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The development of polyploidy in two classes of rat liver nuclei
306
Biochimica et Biophysica Acta, 519 (1978) 306--316 © Elsevier/North-Holland Biomedical Press
BBA 99187
THE DEVELOPMENT OF POLYPLOIDY IN TWO CLASSES OF RAT LIVER NUCLEI
JAMES A. ROSZELL, JOSEPH L. FREDI and CHARLES C. IRVING Veterans Administration Hospital and Department of Urology, University of Tennessee Center for the Health Sciences, Memphis, Tenn. 38104 (U.S.A.)
(Received November 7th, 1977)
Summary Two classes of nuclei from livers of Sprague-Dawley ra~ were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.
Introduction Liver is a complex tissue performing numerous biochemical and physiological functions. In the fetus, liverserves as a hematopoietic organ, while in adults the liver serves as a secretory organ as well as being the main tissue for detoxifying harmful chemicals [1,2]. Parallel with the development of adult liver function is the development of extensive polyploidy in the liver cells [3--8] with 6 5 % of the hepatocyte population becoming tetraploid in 1-year-old rats [7]. Methods for studying the development of polyploidy have been Feulgen
307 staining for DNA [3,4,7] or interference microscopy [5]. Isolated nuclei have also been used in developmental studies which involved measurements of size and volume of the nuclei [5,9]. Bushnell et al. [10] and Sneider et al. [11] have published a method for isolating two classes of nuclei from rat liver based on sedimentation through dense sucrose. We have used flow microfluorometry with propidium iodide as a DNA stain [12] to characterize two nuclear fractions obtained by a modification of the procedure of Sneider et al. [10] to study the development of polyploidy in these two nuclear fractions as a function of age. Materials and Methods
Animals. Sperm-positive female Sprague-Dawley rats were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Newborn rats from these animals were weaned and separated by sex at 3 weeks of age. They were maintained on Purina lab chow and water ad libitum in animal quarters that provided 12 h of light and dark throughout the duration of the experiment. Isolation o f nuclear fractions. All operations were carried out at 4°C. In some experiments, N-I and N-II nuclear fractions were isolated exactly as described by Sneider et al. [11]. Briefly, the liver was homogenized in 2 vols 0.23 M sucrose in TKM buffer (0.05 M Tris • HC1, pH 7.5/0.025 M KC1/0.05 M MgCI:) with a motor
308 determined as described previously [13]. The incorporation of labeled orotic acid or cytidine into RNA of the nuclear fractions following i.p. injection of the labeled precursors was determined by the method of Whittle et ai. [14]. Determination of nuclear ploidy. Nuclei were fixed in 50% methanol for 30 min. The fixed nuclei were recovered by centrifugation at 2000 × g for 15 min and resuspended in TS buffer containing 1% Triton X-100. An aliquot of the nuclear suspension was stained with propidium iodide [12]. To 1 ml of a cold solution of propidium iodide (0.05 mg/ml in 1.12% sodium citrate), 0.025-0.1 ml of nuclear suspension was added. The mixture was stirred vigorously and allowed to stand in an ice-bath in the dark for 15 min, then diluted with 2 ml cold 1.12% sodium citrate to yield a suspension containing 3 . l 0 s - - 5 . l 0 s nuclei/ml. Nuclear fluorescence was measured on a Bio/Physics Model 4800A Cytofluorograph, coupled to a Model 2102 Multichannel Distribution Analyzer (Ortho Instruments, Westwood, Mass.). Chemicals. Spermidine and Tris were purchased from Sigma Chemical Co., St. Louis, Mo., and propidium iodide was obtained from Calbiochem, San Diego, Calif. Triton X°100 was from Eastman Chemical Co., Rochester, N.Y. [5-3H]Orotic acid and [5-3H]cytidine were obtained from New England Nuclear Corp., Boston, Mass. All other chemicals and reagents were from Mailinckrodt, Inc., St. Louis, Mo. Results and Discussion
Isolation of heavy (H) and light (L ) nuclear fractions from rat liver It was necessary for us to modify the procedure of Sneider et al. [11] for isolation of the two classes of nuclei from rat liver in order to obtain nuclear fractions suitable for study of ploidy by flow microfluorometric techniques. We have designated the fraction that pelleted in 2.3 M sucrose as heavy nuclei (H nuclei) and the fraction collected at the 1.8/2.3 M sucrose interface as light nuclei (L nuclei) to avoid the possibility of misinterpretation of these two nuclear fractions as hepatocyte nuclei (NJ class) and non-hepatocyte nuclei (N-II class) according to the terminology of Bushnell et ai. [10]. With our modification, the yield of homogenate DNA recovered in the combined H and L nuclear fractions was 93.4 -+ 1.2%. Furthermore, in normal adult rats, the distribution of DNA in the H and L nuclear fractions was similar to that we found in normai adult rats in the N J and NJI nuclear fractions using the procedure of Sneider et al. [11], i.e., 80--85% of the DNA was found in the H fraction. Extending the duration of the centrifugation from I to 2 h did not alter the distribution of nuclei between the two fractions. Differential utilization of [aH]orotic acid and [aH]cytidine for RNA synthesis by N-I and N-II nuclear fractions led Bushnell et al. [10] to conclude that N4 nuclei were derived chiefly from hepatocytes and N-II nuclei from stromal or non-hepatocytic cells. N-I nuclei incorporated 3.4 times as much radioactivity from [aH]orotic acid into RNA per gg DNA as did the N-II class nuclei [10]. On the other hand, with [aH]cytidine as a precursor, Bushnell et al. found that N-I nuclei incorporated slightly less radioactivity into RNA per gg DNA than did the N-II nuclei. We obtained similar results (Table I) when N J and N-II liver nuclei were isolated from male 8-week-old Sprague-Dawley rats
309 TABLE I INCORPORATION OF LABELED OROTIC ACID OR CYTIDINE INTO RNA OF RAT LIVER NUCLEI
Rats w e r e injected i n t r a p e r i t o n e a l l y w i t h either [ 5 - 3 H ] o r o t i c acid ( 1 4 0 C i / m o l ) or [ 5 - 3 H ] c y t i d i n e ( 2 5 . 8 C i / m m o l ) at d o s e s o f 5 0 / J C i / 1 0 0 g b o d y weight. T h e rats w e r e killed after 3 0 m i n and t h e nuclear fractions w e r e isolated f r o m t h e livers. I n c o r p o r a t i o n o f t h e labeled precursors i n t o nuclear R N A w a s d e t e r m i n e d as described b y Whittle et al. [ 1 4 ] . V a l u e s given are t h e m e a n s ± S.E. for separate d e t e r m i n a t i o n s o n three rats. Method of isolation o f nuclei
Age of animals (weeks)
T w o - s t e p gradient * N-I fraction N-II fraction R a t i o , N-I/N-II
8
Three-step gradient ** H fraction L fraction Ratio, H / L
8
Three-step gradient ** H fraction L fraction Ratio, H/L
3
d p m in R N A per # g D N A [ 3 H ] O r o t i c acid
[3 H ] C y t i d i n e
671 -+ 6 0 2 2 7 -+ 2 8 2.96
1 3 0 ± 54 1 4 1 -+ 5 0 0.92
5 2 7 ± 31 2 4 2 -+ 16 2.18
1 3 0 -+ 3 5 1 1 5 ± 23 1.13
6 0 2 -+ 2 0 5 0 9 -+ 46 1.18
2 7 2 -+ 41 2 3 7 ± 29 1.15
* M e t h o d o f Schneider et al. [ 1 1 ] . ** A s d e s c r i b e d in Materials and M e t h o d s .
