EXPERIMENTAL

CELL

RESEARCH

46-5 1 ( 1991)

192,

Reversible Effects of Sodium Butyrate on the Differentiation of F9 Embryonal Carcinoma Cells MITSUKO

KOSAKA, YUKIO NISHINA, Research Institute *Department

for of

MASASHI TAKEDA,*

Microbial Pathology,

Diseases, Osaka University, Osaka University School

Press,

Inc.

INTRODUCTION Embryonal carcinoma (EC)2 cells can be induced to differentiate into endoderm-like cells by altering the culture conditions [I-4], or by exposing the cells to various agents [3, 5, 61. One of the most potent and widely used such inducers is retinoic acid (RA)2 [5]. Addition of RA to F9 teratocarcinoma stem cell cultures results in dramatic phenotypic changes, i.e., morphological alterations and increases in the synthesis and secretion of proteins characteristic of the differentiated phenotypes such as laminin, endoA, and plasminogen activator (PA)2 [5, 7-91. However, these phenotypic changes are believed to be irreversible. It is known that sodium butyrate induces a wide variety of effects on mammalian cells in culture, including a decrease in DNA replication leading to an arrest of cell

Suita Osaka Ei6.5, Japan; Kitaku Osaka 530, Japan

NISHIMUNE’

and

AND METHODS

Cell culture. The stock culture of F9 cells was grown on gelatincoated culture dishes in Eagle’s minimal essential medium (MEM) containing 5 mM glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum (FCS) at 37°C. For experimental cultivation, the stock culture was treated with 0.125% trypsin and 0.5 mA4 EDTA in phosphate-buffered saline (PBS) at 37°C for 10 min. The cells were then seeded at an appropriate concentration per culture dish. Treatment of cells with sodium butyrate was accomplished by adding small volumes of sterile concentrated solutions into the media more than 18 h after cell inoculation. In order to remove sodium butyrate, the culture 46

Inc. reserved.

Yamada-Oka Medicine,

MATERIALS

’ To whom reprint requests should be addressed. ’ Abbreviations used: AD, actinomycin D; CHX, cycloheximide; EC, embryonal carcinoma; PA, plasminogen activator; RA, retinoic acid, tPA, tissue-type plasminogen activator.

0014.4827/91 $3.00 Copyright Q 1991 by Academic Press, All rights of reproduction in any form

of

AND YOSHITAKE

division, modification of cell morphology, and alterations in the level of certain gene products [10,111. Contradicting results concerning its effects on teratocarcinoma cell differentiation have been reported. Some investigations demonstrated that sodium butyrate was able to induce EC cell differentiation [6, 121, while others observed that sodium butyrate was unable to induce F9 EC cell differentiation [13] or rather inhibited RA-induced cell differentiation [ 141. Furthermore, dibutyryl CAMP, when added into culture medium along with RA, was shown to enhance the phenotypic differentiation between EC cells and the ultimate differentiated derivatives. This enhancing effect was presumably due to the CAMP rather than to the butyrate moiety since agents such as cholera toxin elicited the same response [5]. However, the butyrate moiety may have a function to stimulate cellular retinoic acid-binding protein activity [13]. Thus, the effects of sodium butyrate on teratocarcinoma stem cells have remained to be determined by a systematic analysis. Cells derived from some EC cell lines differentiate in response to sodium butyrate as well as to RA, and the differentiation is reported to be irreversible [I2]. Although a reversible step has been detected before irreversible changes taking place during differentiation of some teratocarcinoma cell lines [15], many investigators reported that the induction of teratocarcinoma cell differentiation caused an irreversible change [5,14,1620]. Here we report that sodium butyrate induces rapid dramatic alterations in various phenotypes of F9 cells, and that its effects are reversible.

