[CANCER RESEARCtt 35, 2954-2958, November 1975]

Fractionation of Nuclei and Analysis of Nuclear Proteins of Rat Liver and Morris Hepatoma 7777 x

Brian Wi lson, ~ Michae l A. Lea, 3 Giorgio Vidali , and Vincent G. AII frey 4

The Rockefeller University, New York, New York 10021

S U M M A R Y

The contributions of nuclear populations to the total profile of nuclear proteins in a tissue were examined in normal rat liver and Morris hepatoma 7777. Comparison by sodium dodecyl sulfate polyacrylamide gel electrophoresis of phenol-soluble nuclear proteins from tumor and control liver revealed additional proteins of molecular weight 60,000, 100,000, and 135,000 and the loss of proteins of about 45,000 and 55,000 in the tumor. Subfractionation of liver nuclei on a 30 to 50% sucrose gradient yielded three nuclear classes with nearly identical complements of the phenol-soluble proteins. Similar fractionation performed on the hepatoma nuclei also produced three nuclear popula- tions. In the hepatoma nuclei, several differences in the phenol-soluble proteins were found between the minor, slowly sedimenting nuclear fraction, and the two major fractions, while the two latter fractions were very similar in their protein composition. Histones derived from both tissues were also compared electrophoretically, indicating a decrease in the concentration of histone H i " in all nuclear classes derived from the tumor.

I N T R O D U C T I O N

The postulated role of chromosomal proteins as regula- tors of genetic expression has prompted many investigations of the tissue specificity of these proteins (1). The growth of neoplastic cells might conceivably be influenced by changes in the relative amounts of different nuclear proteins, and this possibility has led several investigators to examine chromosomal proteins in cancer cells. The Morris series of hepatomas show a spectrum of growth rates that provide a useful model for the correlation of biochemical changes with tissue growth. Differences in the electrophoretic patterns of

'This work was supported by USPHS Grants CA-12933, CA-14908, and CA-16274 and by American Cancer Society Grant VC-114D.

2 On sabbatical leave from the MRC Iodine Metabolism Research Unit, Department of Pharmacology, University of Stellenbosch Medical School, Ti'ervlei, South Africa.

Present address: Department of Biochemistry, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, N. J. 07103.

' To whom reprint requests should be addressed. Received April 29, 1975; accepted July 18, 1975.

nonhistone nuclear proteins in liver and Morris hepatomas were first reported by Chae et al . (8). These observations were extended in other reports (2, 9, 14, 22, 24, 26) which agreed on the presence of changes in hepatomas but differed on their extent. Less notable changes have been recorded for histones in liver neoplasms but alterations in side-chain modification, rates of synthesis, and the amount of HI" histone have been reported (3, 7, 15).

In view of the greater cellular heterogeneity in normal liver than in hepatomas, it was possible that reported differences in nuclear proteins reflected altered nuclear populations rather than changes in a given type of nucleus. Gonzalez-Mujica and Mathias (12) have shown by means of sucrose gradient centrifugation that normal liver nuclei can be separated according to ploidy and cell type, and these workers have reported that the individual nuclear types have nonhistone chromosomal proteins giving different electro- phoretic patterns. We have undertaken studies of this type with normal liver and Morris hepatoma 7777 in order to obtain information on the nuclear types in hepatoma which may be responsible for overall changes in nuclear proteins.

M A T E R I A L S AND M E T H O D S

Animals and Tumors. Experiments were performed with male Buffalo rats. Morris hepatoma 7777 was maintained as s.c. transplants. The induction, histology, and growth properties of this tumor have been described (19).

Isolation of Nuclei. Rats weighing 160 to 200 g were killed by decapitation. Livers and tumors were immediately removed and chilled. All adhering connective tissue and grossly necrotic regions of the tumor were removed before the tissues were finely minced with scissors. Twenty g of minced tissue were homogenized in 100 ml of 50 mM Tris-HCl, pH 7.7, containing 0.32 M sucrose and 25 mM MgCI2, using sequential shearing in a Teflon-to-glass ho- mogenizer with pestle clearances of 0.20 and 0.30 ram. Six to 8 strokes were applied with the loose pestle, and 10 to 12 strokes were applied with the tight pestle at a rotation speed of 1200 rpm. The homogenates were filtered through 8 layers of cheese cloth and centrifuged at 750 • g for 6 min. The crude nuclear pellet was resuspended in 15 volumes 2.4 M sucrose containing 3 mM MgCI2 and was centrifuged at 130,000 x g for 75 min. The 2.4 M sucrose solution and all subsequent sucrose solutions used were adjusted to pH 7.4 with i M NaHCO3. The purified nuclei were washed in 10

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volumes of buffered 15% sucrose containing 1 mM MgCI2 and were centrifuged at 500 • g for 7 min. The nuclear pellet obtained was gently suspended in a final volume of 5 ml of 15% sucrose with a Vortex mixer.