by the procedure of Sneider et al. [11]. Approximately three times as much radioactivity from [3H]orotic acid was found in RNA of the class N-I nuclei, whereas, the specific radioactivities of the RNA in the two classes of nuclei were nearly identical after injection of [3H]cytidine (Table I). Data on the labeling of nuclear RNA in the heavy (H) and light (L) nuclear fractions isolated from 8-week-old rats by the modified three-step gradient method after i.p. injection of [3H]orotic acid or [3H]cytidine are shown in Table I. There was no significant difference between the specific radioactivities of RNA in the H fraction and the N-I fraction, or in the L fraction and the N-II fraction; and there was a preferential utilization of [3H]orotic acid in the H fraction compared to the L fraction (Table I), in agreement with results obtained with the N-I and N-II nuclear fractions. Incorporation of [3H]cytidine into nuclear RNA of the H- and L-fractions were nearly identical, and the specific radioactivities of the RNA were about the same as those found in the N-I and N-II nuclei (Table I). In the 8-week-old rats used in the experiments described in Table I, the percent distribution of homogenate DNA in the nuclear fractions was: two-step gradient, N-I fraction, 81.5 + 1.6%; N-II fraction, 15.7-+ 1.3%; and three-step gradient, H fraction, 77.5 + 0.9%; L fraction, 12.9-+ 0.3%. These results are consistent with the interpretation that our H nuclei and L nuclei are similar to the N-I and N-II nuclei o f Sneider et al. [11], respectively. In younger rats, we have found that there is a higher percentage of the liver nuclei in the L fraction. For example, in 3-week-old male Sprague-Dawley rats,
310 33.0 + 2.5% of the homogenate DNA was recovered in the H fraction and 67.1 + 2.2% in the L fraction; similar results were obtained if N-I and N-II nuclei were isolated from 3-week-old rats by the method of Sneider et al. [11]. Furthermore, in the 3-week-old rats, there was no significant difference in the in vivo incorporation of [3H]orotic acid into RNA of nuclei recovered in the H and L fractions (Table I). The lower ratio of specific radioactivities of RNA in the two nuclear fractions in the 3-week~ld rats was due to an increase in the specific radioactivity of RNA in the L fraction (Table I). Although RNA of liver nuclei from the younger animals had a higher specific radioactivity after injection of [3H]cytldine compared to the 8-week-old rats, the ratio of specific radioactivities in the H and L nuclear fractions from the 3-week~ld rats was still about 1 (Table I). It is clear from these data that there can be dramatic changes in the sedimentation properties of rat liver nuclei in dense sucrose solutions and that failure of rat liver nuclei to sediment through 2.3 M sucrose cannot be used as a criterion to characterize rat liver nuclei as predominantly "non-hepatocytic" nuclei (see, e.g., ref. 15). This conclusion is strengthened by the data obtained after feeding rats the carcinogens 3'-methyl-4
311
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Fig. i . F r e q u e n c y d i s t r i b u t i o n s of liver cell nuclei in heavy (H) and light (L) nuclear fractions from SpragueoDawley rats in rc]atlon to relative DNA c o n t e n t (fluorescence i n t e n s i t y ) as d e t e r m i n e d by flow m l e r o f l u o r o m e t r y , Nuclei were isolated and stained wish l ~ o p i d i u m iodide as described in Materials and Methods. A. H f~action of l ~ l a y - o l d rat. B. L fraction of 1-day-old rat. C. H fraction of 14-week-old male rat. D. L fraction of 14-week-old male rat.
of age are shown in Figs. 1A and 1B, respectively. Both H and L nuclear fractions from newborn rat liver show nuclei in S phase (Fig. 1A, 1B). S phase decreased more rapidly in L nuclei than in H nuclei. S phase was 14 + 3.7% in L nuclei of the 14~lay embryo and rapidly decreased to 3.7 + 0.5% by 1 week after birth. The H nuclei had 16 + 2.9% S phase in the 14
312 cells for analysis of cellular ploidy, which would be essential for this type o f study. Pretlow and Williams [21] described a method for separation of hepatocytes from suspensions of mouse liver cells in Ficoll gradients, but the yields of hepatocytes were only 5--10%. Separation of intact hepatocytes or nuclei into ploidy classes by velocity sedimentation in Ficoll [22] gradients at unit gravity was recently reported and this technique might be more promising. Influence of age on the distribution of nuclei in the H and L fractions and on nuclear ploidy The distribution of nuclei in the H and L fractions and the development of polyploidy in these two fractions as a function of age in male and female Sprague-Dawley rats was examined. Fig. 2A shows the changes in total 2n, 4n and 8n nuclear populations of male rat liver from 14 days of gestation through 24 weeks of age. Diploid nuclei showed large decreases from birth through the 4th week. Our analysis of nuclei using propidium iodide as a DNA stain showed that diploid nuclei in the (G2 + M) phase o f the cell cycle appeared at approxi-
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Weeks of Aqe Fig. 2. C h a n g e s in t o t a l n u m b e r s o f n u c l e i / g liver and p e r c e n t o f t o t a l n u c l e i f o u n d in t h e H n u c l e a r fract i o n o f liver f r o m m a l e rats as a f u n c t i o n o f age. P o i n t s and bars are t h e m e a n s + S.E. f o r t h r e e i n d e p e n d e n t d e t e r m i n a t i o n s . A . L o g a r i t h m o f t h e n u m b e r o f 2n (o o), 4n ( e ~) and 8n (o o) n u c l e i in t o t a l liver ( c o m b i n e d H and L f r a c t i o n s ) . B. T h e p e r c e n t o f t o t a l n u c l e i in t h e H f r a c t i o n . T h e average r e c o v e r y o f h o m o g e n a t e D N A in t h e c o m b i n e d H a n d L n u c l e a r f r a c t i o n s w a s a p p r o x . 9 3 % .