We have studied effects of sodium butyrate on embryonal carcinoma F9 cell differentiation. In the presence of sodium butyrate, F9 cells underwent rapid and drastic morphological changes and expressed marked increases in mRNA levels of various differentiation markers. When sodium butyrate was removed from the cultures, all the examined phenotypes of F9 cell differentiation rapidly reverted to the characteristics of undifferentiated stem cells. However, under the same conditions, when cycloheximide or actinomycin D was added to the cultures, such phenotypic reversion was not observed, but high mRNA levels of the differentiation markers as well as altered cell morphology were retained. These results indicated that the effects of sodium butyrate on induction of teratocarcinoma cell differentiation were reversible and that de novo syntheses of some mRNA(s) and protein(s) were necessary for the reversion of differentiated cells to stem cells. c 1991 Academic

KEISHI MATSUMOTO,*

FIG. 1. mM sodium Bar: 50 pm

medium washed

Changes hutyrate

containing with MEM,

in morphology ofF9 EC cells in response to sodium hutyrate. Three days after for 0 h (A), 8 h (B), 24 h (C), 24 h exposed to the drug followed by an incubation

the inducer was removed by aspiration and fresh culture medium was added.

and

A.ssay of PA production. Cells were seeded at 10’ cells per 35mm culture dish. Medium change was carried out on the third day of cultivation and sodium hutyrate was added on the fourth day. After appropriate intervals the medium was removed and the cells were washed with PBS and assayed for the production of PA by an agaroverlay method [6, 211. The agar overlay consisted of Eagle’s MEM containing 0.75% purified agar (Difco), 2.5% commercial nonfat dried milk sterilized with y irradiation, and 0.2 U of bovine plasminogen (Daiichi Chemical) in a total volume of 1 ml. PA production was estimated by means of scoring plasmin-mediated caseinolysis (evidenced by the formation of plaques). The plaques were counted 24 h after the agar overlay. RNA isolation and gel blot anaL.ysis. Cells were inoculated at l-2 X lo6 cells per loo-mm culture dish. After incubation for 18-24 h, a drug was added for an appropriate time. Total cellular RNA was isolated by guanidine isothiocyanate extraction and cesium chloride gradient centrifugation. For RNA gel blot analysis, RNA was fractionated on formaldehyde-agarose gels in MOPS buffer and transferred to nitrocellulose filters in 20x SW as described by Maniatis et al. [22]. The filters were prehyhridized in 50% formamide, 4~ SSC, 5~ Denhardts’ solution, 0.2% SDS, and denatured sonicated salmon sperm DNA (120 pg/ml), at 42°C for 2 h and hybridized with radiolaheled DNA probes (l-5 X lo6 cpm/ml) under the same conditions for 24 h. After hybridization, the filters were washed at 55°C with 0.1X SW, containing 0.1% SDS. The probe used here was cDNA of mouse

cell inoculation, cultures were exposed for 24 h in medium without the drug

to 5 CD).

tissue-type PA (t-PA)* [23], mouse cytokeratin endoA [24], mouse laminin Bl [25], human c-myc [26], and mouse cytoskeletal /+actin [27]. They were nick translated and labeled with [“*P]dCTP by standard techniques.

RESULTS

Morphological Observations The monolayer culture of F9 cells on a gelatin-coated dish shows the typical morphology of EC stem cells as tightly packed colonies (Fig. 1A). Soon after the addition of 5 mM sodium butyrate, it changes to a slightly dispersed arrangement of cells. Each cell treated with sodium butyrate underwent a morphologic change from t,he typical stem cell morphology to the flat polygonal shape having process at each angle (Fig. 1B). This change is rapid and became apparent within 5-6 h after the addit,ion of sodium butyrate and accomplished in the majority of cells within 24 h after the addition (Fig. 1C). This morphological change was stable as long as sodium butyrate was present in the cultures and if the cells did not detach from culture dish. However, after removal of the inducer from culture medium, the cells

48

KOSAKA

(A)

CONC.(mM)

TIME (hours)

FIG. 2. Effect ol’ sodium hutyrwte on PA production in F9 cells. PA production was estimated by the formation of caseinolytic plaques, as described under Materials and Methods and was expressed as the percentage of the total colonies in cultures. More than three plates, each containing X-100 colonies, were examined; and averages of their plaque forming percentages and their standard deviations are shown. (A) Dose effects of sodium butyrate on PA production. The colonies of’F9 cells were incubated with various concentrations of the drug for 16 h. (B) Time course of PA production. The colonies of F9 cells were incubated with 10 mM sodium hutyrate for appropriate periods of time.

with the altered shapes were able to revert to a morphology similar to the undifferentiated phenotype of the stem cells within 12 h (Fig. ID). Thus, the morphological change induced with sodium butyrate appears to be reversible if the inducer is eliminated. Production

of Plasminogen

ET

Al,.