Fractionation of Nuclei. A modification of the method of Johnston et al. (13) was used for the fractionation of nuclei. One-ml aliquots of the nuclear suspension were layered on 30-ml linear sucrose gradients (30 to 50%) in 1- x 3-inch cellulose nitrate tubes. The gradients were immediately centrifuged at 1250 rpm (200 • g) for 10 min at 5 ~ in a Sorvall centrifuge (Model RC2-B, equipped with zonal rotor controls) with a HS-4 rotor. Both autobrake and compressor were switched off during the centrifugation. Once the purified nuclei were suspended in the 15% sucrose, it was found necessary to perform the gradient centrifuga- tion as quickly as possible to prevent aggregation of the nuclei. The gradients were fractionated into l-ml fractions, and the absorbance at 280 nm was read. In some cases, gradients were monitored at 600 nm or by the diphenyla- mine assay for DNA (6). The profiles obtained were found to be essentially the same as those read at 280 nm. The fractions constituting the individual zones were pooled as indicated in Chart I.

Extraction of Proteins from Nuclear Fractions. The different classes of nuclei separated by sucrose gradients were washed 3 times with 0.25 M HCi. The 1st 2 washes were combined, and the extracted histones were precipitated by adding 10 volumes of acetone. The nuclei were then washed once with 1:1 (v/v) chloroform:methanol containing 0.2 M HC! and once with 2:i (v/v) chloroform:methanol contain- ing 0.2 M HCI. Phenol-soluble proteins were extracted from the washed nuclei according to the procedure of Teng et al. (25). The extracted proteins were restored to the aqueous phase as described by Shelton and Allfrey (23).

Electrophoresis of Nuclear Proteins. Histones were char- acterized by polyacrylamide gel electrophoresis in 0.9 y acetic acid and 6.25 M urea by the method of Panyim and Chaikley (20). Fifty ,ug protein, measured by the method of Lowry et al. (18), were applied to each gel. The protein bands were stained with 0.1% Amido black in methanol: acetic acid:water (3:1:6). The gels were destained in the same solvent and scanned in a densitometer, measuring ab- sorbance at 615 rim. Nonhistone proteins were analyzed by electrophoresis in 8.8% polyacrylamide gels containing 0.1% SDS 5 as described by LeStourgeon and Rusch (17).

Prior to the electrophoresis, the nonhistone proteins were dialyzed for 24 hr against 2 changes of 0.01 M sodium phosphate buffer, pH 7.4, containing 0.1% SDS and 0.14 M 2-mercaptoethanol. Approximately 90 ~ag protein, deter- mined by the method of Christian and Warburg (10), were applied per gel, and electrophoresis was carried out at 2.25 ma/gel for about 6 hr. The gels were stained with 0.1% Coomassie brilliant blue R in isopropyl alcohol:acetic acid:water (2.5:i:6.5). The protein bands were analyzed by densitometry at 590 nm. Estimates of the molecular weights of the individual nonhistone protein bands were based on

s The abbrev ia t ion used is: SDS, sodium dodecyl sulfate.

Nuc lear Prote ins o f H e p a t o m a

1.5

1.0

0.5

E

I_U C) Z

m rY 0 oo r n

I.,5

l.O

0.5

(a)

/,

I 2 3

--7 f - i

7

(b) I 2 3

i i 1 1 1 [ 0 I0 15 20 25 30

FRACTION NOI Char t 1. A b s o r b a n c e profiles of nuclei separa ted by cent r i fuga t ion on

30 to 50% sucrose gradients . The nuclei were obta ined f rom (a) control rat

liver or (b) h e p a t o m a 7777. The f ract ions that were pooled to give the 3

nuclear classes are indicated by a r r o w s .

mobility versus molecular weight plots for proteins of known molecular weight under identical conditions.