313
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-I B 2 4 6 8 I() 12 t4 16 18 2'0 2'2 24 Weeks of Age Fig. 3. Changes in nuclear p l o l d y in the heavy (H) and light (T,) nueleax tractions of male zat livez. A. T,oga r i t h m o f t h e n u m b e z o f H n u c l e i / g liver. B. L o g a r i t h m o f t h e n u m b e z o f L n u c l e i / g liver, o. nuclei; • -', 4n n u c l e i ; a ~ , 8n n u c l e i .
o, 2n
mately the same fluorescence intensity as true tetraploid nuclei. For this reason, " 4 n " nuclei seemed to decrease from birth through 4 weeks of age (Figs. 2A and 4A). The presence of these " 4 n " nuclei in 14~iay embryos through 4 weeks is accompanied by elevated S phase, making it apparent that these " 4 n " nuclei are actually G2 nuclei. When 4n nuclei developed after 4 weeks (Fig. 2A), there was no detectable increase in S phase, indicating that these later 4n nuclei are true tetraploid nuclei. By the 6th week, 2n nuclei have stabilized at 3 - 1 0 v nuclei/g liver and remain essentially unchanged for the remaining 18 weeks. Tetraploid nuclei increased from the 4th week until they were equal to 2n nuclei by the 12th week. By the 18th week, 4n nuclei exceeded the number of 2n nuclei. Octaploid nuclei were not present during early development and appeared at weaning. Octaploid nuclei increased over a 4-week period to 3 • 10 ~ nuclei/g liver by 12 weeks. Fig. 2B shows the percentage of total nuclei isolated in the H fraction from male rat liver. Approximately 30% of the nuclei were in the H fraction in 14day embryos. This level was maintained until 5 weeks of age. The percentage of nuclei in the H fraction then increased rapidly until, at 7 weeks, 67% of the nuclei were in the H fraction. During the remaining 17 weeks of the experiment, the percentage of nuclei in the H fraction fluctuated in cycles from 70 to 45%. The development of 2n, 4n and 8n nuclei in the H fraction is shown in Fig. 3A. Most striking was the rapid increase in 4n and 8n nuclei in the H fraction from 3 to 7 weeks. Tetraploid H nuclei increased from 4 • 106/g liver at 3 weeks
314 T A B L E II SELECTED DATA ON THE PERCENTAGE DISTRIBUTION OF NUCLEAR FRACTIONS IN DEVELOPING MALE RAT LIVER N u c l e i w e r e i s o l a t e d f r o m t h r e e livers f r o m m a l e rats as d e s c r i b e d in Materials and M e t h o d s . T h e v a l u e s r e p o r t e d are t h e m e a n p e r c e n t a g e s o f t h e t o t a l n u c l e i in e a c h s u b f r a c t i o n and t h e S.E. for three i n d e p e n d e n t d e t e r m i n a t i o n s . T o t a l v a l u e s less t h a n 1 0 0 % are a c c o u n t e d for b y debris, c l u m p s o f nuclei, n u c l e i in S p h a s e a n d f l u o r e s c e n t m a t e r i a l in c h a n n e l s greater t h a n 8n. Age (weeks)
4 12 22
H fraction
L fraction
2n
4n
8n
2n
4n
8n
24.4±1.1 13.0±0.5 11.5±1.1
6.3±0.5 26.0±1.5 49.1±1.7
I.I±0.I 5.5±0.3 6.4±0.1
45.0±1.4 34.0±2.3 20.2±2.3
8.0±0.6 9.1±1.0 4.7±0.5
1.2±0.1 1.2±0.2 0.4±0.1
to 16 • 106/g liver at 7 weeks, while 8n-H nuclei increased from 3.5 • 10S/g liver at 3 weeks to 30 • 10S/g liver at 7 weeks. This rapid and extensive increase in ploidy of liver nuclei coincided with the increase in the percent of total nuclei in the H fraction as seen in Fig. 2B. Further increases in ploidy occurred over the remaining 18 weeks of the experiment resulting in 4n-H nuclei being the single largest population in the liver, accounting for 5G G0% of the total liver nuclei at 24 weeks of age. Corresponding data for the changes occurring in 2n, 4n and 8n nuclear populations in the L fraction are shown in Fig. 3B. Generally, there was a progressive, if cyclical, decrease in the n u m b e r of L nuclei per g liver during the development. In the fetal and neonatal rat liver, 2n-L nuclei were the predominant population of nuclei. After 22 weeks, 2n-L nuclei constit u t e d only 20% o f the total liver nuclei. Table II shows the percentage changes in nuclear populations at 4, 12 and 22 weeks. It can be seen that there is a progressive increase in 4n-H and 8n-H nuclei with corresponding decreases in other nuclear populations. F r o m Figs. 2B, 3A and 3B some general trends were apparent. When the percent H nuclei (Fig. 2B) decreased at 10 weeks and 18 weeks, 2n-H and 8n-H populations were lowered (Fig. 3A), and the 4n-H population remained constant. In the L nuclear population, 4n-L and 8n-L nuclei increased as the percent of H nuclei in liver decreased (Fig. 3B), whereas the 2n-L population showed only slight changes at the same times. Since increases in 4n-L and 8n-L populations preceded increases in 4n-H and 8n-H populations, it is compelling t ° propose that 4n-L and 8n-L populations are in some w a y precursors or intermediates in the development o f 4n-H and 8n-H nuclei. Female rat liver showed the same pattern of development as males. The total numbers of 2n and 4n nuclei were higher in females throughout the experiment than in males, and the n u m b e r o f 8n nuclei was slightly lower than in males (Fig. 4A). The same cyclical changes observed in male rat liver in the percent of nuclei in the H fraction (Fig. 2B) was seen in female rat liver (Fig. 4B). The values for percent H nuclei were similar in males and females and the high and low points of the cycles showed a phenomenal degree o f synchrony in the t w o sexes. Changes in ploidy of nuclei in the H and L fractions also showed the same
315
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Fig. 4. Changes in t o t a l n u m b e r s o f nuclei/g liver and p e r c e n t o f t o t a l nuclei f o u n d in t h e H nuclear fract i o n o f liver f r o m female rats as a f u n c t i o n o f age. Points a n d bars are the m e a n s + S.E. for three i n d e p e n d e n t d e t e r m i n a t i o n s . A . L o g a r i t h m o f t h e n u m b e r o f 2n (o o ) , 4n ( • e ) a n d Sn (o o) nuclei in t o t a l liver ( c o m b i n e d H and L f r a c t i o n s ) . B. T h e pexcent o f t o t a l nuclei in t h e H f r a c t i o n . T h e average r e c o v e ~ ] o f h o m o g e n a t e D N A in the c o m b i n e d H a n d L nuclear fractions w a s a p p r o x . 93%. Fig. 5. Changes in nuclear p l o i d y in the h e a v y (H) a n d light (L) nuclear fractions o f female rat liver. A . L o g a r i t h m o f t h e n u m b e r o f H nuclei]g liver. B. L o g a r i t h m o f t h e n u m b e r o f L nuclei]g liver, o o, 2n nuclei; • e , 4n nuclei; [] o , 8n nuclei.