t-PA of 2.8 kb [23], endoA of 1.8 kb [24], laminin transcripts of 6.0 kb [25], and c-myc of 2.3 kb [26]. Treatment with sodium butyrate markedly enhanced mRNA levels of laminin Bl, t-PA, and endoA by 8 h after addition of butyrate and continued to increase for 36-48 h after the addition (Fig. 4). In contrast, c-myc mRNA decreased rapidly after butyrate treatment. Removal of the inducer, however, resulted in reversion of mRNAs of all examined marker genes to the control levels within 6-12 h. The mRNAs of t-PA, laminin Bl, and endoA almost disappeared, and the levels of these marker genes were the same as those observed in the noninduced F9 cells. On the other hand, the cmyc mRNA level recovered to that of noninduced stem cells. These reversible changes in mRNA levels of examined genes were accompanied by the reversible changes of cell morphology (Fig. 1) and the production of PA (Fig. 2, Fig. 3). Effects of Cycloheximide (CHX) and Actinomycin D2 (AD) on the Levels of c-myc and a Differentiation Marker Gene, t-PA Levels of c-myc mRNA are regulated mainly by posttranscriptional control mechanisms, and inhibition of protein synthesis by CHX causes stabilization of c-myc mRNA in F9 stem cells [28] (see Fig. 5, lane 2). In order to further characterize sodium butyrate-induced differentiation, we have examined effects of CHX on mRNA

Activator

In order to further characterize differentiation induced with sodium butyrate, the activity of PA secreted into the media was determined. After the addition of sodium butyrate to the culture, a significant number of colonies producing PA were observed by 8 h. The number of PA-producing colonies increased in the presence of the drug in a dose-dependent manner (Fig. 2A) and in a time-dependent manner (Fig. 2B). It was observed that PA activity induced with sodium butyrate was reversible, as shown in Fig. 3. Although exposure to sodium butyrate for 16 h caused a great increase in the number of PA-secreting colonies, when the drug was removed from the culture medium, PA production declined rapidly and essentially returned to control levels within 12 h. Effects of Butyrate on the Expression of Marker in Teratocarcinoma Cell Differentiation

Genes

In order to study the effect of butyrate on the expression of differentiation associated genes, mRNA levels of t-PA, laminin Bl, endoA, and c-myc were measured by Northern blot analysis. In this experiment, each transcripts was detected as a single band of expected size:

100

r 1 50

-

0

0 Incubation

4

8 Time

12 ( hr )

Reversibility of the effect of sodium butyrate on PA production in F9 cells. The colonies of FY cells were incubated with 10 mM sodium hutyrate for 16 h (solid line on the abscissa, O-16 h) and washed and incubated without the drug for an appropriate time (hroken line on the abscissa, O-12 h). PA production was estimated by the formation of caseinolytic plaques in the same manner as was described in the legend to Fig. 2 and is shown as a percentage on the ordinate.

REVERSIBLE

A

DIFFERENTIATION

OF

F9

49

CELLS

12345678

B -actin

p-actin

FIG. 4. Effects of sodium butyrate on the expression of various genes in F9 cells, (A) Changes in mRNA levels of t-PA, laminin Bl, and endo A. F9 cells treated with 5 mM sodium butyrate for 0 h (lane l), 8 h (lane 2), 16 h (lane 3), 24 h (lane 4), 36 h (lane 5), 48 h (lane 6); after 16 h of exposure to the drug, the cultures were further incubated in the absence of sodium butyrate, for 6 h (lane 7) and 12 h (lane 8). (B) Changes in c-myc mRNA levels. F9 cells treated with the drug for 0 h (lane 1). 4 h (lane 2), 8 h ilane 3), 24 h (lane 4); after 16 h exposure to the drug, incubated for 6 h (lane 5) and 12 h (lane 6) without drug. Total RNA was isolated and examined by Northern blot analysis with “P-labeled DNA probes as described under Materials and Methods. Each lane contains 35 pg of total RNA. The blots were rehybridized with p-actin cDNA probe for control. Ribosomal RNA subunits were used as molecular size standards (arrowheads).

