Dye Carcinogenesis*

BOHDAN BAKAY AND SAM SOROF

(The Institute for Cancer Research, Philadelphia, Pennsylvania)

SUMMARY

With the use of 2.1 M sucrose solution, nuclei were isolated from normal, control, andazo preneoplastic livers, and from azo dye-inducedliver tumors of rats. Four consecutive extractions of nuclear sap in isotonic saline-phosphate buffer solubiized 49 and37 per cent of the nitrogen of liver and tumor nuclei, respectively.

Velocity sedimentation analyses revealed that the soluble nuclear macromoleculesconsist of the following size classes: 41 S, 18 S, 14 8, 8 2, 6 8, 4 S, and 2 S, and twodiffuse groups sedimenting at greater than 41 8 and at 18—41S. No significant diiference in sedimentation could be attributed to early preneoplasia induced by theliver carcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). However,nuclear extracts of 3'-Me-DAB-induced liver tumor contain considerably more 4 2 andmuch less 6 8 and 8 2 proteins than do nuclear extracts of liver.

Extensive resolution of the soluble macromolecules of liver nuclei by free boundaryelectrophoresis yielded profiles which have been extrapolated into eighteen hypothetical classes. These have been grouped into five charge types : basic, near-neutral,weakly acidic, highly acidic, and strongly polyanionic. The mobilities of most of theclasses coincide with those previously found in liver extracts of essentially cytoplasmicorigin. This applies in particular to the five near-neutral nuclear components andthe five cytoplasmic h components, which previously have been implicated in thecausal mechanisms of three different types of chemical carcinogeneses.

The only difference in the electrophoresis of liver nuclear extracts which could be attributed to early hepatic preneoplasia induced by azocarcinogen was the frequencyof absence of the most basic component. In contrast, nuclear extracts of dye-inducedliver tumors exhibit a marked reduction of near-neutral (h-like) and basic proteinsand an increase in amount of highly acidic components. Parallel alterations havepreviously been found with cytoplasmic extracts.

On the basis of present and past findings, a causal mechanism of carcinogenesis bycertain chemicals is discussed, involving carcinogen binding to the h@type of proteinsand the subsequent deletion of h (near-neutral) and basic proteins in nucleus and cytoplasm of target cells.

Certain macromolecules of the cell nucleus are soluble‘IIisotonic salt solution. These proteins (“globulins― or

“albumin-globulins―)appear to be derived predominantlyfrom the nuclear sap (13, 27, 28, 42, 64). Compared withthe considerable study @nthe other proteins of nuclei,the soluble nuclear proteins have received little attentionregarding their physical and chemical composition, andeven less concerning their biological roles (see reviews[14, 17, 63J). Nevertheless, it has been reported that thisfraction contains nuclear ribosonies (3, 41, 61), macromole

* This investigation was supported in part by Public HealthService Research Grant CA-05945 from the National CancerInstitute. Presented (5) in part at the 47th Annual Meeting ofThe Federation of American Societies for Experimental Biology,April, 1963.

Received for publication June 22, 1964.

cules of many charges (6—9,20, 21, 24, 32, 39, 43, 44) andsizes (45, 59, 60), and specific ability to arrest developmentof the frog embryo (35).

This report characterizes in greater detail the electrophoretic and ultracentrifugal properties of the solublenuclear macromolecules of normal rat liver. In addition,in a search for intranuclear alterations associated withliver carcinogenesis, normal and preneoplastic livers andtumors induced by an aminoazo dye have been compared.Present findings are also correlated with those of equivalentcytoplasmic soluble proteins, which previously have beenextensively studied in this laboratory under conditionsclosely comparable to those used here (49, 54, 55). Vanous implications regarding nucleo-cytoplasmic interrelationships and carcinogenesis are discussed.

1814

Soluble Nuclear Proteins of Liver and Tumor in Azo

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B&ic&y AND S0R0F—Soluble Nuclear Proteins of Liver and Tumor 1815

MATERIALS AND METHODS

Insofar as possible, experiments were carried out underconditions used in previous studies on the soluble cytoplasmic proteins of rat liver (54, 55).