developmental pattern in female rats (Fig. 5) as described for males (Fig. 3). The magnitude o f the changes differed, but the trend to produce 4n nuclei in the H fraction was the same. These data are consistent with the known sequence o f events for the develo p m e n t of polyploidy in tat'liver [1,3,4,7,8]. The cyclical changes of 2n, 4n and 8n nuclei in the H and L fractions was an interesting aspect o f our study. If 4n-L and 8n-L nuclei are precursors for 4n-H and 8n-H nuclei, t w o possible mechanisms for production o f 4n-H and 8n-H nuclei would seem apparent. The first involves the shift o f 2n-H and 8n-H to the L fraction, where 2n-L, 4n-L and 8n-L undergo D N A synthesis and nuclear and/or cellular division. When 4n-L and 8n-L have increased in number, they are shifted to the H fraction. The second mechanism involves only an increase of 4n-L and 8n-L fractions to a level such that the percent nuclei in H appears lowered. The 4n-L and 8n-L nuclei then shift to the H fraction. Since the number o f nuclei per g liver was a relatively constan$ value, but the mass o f the liver was continuously increasing, we cannot determine which o f these mechanisms might be involved in the development o f polyploidy in the liver. Fractionation of liver nuclei into heavy and light'fractions, based on sedi-
316 mentation in dense sucrose solutions, not only revealed that 2n, 4n and 8n nuclei were distributed in both nuclear fractions, but that there could be rather dramatic shifts in the distribution of nuclei between the two fractions. The changes in distribution o f nuclei in the heavy and light fractions appears to be a reflection of changes in the internal organization of the nuclei, resulting in an altered sedimentation in dense sucrose. Although the nature of these changes is not known, we do know that the separation of rat liver nuclei into the heavy and light fractions does not correspond to a separation based on nuclear ploidy or on whether the nuclei are of parenchymal or non-parenchymal origin as proposed [10,11] or suggested [15] by others. Acknowledgments This work was supported by the United States Veterans Administration and by USPHS Research Grant CA-05490 from the National Cancer Institute. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Carriere, R. (1969) Int. Rev. Cytol. 25,201--277 Pogo, A.O., Cordero Funes, J.R. and Mordoh, J. (1960) Exp. Cell Res. 21,482--497 Gerzell, G. and Barnl, S. (1976) Cell Tissue Kinet. 9 , 2 6 7 - - 2 7 2 Gerzeli, G. and Barni, S. (1976) Riv. Istoeh. Norm. Pat. 20, 67--78 Tongiani, R., Malvaldi, G., Lopez, M. and Pueinelli, E. (1976) Histochem. 47, 1--21 Sell, S , Becket, F.F., Leffert, H.L. and Watabe, H. (1976) Cancer Res. 36, 4239---4249 Alfert, M. and Gesehwind, I.I. (1958) Exp. Cell Res. 15, 230--232 Naora, H. (1967) J. Biophys. Biochem Cytol. 3,949--975 Durban, E. and Durban, E.M. (1976) Eur. J. Cancer 12, 651--655 Bushnell, D.E., Whittle, E.D. and Potter, V.R. (1969) Biochim. Biophys. Aeta 179,497---499 Sneider, T.W., Bushnell, D.E. and Potter, V.R. (1970) Cancer Res. 30,1867--1873 Crissman, H.A., Mul1~ney, P.F. and S t e i n k a m p , J.A. (1975) Methods Cell. Biol. 9 , 1 7 9 - - 2 4 6 Jackson, C.D. and Irving, C.C. (1972) Cancer Res. 32, 1590--1594 Whittle, E.D., Bushnell, D.E. and Potter, V.R. (1968) Bioehim. Biophys. Acta 161, 41--50 Glazer, R.I. (1974) Biochim. Blophys. Aeta 361,361--366 Rabes, H.M. Hartenstein, R. and R i n g e l m a n , W. (1972) Cancer Res. 32, 83--89 Roszell, J.A., Fredi, J.L. and Irving, C.C. (1977) Proc. Am. Ass. Cancer Res. 1 8 , 1 6 7 Irving, C.C., Roszell, J.A. and Fredl, J.L. (1978) Adv. Enzyme Regul. 16, in the press Albrecht, C.F. (1968) Exp. Cell Res. 4 9 , 3 7 3 - - 3 7 8 J o h n s t o n , I.R., Mathlas, A.P., Pennington, F. and Ridge, D. (1968) Bioehem. J. 1 0 9 , 1 2 7 - - 1 3 5 Pretlow, T.G. and Williams, E.E. (1973) Anal. Biochem. 65, 114--122 Tulp, A., Welagen, J~J.M.N. and Emmelot, P. (1976) Bi~chim. Biophys. Acta 451,567---582
Biochimica et Biophysica Acta, 519 (1978) 306--316 © Elsevier/North-Holland Biomedical Press
BBA 99187
THE DEVELOPMENT OF POLYPLOIDY IN TWO CLASSES OF RAT LIVER NUCLEI
JAMES A. ROSZELL, JOSEPH L. FREDI and CHARLES C. IRVING Veterans Administration Hospital and Department of Urology, University of Tennessee Center for the Health Sciences, Memphis, Tenn. 38104 (U.S.A.)