levels of c-myc and t-PA during the induction of differentiation and during the reversion after the removal of sodium butyrate from cultures. In the presence of sodium butyrate, a rapid decrease in the level of c-myc mRNA was observed. However, CHX had no stabilizing effect on this decay in contrast to the case with F9 stem cells. Furthermore, CHX inhibited the accumulation of t-PA mRNA (Fig. 5) and also prevented the butyrate-associated increase in laminin Bl and endoA mRNA (data not shown). When sodium butyrate was washed out, differentiated cells were able to revert to undifferentiated stem cells, as was described. However, the addition of CHX or AD to such cultures caused retardation of the reversion, resulting in no degradation of mRNA of differentiation markers such as t-PA. Furthermore, this retardation was accompanied by the retardation of morphological reversion (not shown) and also by no significant recovery of c-myc mRNA levels (Fig. 5). These results indicate that new mRNA and protein syntheses are neces-

sary for the reversion of differentiated stem cells.

cells back to the

DISCUSSION

Sodium butyrate has various effects on cell differentiation of many kinds of cells. It was suggested, however, that sodium butyrate was unable to induce F9 cell differentiation (concentration unspecified) [5], or that sodium butyrate inhibited the differentiation induced by RA [ 141. In contrast, cells from EC lines 6050 AJ and PCC4.azalR were demonstrated to differentiate irreversibly in response to treatment with sodium butyrate [la]. Our results showed that F9 cells underwent dramatic changes in response to butyrate. Their cell shape was altered very rapidly, though their differentiated cell morphology was slightly different from that of the endodermal cells induced with RA (Fig. I). Differentiated F9 cells also showed a large increase in secreted level of t-PA (Fig. 2), disappearance of SSEA-1 surface antigen

50

KOSAKA

1 2 3 4 5

6 7 8

9 1011

121314

t-PA 4

c-myc

,&actin

(CHX) and actinomycin ID (AD) FIG. 5. Effects ofcycloheximide on the mRNA levels oft-PA and c-myc induced with sodium butyrate. Uninduced stem cells (lane 1); stem cells treated with CHX (10 pg/ ml) for 3 h (lane 2); cells treated with 5 mM sodium butyrate for 4 h (lane 3). 8 h (lanes 4 and 5), 16 h (lanes 6 and 7) with (lanes 5 and 7) or without (lanes 3,4, and 6) CHX (5 pg/ml). After treatment of sodium butyrate for 16 h, the cells were washed and incubated for an additional 6 h or 12 h in the butyrate-free fresh culture medium (lanes 8-14) with (lanes 9 and 11) or without (lanes 8 and 10) CHX (5 Fg/ ml). After washing out sodium butyrate, cells were incubated for 3 h (lane 12), 6 h (lane 13) and 9 h (lane 14) in the presence of AD (1 pg/ml). Total RNA was isolated and Northern blot analysis was performed as described in the legend to Fig. 4.

(data not shown), markedly enhanced mRNA levels of t-PA, laminin Bl, and endoA and decreased level of cmyc mRNA (Fig. 4). All of these changes are consistent with the point of view that F9 cells differentiate in response to butyrate; moreover, their responses were very rapid and pronounced. For example, sodium butyrate induces extremely high level of t-PA mRNA (Fig. 4) and the induction of t-PA mRNA occurs within 8 h, which is very short as compared with 2-3 days for RA induction [23]. In connection with the toxicity of 5 mM sodium butyrate, we have observed no difference in the plating efficiency between F9 cells treated for 16 h and nontreated control cells. Furthermore, following removal of the butyrate, the cells reverted to a typical EC-like phe-

ET

Al,.