Rats, diets, and tissues.—Each of the following four tissues was studied in five to six experiments, in each of whichwere used twelve to fifteen Carworth CFN male ratsweighing 350—425 gm.:

a) Normal livers of rats grown on stock Wayne LabBlox;

b) Controlliversof rats fed ad libitum the basaldiet no. 3of Mifier et at. (37) containing 180 gm. ca.seinand 1.0 mg. ofriboflavin per kilogram of diet for 18 or 24 days;

c) 3'-Me-DAB preneoplasti.c livers of rats fed the abovebasal diet and the hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), as 580 mg/kg ofdiet for 18 or 24 days;

d) 3'-Me-DAB liver tumor8 induced by feeding the3'-Me-DAB diet until sacrifice at 135—152days.

Rats fed stock diet received tap water; the others weregiven distified water.

Isolation of nuclei.—All rats except tumor bearers werefasted for 16—20hr. prior to sacrifice. Livers were thenimmediately perfused with cold 0.25 M sucrose (BakerAnalyzed Reagent no. 4072) and collected in an ice-cooledbeaker. No attempt was made to perfuse the tumors.Tumors 0.5—3cm. in diameter were quickly excised, chilled,trimmed free of liver, cut into pieces, examined macroscopically for firmness and absence of necrosis, and collected in an ice-cooled beaker. Pools of 130—140gm. ofliver or 160—170gm. of tumor were rinsed in cold 0.25 Msucrose, blotted on paper towels, and thereafter processedin a room kept at 2O@4OC.

Livers were minced with scissors, mixed with 2 ml. ofcold 2.1 M sucrose solution per gram of tissue, and squeezedthrough a 50-mi. syringe into a Lucite Potter-Elvehjemhomogenizer (3.7 X 27 cm., internal diameter). Tumor,being considerably harder, was niinced, mixed similarlywith sucrose solution, and forced through enlarged holes(1.7 mm.) of a garlic press.

Nuclei were then isolated in 2.1 @isucrose solution (d2. c.= 1.276 gm/ml), according to a modification of the method

of Chaveau et al. (19). The tissue was homogenized bysix to eight passes with a pestle having a clearance of 200—250@ and rotating at 300 r.p.m. The homogenate wasfiltered in a piston-type tissue press through a series ofstainless steel wire screens of 32, 42, 60, 90, 120, and 200meshes under mild manual pressure. Passage throughthe finest screen (74-@@openings) removed the connectivetissue strands and ruptured all cells. Filtrates were diluted with 2.1 M sucrose solution to a volume (milliliters)6.8 times that of the weight of the tissue (grains) and centnifuged at 40,000 X g (average) for 60 mm. in a refnigerated Spinco model L ultracentnifuge with rotor 21 of940-mi. capacity. Supernatant fluids were decanted,and the floating layers which adhered to the tops of thecentrifuge tubes were removed with a straight-edgedspatula. The tubes were drained and thoroughly rinsedwith cold 0.12 Msodium chloride solution containing 0.01 amsodium phosphate buffer at pH 7.4 Care was taken notto disturb the pellet of nuclei at the bottom of the tubes.

Specks of pellets were stained with aceto-orcein-fastgreen (33) and examined microscopically under oil immersion. Liver nuclei were usually well preserved andalmost totally free of visible cytoplasmic contamination.In contrast, a small fraction of the tumor nuclei containedcytoplasmic coats or tags. Approximately 10 per centof the tumor nuclei in the pellet were fragmented. Difliculties in isolating pure whole nuclei from tumors havebeen encountered by others (23, 29).

Extraction of nuclei.—Nuclei were extracted in 8—10-mi.aliquots of 0.12 M NaCl containing 0.01 M sodium phosphate, pH 7.4, in a Lucite Potter-Elvehjem homogenizer(16 X 1.6 cm., internal diameter) with a pestle clearance of100-125 @Land rotating at 300 r.p.m. Centrifugation at20,000 X g (average) for 10 mm. yielded a clear extract.Three additional extractions followed by sedimentationswere required to obtain the bulk of the saline-phosphatesoluble substances.' In order to remove small amountsof transferred insoluble material, the pooled extracts (40—50 ml.) were centrifuged at 20,000 X g (average) for 20mm., yielding the soluble nuclear extract.