(Received November 7th, 1977)
Summary Two classes of nuclei from livers of Sprague-Dawley ra~ were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.
Introduction Liver is a complex tissue performing numerous biochemical and physiological functions. In the fetus, liverserves as a hematopoietic organ, while in adults the liver serves as a secretory organ as well as being the main tissue for detoxifying harmful chemicals [1,2]. Parallel with the development of adult liver function is the development of extensive polyploidy in the liver cells [3--8] with 6 5 % of the hepatocyte population becoming tetraploid in 1-year-old rats [7]. Methods for studying the development of polyploidy have been Feulgen
307 staining for DNA [3,4,7] or interference microscopy [5]. Isolated nuclei have also been used in developmental studies which involved measurements of size and volume of the nuclei [5,9]. Bushnell et al. [10] and Sneider et al. [11] have published a method for isolating two classes of nuclei from rat liver based on sedimentation through dense sucrose. We have used flow microfluorometry with propidium iodide as a DNA stain [12] to characterize two nuclear fractions obtained by a modification of the procedure of Sneider et al. [10] to study the development of polyploidy in these two nuclear fractions as a function of age. Materials and Methods
Animals. Sperm-positive female Sprague-Dawley rats were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Newborn rats from these animals were weaned and separated by sex at 3 weeks of age. They were maintained on Purina lab chow and water ad libitum in animal quarters that provided 12 h of light and dark throughout the duration of the experiment. Isolation o f nuclear fractions. All operations were carried out at 4°C. In some experiments, N-I and N-II nuclear fractions were isolated exactly as described by Sneider et al. [11]. Briefly, the liver was homogenized in 2 vols 0.23 M sucrose in TKM buffer (0.05 M Tris • HC1, pH 7.5/0.025 M KC1/0.05 M MgCI:) with a motor
308 determined as described previously [13]. The incorporation of labeled orotic acid or cytidine into RNA of the nuclear fractions following i.p. injection of the labeled precursors was determined by the method of Whittle et ai. [14]. Determination of nuclear ploidy. Nuclei were fixed in 50% methanol for 30 min. The fixed nuclei were recovered by centrifugation at 2000 × g for 15 min and resuspended in TS buffer containing 1% Triton X-100. An aliquot of the nuclear suspension was stained with propidium iodide [12]. To 1 ml of a cold solution of propidium iodide (0.05 mg/ml in 1.12% sodium citrate), 0.025-0.1 ml of nuclear suspension was added. The mixture was stirred vigorously and allowed to stand in an ice-bath in the dark for 15 min, then diluted with 2 ml cold 1.12% sodium citrate to yield a suspension containing 3 . l 0 s - - 5 . l 0 s nuclei/ml. Nuclear fluorescence was measured on a Bio/Physics Model 4800A Cytofluorograph, coupled to a Model 2102 Multichannel Distribution Analyzer (Ortho Instruments, Westwood, Mass.). Chemicals. Spermidine and Tris were purchased from Sigma Chemical Co., St. Louis, Mo., and propidium iodide was obtained from Calbiochem, San Diego, Calif. Triton X°100 was from Eastman Chemical Co., Rochester, N.Y. [5-3H]Orotic acid and [5-3H]cytidine were obtained from New England Nuclear Corp., Boston, Mass. All other chemicals and reagents were from Mailinckrodt, Inc., St. Louis, Mo. Results and Discussion
Isolation of heavy (H) and light (L ) nuclear fractions from rat liver It was necessary for us to modify the procedure of Sneider et al. [11] for isolation of the two classes of nuclei from rat liver in order to obtain nuclear fractions suitable for study of ploidy by flow microfluorometric techniques. We have designated the fraction that pelleted in 2.3 M sucrose as heavy nuclei (H nuclei) and the fraction collected at the 1.8/2.3 M sucrose interface as light nuclei (L nuclei) to avoid the possibility of misinterpretation of these two nuclear fractions as hepatocyte nuclei (NJ class) and non-hepatocyte nuclei (N-II class) according to the terminology of Bushnell et ai. [10]. With our modification, the yield of homogenate DNA recovered in the combined H and L nuclear fractions was 93.4 -+ 1.2%. Furthermore, in normal adult rats, the distribution of DNA in the H and L nuclear fractions was similar to that we found in normai adult rats in the N J and NJI nuclear fractions using the procedure of Sneider et al. [11], i.e., 80--85% of the DNA was found in the H fraction. Extending the duration of the centrifugation from I to 2 h did not alter the distribution of nuclei between the two fractions. Differential utilization of [aH]orotic acid and [aH]cytidine for RNA synthesis by N-I and N-II nuclear fractions led Bushnell et al. [10] to conclude that N4 nuclei were derived chiefly from hepatocytes and N-II nuclei from stromal or non-hepatocytic cells. N-I nuclei incorporated 3.4 times as much radioactivity from [aH]orotic acid into RNA per gg DNA as did the N-II class nuclei [10]. On the other hand, with [aH]cytidine as a precursor, Bushnell et al. found that N-I nuclei incorporated slightly less radioactivity into RNA per gg DNA than did the N-II nuclei. We obtained similar results (Table I) when N J and N-II liver nuclei were isolated from male 8-week-old Sprague-Dawley rats
309 TABLE I INCORPORATION OF LABELED OROTIC ACID OR CYTIDINE INTO RNA OF RAT LIVER NUCLEI
Rats w e r e injected i n t r a p e r i t o n e a l l y w i t h either [ 5 - 3 H ] o r o t i c acid ( 1 4 0 C i / m o l ) or [ 5 - 3 H ] c y t i d i n e ( 2 5 . 8 C i / m m o l ) at d o s e s o f 5 0 / J C i / 1 0 0 g b o d y weight. T h e rats w e r e killed after 3 0 m i n and t h e nuclear fractions w e r e isolated f r o m t h e livers. I n c o r p o r a t i o n o f t h e labeled precursors i n t o nuclear R N A w a s d e t e r m i n e d as described b y Whittle et al. [ 1 4 ] . V a l u e s given are t h e m e a n s ± S.E. for separate d e t e r m i n a t i o n s o n three rats. Method of isolation o f nuclei
Age of animals (weeks)
T w o - s t e p gradient * N-I fraction N-II fraction R a t i o , N-I/N-II
8
Three-step gradient ** H fraction L fraction Ratio, H / L
8
Three-step gradient ** H fraction L fraction Ratio, H/L
3
d p m in R N A per # g D N A [ 3 H ] O r o t i c acid
[3 H ] C y t i d i n e
671 -+ 6 0 2 2 7 -+ 2 8 2.96
1 3 0 ± 54 1 4 1 -+ 5 0 0.92
5 2 7 ± 31 2 4 2 -+ 16 2.18
1 3 0 -+ 3 5 1 1 5 ± 23 1.13
6 0 2 -+ 2 0 5 0 9 -+ 46 1.18
2 7 2 -+ 41 2 3 7 ± 29 1.15
* M e t h o d o f Schneider et al. [ 1 1 ] . ** A s d e s c r i b e d in Materials and M e t h o d s .