notype (Fig. 1, Fig. 3). This reversal of morphology was also rapid and was observed in the vast majority of cells in the culture. Similarly, only 6-12 h after the removal of the drug, the expression of differentiation marker genes decreased to each control level of undifferentiated F9 stem cells (Fig. 4). These events argue against the possibility that the culture contains a small population of cells refractory to butyrate which eventually overgrow the differentiated progeny following removal of the butyrate. Although a few reports recognized the “reversible phase” of phenotypic changes in EC cell differentiation [15, 171, irreversible differentiation induced with sodium butyrate has been demonstrated [12]. However, our study showed that the effects of the butyrate were reversible and that sodium butyrate was unable to induce irreversible differentiation (Fig. 1, 3,4), even after a prolonged exposure (48 h) to sodium butyrate (data not shown). This discrepancy may have resulted from the difference in EC cell lines and the different criteria used to evaluate differentiation. Furthermore, our results strongly support the notion that the “reversible phase” exists in an early stage of EC cell differentiation. However, it has been reported that in response to RA most cells in PC13, F9, and Nulli SSCl cultures committed to differentiate irreversibly within 24 h and that some cells appeared to be irreversibly affected within 6 h [ 18-201. Thus, in this RA system, the precommitment period could be too short and it may be difficult to detect the reversible phase. In the case of the sodium butyrate system, however, such a reversible stage of cell differentiation seems to be prolonged though the induction of differentiation is very rapid. If the commitment is defined as the ability of cells to achieve terminal differentiation after removal of inducer, then F9 cell differentiation induced with sodium butyrate may be at the precommitment stage and could be useful for analysis of early events of differentiation. F9 cells do not undergo permanent phenotypic changes in response to butyrate and fail to progress beyond the precommitment stage of differentiation mentioned above. The existence of such a precommitment stage is consistent with various observations made by studies of embryogenesis [29], in that differentiation in early mammalian embryogenesis proceeds as a series of events beginning prior to cell commitment that is attained only after the cascade of molecular reactions reaches an irreversible stage. Sodium butyrate is known to induce hyperacetylation of histones by inhibiting histone deacetylation [ 12, 30321, and alteration of histone acetylation may affect transcription of some genes. Although the mechanism by which butyrate induces EC cell differentiation remains to be understood, histone hyperacetylation was observed in EC line 6050AJ cells [12] and F9 cells [14]. However, many studies suggest that the effects of buty-

REVERSIBLE

DIFFERENTIATION

rate treatment may be far more complicated than the simple inhibition of histone deacetylase activities, since butyrate is also able to induce alterations in the phosphorylation and acetylation of nuclear proteins other than histones [33, 341 and in the methylation of DNA [35]. Resultant modifications at the nucleosome level might be partially or fully responsible for the alterations in chromatin morphology and gene expression that yield the arrest of the cell cycle and the observed phenotypic changes induced with sodium butyrate. The mRNA level of c-myc in F9 cells is known to be controlled post-transcriptionally and the induction with RA causes the stimulation of c-myc mRNA degradation, resulting in a low level of c-myc mRNA [36, 371. The sodium butyrate induction stimulated c-myc mRNA degradation, as was the case with RA. Although the inhibition of protein syntheses by CHX causes stabilization of c-myc mRNA both in RA-induced differentiated cells [36] and in noninduced stem cells (Fig. 5), CHX did not prevent but rather stimulated the degradation of c-myc mRNA in the cells treated with sodium butyrate. Since this CHX eff’ect was different from that observed with RA, it is possible that the mechanism through which sodium butyrate affects the c-myc mRNA level could be different from that operating in RA-induced cells. As was mentioned, sodium butyrate increased mRNA levels of differentiation marker genes in F9 EC cells. However, CHX prevented this sodium butyrate inducing ability, resulting in very low levels of the differentiation-specific mRNAs (Fig. 5). These observations suggest that mRNA expressions of differentiation-specific genes are indirectly regulated by butyrate via some gene product(s) whose expression can be inhibited by the protein synthesis inhibitor. In contrast, during the reversing step after the removal of sodium butyrate from cultures, degradation of mRNAs of marker genes was inhibited, and stabilization of their mRNAs was observed in the presence of CHX or AD (Fig. 5). These results indicate that de nouo syntheses of some mRNAs and proteins are required for the degradation of specific mRNA and regulation of gene expression. We are interested in the regulation system by these same molecules that must be important in turnover of the specific mRNA. We thank Dr. S. Strickland, Dr. T. Morita, Dr. Y. Yamada, and Dr. S. Sakiyama, for the gifts of the mouse t-PA, endoA, laminin Bl, and ij-actin r-DNA clones, respectively. We thank Dr. A. Tanaka for valuable discussions and suggestions for preparing the manuscript,

OF Strickland,

6.

Nishimune, Y., Kume, A., Ogiso, Exp. Cell Rex 146, 439-444.

7.

Sherman, M. I., Strickland, 36, 4208-4216.

8.

Knowles, B. B., Pan, S., Solter, D., Linnenbach, A., Croce, C., and Huebner, K. (1980) Nature (London) 288, 615-618. Strickland, S., Smith, K. K., and Marotti. K. R. (1980) Cell 21, 347-355.

9.

2. 3. 4.