RESULTS

Isolation and Fractionation of Nuclei. Sucrose gradient profiles showed that normal liver nuclei isolated from Buffalo rats weighing 160 to 200 g separated into 3 distinct classes on 30 to 50% sucrose gradients (Chart la). Similar findings were previously reported for Norwegian hooded rats by Johnston et al. (13) who described Zone 1 as stromal diploid nuclei, Zone 2 as parenchymal diploid nuclei, and Zone 3 as parenchymal tetraploid nuclei. Nuclei isolated from the rapid growing and poorly differentiated Morris

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B. W i l s o n et al.

hepa toma 7777 were s imi lar ly found to separate into 3 dist inct zones (Char t 1B). The inclusion of 2 mM CaCI2 in the homogeniz ing medium was observed to increase the yield of tumor nuclei. Al though Zone 1 of the hepa toma cosedimented with Zone 1 of the liver nuclei, Zones 2 and 3 of the hepa toma nuclei sedimented a little faster than the corresponding zones of liver nuclei. The proport ion of nuclei found in Zone 1 was very much less in the hepa toma than in no rma l liver. On the other hand, a greater proport ion of nuclei was observed in Zone 3 in the case of the tumor. Nuc lea r profiles obtained from host liver were the same as those from the livers of non- tumor-bear ing rats.

Acid-soluble Proteins. Char t 2 records the densi tometr ic t racings for u rea-po lyacry lamide gel electrophoresis of acid-soluble proteins extracted from the 3 different classes of liver and hepa toma 7777 nuclei separated on 30 to 50% sucrose gradients. In all cases, the major histone compo- nents exhibited s imilar pat terns with respect to amount and relative migrat ion. The minor histone fraction H1 ~ was present at a s imilar concentrat ion in all the nuclear fractions obtained from normal liver, but was greatly reduced or not detectable in the 3 nuclear classes f rom the tumor.

Phenol-soluble Proteins. The phenol-soluble proteins ex- tracted from the residue remain ing after acid extraction of the nuclei were analyzed by electrophoresis according to molecular weight on SDS-po lyac ry l amide gels. Char t 3 shows the gel patterns of the proteins extracted from

A D

B E

J

o

~z JL

JL F

Chart 2. Polyacrylamide gel electrophoretic patterns of histones ob- tained from hepatoma 7777 (A, B, and C) and control rat liver (D, E, and F). The histories were extracted from nuclei of Class 1 (A and D), Class 2 (B and E), and Class 3 (C and F). Where present in significant amounts, the position of histone H l ~ is indicated by an arrow.

2 5

1:55,000

I 0 0 , 0 0 0

80,000

6 5 , 0 0 0

5 5 , 0 0 0 - ~

4 5 , 0 0 0

Chart 3. SDS-polyacrylamide gel electrophoretic patterns of phenol- soluble proteins extracted from unfractionated nuclei of livers from non-tumor-bearing rats (I), host livers (2), and Morris hepatoma 7777 (3). Values for molecular weights are indicated to the left of the gels.

unfract ionated nuclei of control and host liver and also of Morr is hepa toma 7777. No major differences were observed in the overall protein patterns. However, in the tumor, there were both addit ions and deletions of single bands. In the high-molecular-weight region, several addi t ional protein bands were evident. At 135,000 daltons and also at about 100,000 daltons, addit ional bands were observed. In the lower-molecular-weight range, addit ional bands were found at approximate ly 65,000 daltons. Fur thermore , there was the deletion of a protein band at 55,000 dattons; also, at 45,000 daltons, a band appeared to be greatly reduced or absent. These al terat ions were highly reproducible from one preparat ion to another. There were no significant differ- ences between the phenol-soluble proteins f rom the livers of non- tumor-bear ing and host rats.

Rat liver nuclei were separated into the different classes on a 30 to 50% sucrose gradient, and the phenol-soluble proteins were extracted and analyzed on SDS polyacrylam- ide gels, as already described. In Char t 4, the corresponding protein patterns of the individual nuclear classes are com- pared with the proteins extracted from unfract ionated liver nuclei. It appears that the electrophoretic patterns of the proteins from the 3 nuclear classes isolated from liver are very s imilar to the protein pattern of unfract ionated nuclei. Similar ly , the phenol-soluble proteins extracted from the 3 classes of tumor nuclei were compared with unfract ionated tumor nuclei by means of SDS-po lyacry lamide gel electro- phoresis (Char t 5). Except for a few minor quanti tat ive differences, the phenol-soluble protein complemen t of nu- clear Classes 2 and 3 of the tumor seemed to be similar to

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I

Chart 4. SDS-polyacrylamide gel electrophoretic patterns of phenol- soluble proteins extracted from unfractionated liver nuclei (T) and from the 3 classes of liver nuclei fractionated on 30 50% sucrose gradients: 1, Class 1 nuclei; 2, Class 2 nuclei, and 3, Class 3 nuclei. (See Chart 1 for explanation of nuclear classes.)

that extracted from unfractionated tumor nuclei. Although the additional proteins observed in the tumor in the molecular weight regions of 135,000, 100,000, and 65,000 daltons are present to a lesser extent in nuclear Classes 2 and 3, they appear to be enriched in Class 1 nuclei of the tumor. Interestingly, these proteins are the ones lacking in the liver nuclei. Furthermore, proteins of molecular weights of approximately 80,000 and 45,000 that are present in nuclear Classes 2 and 3 are greatly reduced in Class 1 nuclei. These observations contrast with the uniformity of protein patterns derived from the 3 nuclear classes from normal liver.

