons for at least the first 24 h after fusion (11). Our experiments with heterokaryons between cooperating and noncooperating cells eliminate the possibility that the fusion process renders the hybrid membrane nonfunctional for metabolic cooperation. Similarly, those experiments also establish that the introduction of chick membrane components into a mammalian membrane does not extinguish its ability to participate in metabolic cooperation. It is still possible, however, that a heterokaryon between two noncooperating cells may participate in other forms of intercellular communication, as detected by ionic coupling or intercellular fluorescein transfer. While heterokaryons between mouse A9 cells and chick erythrocytes are unable to participate in metabolic cooperation immediately after fusion, many of the heterokaryons acquired this ability 4 days later. Inasmuch as metabolic cooperation is mediated through gap junctions (3) and that with time chick genes are expressed in heterokaryons (11), the most likely explanation for this result is that the chick nucleus directs the synthesis of components required for the formation of gap junctions. Other chick specific gene products have been reported to appear some time after fusion, and their appearance has been correlated with the development of nucleoli in the erythrocyte nuclei (20). In this study the development of metabolic cooperation and nucleoli could not be directly correlated because in the autoradiograms, in which cooperation is measured, grains often interfere with the phase microscopic view of the reactivating nuclei. However, nucleoli are clearly visible in some cooperating heterokaryons. The failure of a majority of the heterokaryons to cooperate could be due to incomplete reactivation of the erythrocyte nucleus. Others have noted incomplete reactivation at 4 days after fusion (13). However, the important point is that at least one third of the heterokaryons underwent metabolic cooperation. This is likely due to the reactivation of the chick genes for gap junctions, although there are two other possible explanations, which are outlined below. First, with reactivation of the chick erythrocyte nucleus, there could be expression of the mouse genes which are responsible for metabolic coopera-
1058
Bols et al.
tion. Expression of suppressed mouse genes has been noted previously by the appearance of HGPRT of mouse origin in hybrids between A9 cells and chick erythrocytes (21) or HGPRT + human cells (22), and in hybrids between mouse IR cells (L cells deficient in HGPRT) and chick fibroblasts (23). It is possible, then, that the genes for HGPRT and metabolic cooperation are linked and reexpressed concomittantly during selection of HAT medium. This is not likely because in hybrids between mouse C1-D1 cells and human Lesch-Nyhan fibroblasts the segregants, which were able to participate in intercellular communication and showed gap junctions, did not necessarily retain HGPRT or TK activity (7). The second possibility is that the ability of heterokaryons between two noncooperating cells to participate in metabolic cooperation is unrelated to nuclear activation and gene expression. Since metabolic cooperation is closely correlated with the occurrence of specialized membrane structures called gap junctions (3), it is possible that in heterokaryons between A9 fibroblasts and chick embryo erythrocytes reorganization of membrane components into gap junctions occurs late after fusion, and independently of nuclear activation. ACKNOWLEDGEMENTS This research was supported by a grant to Dr. Nils R. Ringertz from the Swedish Cancer Society. N. C. Bols was the recipient of a National Research Council of Canada postdoctoral fellowship. A. B. Kane was supported by a National Research Service Award postdoctoral fellowship CA05180 from the National Institutes of Health, U . S . A . N . C . B . would like to thank Dr. C. Peterson of the University of Waterloo for the use of her fluorescence microscope. LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Lowenstein, W. R., (1973). Fed. Proe. 32:60. Subak-Sharpe, J. H., Burk, R. R., and Pitts, J. D. (1966). Heredity 21:342. Gilula, N. B., Reeves, O. R., and Steinbach, A. (1972). Nature 235:262. Pitts, J. D. 1976. In The Developmental Biology o f Plants and Animals. (ed) Graham, C. F., and Waveing, E. F. (Blackwell, Oxford) p. 96. Epstein, M. L., and Gilula, N. B. (1977). J Cell Biol. 75:769. Weinstein, R. S., Merk, F. B., and Alroy, J. (1976). Adv Cancer Res 23:23. Azarnia, R., Larsen, W. J., and Lowenstein, W. R. (1974). Proc Natl. Acad. Sci. U.S.A. 71:880. Clements, G. B., and Subak-Sharpe, J. H. (1975). Exp Cell Res. 95:25. Schramm, M., Orly, J., Eimerl, S., and Korner, M. (1977). Nature 268:310. Ringertz, N. R., and Savage, R. E. (1976). Cell Hybrids, (Academic Press, New York), p. 87. Harris, H., Sidebottom, E., Grace, D. M., and Bramwell, M. E. (1969). J Cell Sci. 4:499. Ringertz, N. R., Carlsson, S. A., Ege, T., and Bolund, L. (1971). Proc Natl. Acad. Sci. U.S.A. 68:3228.
Metabofic Cooperation in Heterokaryons
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
1059
Dendy, P. R., and Harris, H. (1973). J Cell Sci. 12"831. Littlefield, J. W. (1964). Nature 203:1142. Chen, T. R. (1977). Exp CellRes. 104:255. Appels, R., Bolund, L., and Ringertz, N. R. (1974). JMol. Biol. 87:339. Pontecorvo, G. (1975). Somat. Cell Genet. 1:397. Hilwig, I., and Gropp, A. (1975). Exp Cell Res. 91:457. Bols, N. C., and Ringertz, N. R. (1979). Exp Cell Res. 120:15. Sidebottom, E., and Deak, I. I. (1976). Int Rev. Cytol. 44:29. Schwartz, A. G., Cook, P. R., and Harris, H. (1971). Nature New Biol. (London), 230:5. Watson, B., Gormley, I. P., Gardiner, S. E., Evans, H. J., and Harris, H. (1972). Exp Cell Res. 75:401. 23. Bakay, B., Croce, C. M., Koprowski, H., and Nyhan, W. L. (1973). Proc Natl Acad Sci U.S.A. 70:1998.