Martin, G. R., and Evans, M. J. (1975) Proc. N&l. Acad. Sci. 1JSA 72, 1441-1445. McHurney, M. W. (1976) J. Cell. Physiol. 89, 441-456. Nicolas, .J. F., Avner, P., Gaillard, J., Guenet, J. L., Jacob, H., and Jacob, F. (1976) Cancer Res. 36,4224-4231. Ogiso, Y., Kume, A.. Nishimune, Y., and Matsushiro, A. (1982) Difjkentiation 22, 218-221.

Received Revised

July 2, 1990 and accepted August

13, 1990

S., and Mahdavi,

V. (1978)

5.

Cdl

S., and Reich,

E. (1976)

Prasad,

15. 16. 17.

In Vitro

Cancer

Kruh, J. (1982) Mol. Cell. Biochem. 42, 62-82. McCue, P. A., Gubler, M. L., Sherman, M. I., and Cohen, (1984) J. Cell Biol. 98, 602-608. Grippo, J. F., and Gudas, L. d. (1987) J. Biol. Chem. 262, 4500.

14.

P. K. (1976)

A. (1983)

11. 12. 13.

K. N., and Sinha,

15, 3933403.

Y., and Matsushiro,

10.

Res.

12, 125-132. B. N. 4492-

Levine, R. A., Campisi, J., Wang, S. Y., and Gudas, L. J. (1984) Lb. Riol. 105,443-450. Ogiso, Y., Kume, A., Nishimune, Y., and Matsushiro, A. (1982) Enp. Cell Res. 137, 365-372. Sherman, M. I. (1986) Curr. 7’up. Deu. Biol. 20, 345-356. Sherman, M. I., Eglitis, bryol. Bxp. Morphol. 93,

hl. A., and Richard, 179-196.

T. (1986)

J. Em-

18. 19.

Rayner, M. J., and Graham, C. F. (1982) J. Cell Sci. 58,331-334. Rodrigues, M., Balicki, D., Newrock, K. M., and Mukherjee, B. B. (1985) Exp. Cell Res. 156, 22-30.

20.

Sherman, M. I., Gubler, M. L.? Barkai, Il., Harper, pola, G., and Yuan, J. (1985) Ciba Found. Symp. Goldberg,

22.

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Rickles, R. J., Darrow, A. L., and Strickland, S. (1988) J. Riol. Chem. 263, 1563-1569.

23.

A. R. (1974)

M. I., Cop-

113, 42-60.

21.

Morita, Ichinose, 1099117.

25.

Sasaki, M., Kato, S., Kohno, K., Martin, G. R., and Yamada, (1987) Proc. Natl. Acad. Sci. USA 84, 935-939. Amersham International plc. (1985) Amersham Laboratories.

26. 27.

Tokunaga, Sakiyama,

T., Tondella, Y., Nozaki,

Cell 2, 95-102.

24.

M. L. C., Takemoto, N., and Matsushiro,

K., Taniguchi, S. (1986) Nucleic

H., Yoda, Acids Res.

Shim&

M.,

Y.

and

14, 2829.

Michael, D., Levine, 6, 518-524.

29.

Johnson, M. H., Handyside, A. H., and Braude, P. R. (1977) in Development in Mammals (Johnson, M. H., Ed.), Vol. 2, pp. 67-97, Elsevier, Amsterdam/New York.

30.

Vidali, G., Boffa, Biochem. Biophys.

31. 32.

Sealy, L., and Chalkley, R. (1978) Cell 14, 115-121. Candido, E. P. M., Reeves, R., and Davie, J. R. (1978) 105-11X.

33.

Whitlock Jr., J. P., Augustine, ture (London) 287, 74-76.

34.

Boffa, L. C., Gruss, R. J., and Allfrey, V. G. (1981) J. Hiol. Chem. 256, 9612-9621. Parker, M. I., de Haan, J. B., and Gevers, W. (1986) J. Hiol. Chem. 261,2786-2790. Dony, C., Kessel, M., and Gruss, P. (1985) Nature (London) 3 17,636-639. Sejersen, T., Rahm, M., Szabo, G., Ingvarsson, S., and Sumegi, .J. (1987) Exp. Cell Res. 172, 304-317.

35. 36. 37.

R. A., and Campisi,

K.,

Y., Hashido, K., A. (1988) (gene 68,

28.

REFERENCES 1.

51

F9 CELLS

J. (1986)

L. C., Mann, R. C., and Allfrey, Res. Commun. 82, 223-227.

R., and Schulman,

Mol.

Cell. Biol.

V. G. (1978)

Cell 14,

H. (1980)

Nu-