by the procedure of Sneider et al. [11]. Approximately three times as much radioactivity from [3H]orotic acid was found in RNA of the class N-I nuclei, whereas, the specific radioactivities of the RNA in the two classes of nuclei were nearly identical after injection of [3H]cytidine (Table I). Data on the labeling of nuclear RNA in the heavy (H) and light (L) nuclear fractions isolated from 8-week-old rats by the modified three-step gradient method after i.p. injection of [3H]orotic acid or [3H]cytidine are shown in Table I. There was no significant difference between the specific radioactivities of RNA in the H fraction and the N-I fraction, or in the L fraction and the N-II fraction; and there was a preferential utilization of [3H]orotic acid in the H fraction compared to the L fraction (Table I), in agreement with results obtained with the N-I and N-II nuclear fractions. Incorporation of [3H]cytidine into nuclear RNA of the H- and L-fractions were nearly identical, and the specific radioactivities of the RNA were about the same as those found in the N-I and N-II nuclei (Table I). In the 8-week-old rats used in the experiments described in Table I, the percent distribution of homogenate DNA in the nuclear fractions was: two-step gradient, N-I fraction, 81.5 + 1.6%; N-II fraction, 15.7-+ 1.3%; and three-step gradient, H fraction, 77.5 + 0.9%; L fraction, 12.9-+ 0.3%. These results are consistent with the interpretation that our H nuclei and L nuclei are similar to the N-I and N-II nuclei o f Sneider et al. [11], respectively. In younger rats, we have found that there is a higher percentage of the liver nuclei in the L fraction. For example, in 3-week-old male Sprague-Dawley rats,
310 33.0 + 2.5% of the homogenate DNA was recovered in the H fraction and 67.1 + 2.2% in the L fraction; similar results were obtained if N-I and N-II nuclei were isolated from 3-week-old rats by the method of Sneider et al. [11]. Furthermore, in the 3-week-old rats, there was no significant difference in the in vivo incorporation of [3H]orotic acid into RNA of nuclei recovered in the H and L fractions (Table I). The lower ratio of specific radioactivities of RNA in the two nuclear fractions in the 3-week~ld rats was due to an increase in the specific radioactivity of RNA in the L fraction (Table I). Although RNA of liver nuclei from the younger animals had a higher specific radioactivity after injection of [3H]cytldine compared to the 8-week-old rats, the ratio of specific radioactivities in the H and L nuclear fractions from the 3-week~ld rats was still about 1 (Table I). It is clear from these data that there can be dramatic changes in the sedimentation properties of rat liver nuclei in dense sucrose solutions and that failure of rat liver nuclei to sediment through 2.3 M sucrose cannot be used as a criterion to characterize rat liver nuclei as predominantly "non-hepatocytic" nuclei (see, e.g., ref. 15). This conclusion is strengthened by the data obtained after feeding rats the carcinogens 3'-methyl-4
311
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D
z 4000-
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200 400 400 600 200 Chennel Number ( Fluorescence Intensity)
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Fig. i . F r e q u e n c y d i s t r i b u t i o n s of liver cell nuclei in heavy (H) and light (L) nuclear fractions from SpragueoDawley rats in rc]atlon to relative DNA c o n t e n t (fluorescence i n t e n s i t y ) as d e t e r m i n e d by flow m l e r o f l u o r o m e t r y , Nuclei were isolated and stained wish l ~ o p i d i u m iodide as described in Materials and Methods. A. H f~action of l ~ l a y - o l d rat. B. L fraction of 1-day-old rat. C. H fraction of 14-week-old male rat. D. L fraction of 14-week-old male rat.
of age are shown in Figs. 1A and 1B, respectively. Both H and L nuclear fractions from newborn rat liver show nuclei in S phase (Fig. 1A, 1B). S phase decreased more rapidly in L nuclei than in H nuclei. S phase was 14 + 3.7% in L nuclei of the 14~lay embryo and rapidly decreased to 3.7 + 0.5% by 1 week after birth. The H nuclei had 16 + 2.9% S phase in the 14
312 cells for analysis of cellular ploidy, which would be essential for this type o f study. Pretlow and Williams [21] described a method for separation of hepatocytes from suspensions of mouse liver cells in Ficoll gradients, but the yields of hepatocytes were only 5--10%. Separation of intact hepatocytes or nuclei into ploidy classes by velocity sedimentation in Ficoll [22] gradients at unit gravity was recently reported and this technique might be more promising. Influence of age on the distribution of nuclei in the H and L fractions and on nuclear ploidy The distribution of nuclei in the H and L fractions and the development of polyploidy in these two fractions as a function of age in male and female Sprague-Dawley rats was examined. Fig. 2A shows the changes in total 2n, 4n and 8n nuclear populations of male rat liver from 14 days of gestation through 24 weeks of age. Diploid nuclei showed large decreases from birth through the 4th week. Our analysis of nuclei using propidium iodide as a DNA stain showed that diploid nuclei in the (G2 + M) phase o f the cell cycle appeared at approxi-
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Weeks of Aqe Fig. 2. C h a n g e s in t o t a l n u m b e r s o f n u c l e i / g liver and p e r c e n t o f t o t a l n u c l e i f o u n d in t h e H n u c l e a r fract i o n o f liver f r o m m a l e rats as a f u n c t i o n o f age. P o i n t s and bars are t h e m e a n s + S.E. f o r t h r e e i n d e p e n d e n t d e t e r m i n a t i o n s . A . L o g a r i t h m o f t h e n u m b e r o f 2n (o o), 4n ( e ~) and 8n (o o) n u c l e i in t o t a l liver ( c o m b i n e d H and L f r a c t i o n s ) . B. T h e p e r c e n t o f t o t a l n u c l e i in t h e H f r a c t i o n . T h e average r e c o v e r y o f h o m o g e n a t e D N A in t h e c o m b i n e d H a n d L n u c l e a r f r a c t i o n s w a s a p p r o x . 9 3 % .