DISCUSSION

The comparison of SDS electrophoretograms of proteins obtained from total nuclear populations of liver and Morris hepatoma 7777 shows that there are several additional bands appearing in the tumor and also a deletion of at least 1 protein band. These changes cannot be attributed to contamination by membrane or cytoplasmic debris since the differences still exist when nuclei are washed with 1% Triton X-100 (data not shown). It is also unlikely that all these changes are due to differences in contractile proteins, as reported by LeStourgeon et al. (16) for nondividing versus proliferating slime molds. The additional bands in the hepatoma nuclear protein pattern are not in the molecular- weight regions of contractile proteins.

Changes in nonhistone nuclear proteins in liver neoplasia have also been reported by others (2, 9, 14, 22, 24, 26), but

,3s,ooo--

I O 0 , O 0 0 - - -

80,000 --

6 5 , 0 0 0 - -

55,000

45,000

Nuclear Proteins 0[" Hepatoma

Y I 2 3

Chart 5. SDS-polyacrylamide gel electrophoretic patterns of phenol- soluble proteins extracted from unfractionated nuclei of Morris hepatoma 7777 (T) and from the 3 classes of hepatoma nuclei fractionated on 30 to 50% sucrose gradients: I, Class 1 nuclei, 2, Class 2 nuclei, and 3, Class 3 nuclei. (See Chart 1 for explanation of nuclear classes.)

comparison of our results with those of other workers is complicated by the different protein extraction techniques. In studying the biochemistry of liver carcinogenesis, it is important to take into account the complex cellular struc- ture of this organ. While in normal adult rat liver the nuclei from stromal cells are diploid, most of the parenchymal cells may be either diploid or tetraploid, according to the age of the animal (13). It is appropriate, therefore, to assess the influence of different nuclear classes on normal and abnormal biochemical activities of the liver. Gonzalez- Mujica and Mathias (12) have reported that the electropho- retic patterns of nonhistone chromosomal proteins vary within the different classes of liver nuclei fractionated on 20 to 50% sucrose gradients. Furthermore, these workers showed that thioacetamide-induced alterations in nonhis- tone proteins of liver varied according to nuclear class.

Baserga (5) has suggested that the heterogeneity of cell population makes a comparison between liver and hepato- mas somewhat meaningless. Therefore, it would be impor- tant to establish whether the differences previously reported in the electrophoretic patterns of nonhistone proteins from various Morris hepatomas are due to heterogeneity of nuclear type and whether specific alterations are limited to any particular class of nuclei. Accordingly, in our study of the chromosomal proteins from the fast-growing, poorly differentiated Morris hepatoma 7777, we developed a procedure for the fractionation of the hepatoma nuclei into 3 different classes as previously established for liver nuclei (13). Analysis by SDS-polyacrylamide gel electrophoresis of the phenol-soluble proteins extracted from the 3 individ- ual classes of liver nuclei showed similar banding patterns.

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B. W i l s o n e t al .

W i t h i n 1 t i s sue , t h e o n l y n o t a b l e c h a n g e seen in a n u c l e a r f r a c t i o n w a s in t h e m i n o r n u c l e a r f r a c t i o n o f t h e t u m o r ( Z o n e 1) w h i c h w o u l d c o n t r i b u t e l i t t le to t he t o t a l p ro f i l e fo r t he t i s sue . H o w e v e r , o u r e x p e r i m e n t s d o n o t e x c l u d e the p o s s i b i l i t y t h a t t u m o r nuc le i o f Z o n e 1 a r e d e r i v e d f r o m n o n t u m o r cel ls . It m a y be n o t e d t h a t t h e s e n u c l e i co sed i - m e n t w i th t h e s t r o m a l d i p l o i d n u c l e i o f c o n t r o l l iver . T h e a c i d - s o l u b l e p r o t e i n s a l so s h o w e d i n t r a t i s s u e c o n s i s t e n c y , bu t t h e r e w a s an i n t e r t i s s u e d i f f e r e n c e in t he H1 ~ h i s t o n e . T h i s h i s t o n e , w h i c h is f r e q u e n t l y d e c r e a s e d in r a p i d l y d i v i d i n g cel ls (21), ha s a l so been d e s c r i b e d as h i s t o n e fla (15), F r a c t i o n Al l (4), o r A (1 1). In v iew o f t h e e s s e n t i a l s i m i l a r i t y in n u c l e a r p r o t e i n s o b t a i n e d f r o m d i f f e r e n t l iver n u c l e a r p o p u l a t i o n s at t h e level o f r e s o l u t i o n o b t a i n e d u n d e r o u r c o n d i t i o n s , it a p p e a r s t h a t c o m p a r i s o n s c a n be m a d e b e t w e e n l ive r a n d h e p a t o m a s , w h i c h a r e n o t m a d e m e a n i n g - less by t i s sue h e t e r o g e n e i t y .