313
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-I B 2 4 6 8 I() 12 t4 16 18 2'0 2'2 24 Weeks of Age Fig. 3. Changes in nuclear p l o l d y in the heavy (H) and light (T,) nueleax tractions of male zat livez. A. T,oga r i t h m o f t h e n u m b e z o f H n u c l e i / g liver. B. L o g a r i t h m o f t h e n u m b e z o f L n u c l e i / g liver, o. nuclei; • -', 4n n u c l e i ; a ~ , 8n n u c l e i .
o, 2n
mately the same fluorescence intensity as true tetraploid nuclei. For this reason, " 4 n " nuclei seemed to decrease from birth through 4 weeks of age (Figs. 2A and 4A). The presence of these " 4 n " nuclei in 14~iay embryos through 4 weeks is accompanied by elevated S phase, making it apparent that these " 4 n " nuclei are actually G2 nuclei. When 4n nuclei developed after 4 weeks (Fig. 2A), there was no detectable increase in S phase, indicating that these later 4n nuclei are true tetraploid nuclei. By the 6th week, 2n nuclei have stabilized at 3 - 1 0 v nuclei/g liver and remain essentially unchanged for the remaining 18 weeks. Tetraploid nuclei increased from the 4th week until they were equal to 2n nuclei by the 12th week. By the 18th week, 4n nuclei exceeded the number of 2n nuclei. Octaploid nuclei were not present during early development and appeared at weaning. Octaploid nuclei increased over a 4-week period to 3 • 10 ~ nuclei/g liver by 12 weeks. Fig. 2B shows the percentage of total nuclei isolated in the H fraction from male rat liver. Approximately 30% of the nuclei were in the H fraction in 14day embryos. This level was maintained until 5 weeks of age. The percentage of nuclei in the H fraction then increased rapidly until, at 7 weeks, 67% of the nuclei were in the H fraction. During the remaining 17 weeks of the experiment, the percentage of nuclei in the H fraction fluctuated in cycles from 70 to 45%. The development of 2n, 4n and 8n nuclei in the H fraction is shown in Fig. 3A. Most striking was the rapid increase in 4n and 8n nuclei in the H fraction from 3 to 7 weeks. Tetraploid H nuclei increased from 4 • 106/g liver at 3 weeks
314 T A B L E II SELECTED DATA ON THE PERCENTAGE DISTRIBUTION OF NUCLEAR FRACTIONS IN DEVELOPING MALE RAT LIVER N u c l e i w e r e i s o l a t e d f r o m t h r e e livers f r o m m a l e rats as d e s c r i b e d in Materials and M e t h o d s . T h e v a l u e s r e p o r t e d are t h e m e a n p e r c e n t a g e s o f t h e t o t a l n u c l e i in e a c h s u b f r a c t i o n and t h e S.E. for three i n d e p e n d e n t d e t e r m i n a t i o n s . T o t a l v a l u e s less t h a n 1 0 0 % are a c c o u n t e d for b y debris, c l u m p s o f nuclei, n u c l e i in S p h a s e a n d f l u o r e s c e n t m a t e r i a l in c h a n n e l s greater t h a n 8n. Age (weeks)
4 12 22
H fraction
L fraction
2n
4n
8n
2n
4n
8n
24.4±1.1 13.0±0.5 11.5±1.1
6.3±0.5 26.0±1.5 49.1±1.7
I.I±0.I 5.5±0.3 6.4±0.1
45.0±1.4 34.0±2.3 20.2±2.3
8.0±0.6 9.1±1.0 4.7±0.5
1.2±0.1 1.2±0.2 0.4±0.1
to 16 • 106/g liver at 7 weeks, while 8n-H nuclei increased from 3.5 • 10S/g liver at 3 weeks to 30 • 10S/g liver at 7 weeks. This rapid and extensive increase in ploidy of liver nuclei coincided with the increase in the percent of total nuclei in the H fraction as seen in Fig. 2B. Further increases in ploidy occurred over the remaining 18 weeks of the experiment resulting in 4n-H nuclei being the single largest population in the liver, accounting for 5G G0% of the total liver nuclei at 24 weeks of age. Corresponding data for the changes occurring in 2n, 4n and 8n nuclear populations in the L fraction are shown in Fig. 3B. Generally, there was a progressive, if cyclical, decrease in the n u m b e r of L nuclei per g liver during the development. In the fetal and neonatal rat liver, 2n-L nuclei were the predominant population of nuclei. After 22 weeks, 2n-L nuclei constit u t e d only 20% o f the total liver nuclei. Table II shows the percentage changes in nuclear populations at 4, 12 and 22 weeks. It can be seen that there is a progressive increase in 4n-H and 8n-H nuclei with corresponding decreases in other nuclear populations. F r o m Figs. 2B, 3A and 3B some general trends were apparent. When the percent H nuclei (Fig. 2B) decreased at 10 weeks and 18 weeks, 2n-H and 8n-H populations were lowered (Fig. 3A), and the 4n-H population remained constant. In the L nuclear population, 4n-L and 8n-L nuclei increased as the percent of H nuclei in liver decreased (Fig. 3B), whereas the 2n-L population showed only slight changes at the same times. Since increases in 4n-L and 8n-L populations preceded increases in 4n-H and 8n-H populations, it is compelling t ° propose that 4n-L and 8n-L populations are in some w a y precursors or intermediates in the development o f 4n-H and 8n-H nuclei. Female rat liver showed the same pattern of development as males. The total numbers of 2n and 4n nuclei were higher in females throughout the experiment than in males, and the n u m b e r o f 8n nuclei was slightly lower than in males (Fig. 4A). The same cyclical changes observed in male rat liver in the percent of nuclei in the H fraction (Fig. 2B) was seen in female rat liver (Fig. 4B). The values for percent H nuclei were similar in males and females and the high and low points of the cycles showed a phenomenal degree o f synchrony in the t w o sexes. Changes in ploidy of nuclei in the H and L fractions also showed the same
315
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Fig. 4. Changes in t o t a l n u m b e r s o f nuclei/g liver and p e r c e n t o f t o t a l nuclei f o u n d in t h e H nuclear fract i o n o f liver f r o m female rats as a f u n c t i o n o f age. Points a n d bars are the m e a n s + S.E. for three i n d e p e n d e n t d e t e r m i n a t i o n s . A . L o g a r i t h m o f t h e n u m b e r o f 2n (o o ) , 4n ( • e ) a n d Sn (o o) nuclei in t o t a l liver ( c o m b i n e d H and L f r a c t i o n s ) . B. T h e pexcent o f t o t a l nuclei in t h e H f r a c t i o n . T h e average r e c o v e ~ ] o f h o m o g e n a t e D N A in the c o m b i n e d H a n d L nuclear fractions w a s a p p r o x . 93%. Fig. 5. Changes in nuclear p l o i d y in the h e a v y (H) a n d light (L) nuclear fractions o f female rat liver. A . L o g a r i t h m o f t h e n u m b e r o f H nuclei]g liver. B. L o g a r i t h m o f t h e n u m b e r o f L nuclei]g liver, o o, 2n nuclei; • e , 4n nuclei; [] o , 8n nuclei.
developmental pattern in female rats (Fig. 5) as described for males (Fig. 3). The magnitude o f the changes differed, but the trend to produce 4n nuclei in the H fraction was the same. These data are consistent with the known sequence o f events for the develo p m e n t of polyploidy in tat'liver [1,3,4,7,8]. The cyclical changes of 2n, 4n and 8n nuclei in the H and L fractions was an interesting aspect o f our study. If 4n-L and 8n-L nuclei are precursors for 4n-H and 8n-H nuclei, t w o possible mechanisms for production o f 4n-H and 8n-H nuclei would seem apparent. The first involves the shift o f 2n-H and 8n-H to the L fraction, where 2n-L, 4n-L and 8n-L undergo D N A synthesis and nuclear and/or cellular division. When 4n-L and 8n-L have increased in number, they are shifted to the H fraction. The second mechanism involves only an increase of 4n-L and 8n-L fractions to a level such that the percent nuclei in H appears lowered. The 4n-L and 8n-L nuclei then shift to the H fraction. Since the number o f nuclei per g liver was a relatively constan$ value, but the mass o f the liver was continuously increasing, we cannot determine which o f these mechanisms might be involved in the development o f polyploidy in the liver. Fractionation of liver nuclei into heavy and light'fractions, based on sedi-
316 mentation in dense sucrose solutions, not only revealed that 2n, 4n and 8n nuclei were distributed in both nuclear fractions, but that there could be rather dramatic shifts in the distribution of nuclei between the two fractions. The changes in distribution o f nuclei in the heavy and light fractions appears to be a reflection of changes in the internal organization of the nuclei, resulting in an altered sedimentation in dense sucrose. Although the nature of these changes is not known, we do know that the separation of rat liver nuclei into the heavy and light fractions does not correspond to a separation based on nuclear ploidy or on whether the nuclei are of parenchymal or non-parenchymal origin as proposed [10,11] or suggested [15] by others. Acknowledgments This work was supported by the United States Veterans Administration and by USPHS Research Grant CA-05490 from the National Cancer Institute. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Carriere, R. (1969) Int. Rev. Cytol. 25,201--277 Pogo, A.O., Cordero Funes, J.R. and Mordoh, J. (1960) Exp. Cell Res. 21,482--497 Gerzell, G. and Barnl, S. (1976) Cell Tissue Kinet. 9 , 2 6 7 - - 2 7 2 Gerzeli, G. and Barni, S. (1976) Riv. Istoeh. Norm. Pat. 20, 67--78 Tongiani, R., Malvaldi, G., Lopez, M. and Pueinelli, E. (1976) Histochem. 47, 1--21 Sell, S , Becket, F.F., Leffert, H.L. and Watabe, H. (1976) Cancer Res. 36, 4239---4249 Alfert, M. and Gesehwind, I.I. (1958) Exp. Cell Res. 15, 230--232 Naora, H. (1967) J. Biophys. Biochem Cytol. 3,949--975 Durban, E. and Durban, E.M. (1976) Eur. J. Cancer 12, 651--655 Bushnell, D.E., Whittle, E.D. and Potter, V.R. (1969) Biochim. Biophys. Aeta 179,497---499 Sneider, T.W., Bushnell, D.E. and Potter, V.R. (1970) Cancer Res. 30,1867--1873 Crissman, H.A., Mul1~ney, P.F. and S t e i n k a m p , J.A. (1975) Methods Cell. Biol. 9 , 1 7 9 - - 2 4 6 Jackson, C.D. and Irving, C.C. (1972) Cancer Res. 32, 1590--1594 Whittle, E.D., Bushnell, D.E. and Potter, V.R. (1968) Bioehim. Biophys. Acta 161, 41--50 Glazer, R.I. (1974) Biochim. Blophys. Aeta 361,361--366 Rabes, H.M. Hartenstein, R. and R i n g e l m a n , W. (1972) Cancer Res. 32, 83--89 Roszell, J.A., Fredi, J.L. and Irving, C.C. (1977) Proc. Am. Ass. Cancer Res. 1 8 , 1 6 7 Irving, C.C., Roszell, J.A. and Fredl, J.L. (1978) Adv. Enzyme Regul. 16, in the press Albrecht, C.F. (1968) Exp. Cell Res. 4 9 , 3 7 3 - - 3 7 8 J o h n s t o n , I.R., Mathlas, A.P., Pennington, F. and Ridge, D. (1968) Bioehem. J. 1 0 9 , 1 2 7 - - 1 3 5 Pretlow, T.G. and Williams, E.E. (1973) Anal. Biochem. 65, 114--122 Tulp, A., Welagen, J~J.M.N. and Emmelot, P. (1976) Bi~chim. Biophys. Acta 451,567---582