A C K N O W L E D G M E N T S

We are indebted to Dr. Harold P. Morris for providing tumor-bearing rats from which hepatoma 7777 was transplanted in these studies.

R E F E R E N C E S

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2. Arnold, E. A., Buksas, M. M., and Young, K. E. A Comparative Study of Some Properties of Chromatin from Two "Minimal Deviation" Hepatomas. Cancer Res., 33:1169 1176, 1973.

3. Balhorn, R., Balhorn, M., Morris, H. P., and Chalkley, R. Compara- tive High-Resolution Electrophoresis of Tumor Histones: Variation in Phosphorylation as a Function of Cell Replication Rate. Cancer Res., 32. 1775 1784, 1972.

4. Ballal, N. R., and Busch, H. Two-Dimensional Gel Electrophoresis of Acid-soluble Nucleolar Proteins of Walker 256 Carcinosarcoma, Regenerating Liver, and Thioacetamide-treated Liver. Cancer Res., 33." 2737 2743, 1973.

5. Baserga, R. Non-Histone Chromosomal Proteins in Normal and Abnormal Growth. Life Sci., 15:1057 1071, 1974.

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9. Chae, C. B., Smith, M. C., and Morris, H. P. Chromosomal Nonhistone Proteins of Rat Hepatomas and Normal Rat Liver.

Biochem. Biophys. Res. Commun., 60." 1468 1474, 1974. 10. Christian, W., and 9r O. Isolierung and Kristallisation des

G~irungsferments Enolase. Biochem. Z., 310:384 421, 1941. 11. Garrard, W. T., and Bonner, J. Changes in Chromatin Proteins during

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13. Johnston, I. R., Mathias, A. P., Pennington, F., and Ridge, D. The Fractionation of Nuclei from Mammalian Cells by Zonal Centrifuga- tion. Biochem. J., 109:127 135, 1968.

14. Lea, M. A., Koch, M. R., and Morris, H. P. Nuclear Protein Changes in Rat Hepatomas Correlating with Growth Rate. Cancer Res., 35." 1693 1697, 1975.

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16. LeStourgeon, W. M., Forer, A., Yang, Y.-Z., Bertram, J. S., and Rusch, H. P. Contractile Proteins. Major Components of Nuclear and Chromosome Nonhistone Proteins. Biochim. Biophys. Acta, 379. 529 552, 1975.

17. LeStourgeon, W. M., and Rusch, H. P. Localization of Nuclear and Chromatin Residual Acidic Protein Changes during Differentiation in Physarum Polycephalum. Arch. Biochem. Biophys., 155. 144 158, 1973.

18. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265-275, 1951.

19. Morris, H. P., and Meranze, D. R. Induction and Some Characteris- tics of "Minimal Deviation" and Other Transplantable Rat Hepato- mas. In." E. Grundman (ed.), Recent Results Cancer Res., 44." 102 114, 1974.

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22. Reeck, G. R., and Morris, H. P. Nonhistone Chromatin Proteins of Transplantable Rat Hepatomas and Kidney Tumors. Proc. Am. Assoc. Cancer Res., 15." 29, 1974.

23. Shelton, K. R., and Allfrey, V. G. Selective Stimulation of Synthesis of a Nuclear Acidic Protein in Liver Cells Stimulated by Cortisol. Nature, 228." 132 134, 1970.

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26. Yeoman, L. C., Taylor, C. W., and Busch, H. Two-Dimensional Gel Electrophoresis of Acid-extractable Nuclear Proteins of Regenerating and Thioacetamide-treated Rat Liver, Morris 9618A Hepatoma, and Walker 256 Carcinosarcoma. Cancer Res., 34:424 428, 1974.

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