No consistent difference in the amount of nitrogen extractable from liver nuclei could be correlated with thetype of experimental diet. Based on an assumed nitrogencontent of 16 per cent (Kjeldahl assay of whole extractand of component I of Chart 1), nuclear extracts derivedfrom 140 gm. of liver contained 600-680 mg. of protein,equivalent to 0.4—0.5per cent of the wet weight of tissue.Similarly, 170 gm. of tumor yielded 340—510mg. of protein,equivalent to 0.2—0.3per cent of the wet weight of tissue.These amounts of nitrogen represented 49 ±2 per cent ofthe total liver nuclear nitrogen and 37 ±2 per cent of totaltumor nuclear nitrogen. In comparison, normal livernuclei extracted four times with 0.15 M saline alone so!ubiized 43 per cent of the total nuclear nitrogen. Thus itappears that an additional 6 per cent of the nitrogen insaline-phosphate extracts of liver nuclei was liberated byaction of phosphate ions (see Discussion).

Diphenylamine assays (22) on tnichioroacetic acid digests (48) demonstrated that of the DNA of the wholeliver pulp, 80 ± 4 per cent was in the screen filtrates ofhomogenates, 55 ±5 per cent was in the combined nuclearresidues after saline or saline-phosphate extractions, andan insignificant amount was in the soluble nuclear fraction.

Removal of @ucrose.—Thecombined nuclear extractscontained 30-40 per cent sucrose, which had been takenup by the nuclei in the absence of magnesium or calciumion (25). This amount of sucrose seriously interferedwith various chemical analyses, hampered the process ofconcentration, and on prolonged contact caused considerable denaturation of certain proteins during concentration.Therefore, the fresh extracts were immediately ifiteredthrough a gel column (3 X 48 cm.) of Sephadex G-25(medium grade). The column was operated at 3°C. insodium Verona! buffer, pH 8.6, ionic strength 0.02, contaming 0.03 M NaCl, and at 120—140ml/hr, with effluentcollected in a fraction collector refrigerated at 8°C. Themacromolecules of the extracts were thus freed of sucrose

I In one large experiment with 3'-Me-DAB liver, the distribu

tion of protein in three successive extracts was 708, 250, and 150mg.

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1816 Cancer Research Vol. 24, November 1964

284 m@

V

\\

@3Omin.

Kt4Lc I

82mm.

CHART 2.—Sedimentation patterns of solublenuclear macromolecules of normal rat liver.Times at 259,700 X g (average) and direction ofsedimentation indicated. Phase plate angles, topto bottom: 250, @5O,and 30°.

3x48 cmFLOW

700 600 500VOLUME . ML

CHART 1.—Sephadex G—25 elution profileof the nuclear extract of normal rat liver.

and other small molecules and brought into the Veronalchloride buffer.

Examination of the eluate at 284 m@z(12) revealed thatthe constituents of the normal liver nuclear extracts wereresolved ifltA) five components (Chart 1). Component I,which was eluted at the void volume of the column andconsisted of macromolecules excluded from the gel pores,contained approximately 99 per cent of the nitrogen and93 per cent of the absorption at 284 mj@of the appliedliver nuclear extracts. Small molecules, retarded by thegel, migrated with and slower than sucrose as componentsII through V. Judging by negative biuret reaction andcharacteristic absorption spectra, components II throughV appear to contain base constituents of nucleic acids.

Concentration of macromolecules.—Immediately after gelfiltration, fractions of Component I were located visuallyby opalescence and straw color and were pooled. This cut(88—90per cent of the applied 284 mj@absorbance) wassubjected to dialysis in a limp bag of cellulose casing'40 cm. long against 130—140ml. of Veronal-chloride buffer,pH 8.6, containing 33 per cent purified dextran3 in a drum(50 X 4.4 cm., internal diameter) rotating at 25 r.p.m.In 6—8hr., 60 ml. of pool was concentrated to approximately 3 per cent protein concentration (17—18in!.). Ifafter 2—3hr. of dialysis, the concentration of dextran wasrestored to 33 per cent by addition of dry dextran, theprocess of concentration was significantly accelerated.

I The casings used in all dialyses measured 24/32 inches (inflated diameter) and were made by Visking Corp., Chicago, Ill.3Fifteenpercentclinicaldextran,1400ml.(75,000molecularweight ; Commercial Solvent Corp., Terre Haute, md. ; or AbbottLaboratories, North Chicago, Ill.), in distilled water was treatedwith 20 gm. of reactivated Darco G-60 charcoal, sterilized for 1hr. at 120°C., filtered through a layer of cellulose powder, dialyzedtwice (48 hr. total) against 18 1. of distilled water at 2°C. withinternal and external agitations , and lyophilized . I)extranrecoveries averaged 90 per cent.

Analyses of protein.—The concentrated protein was additionally dialyzed2 for 4 hr. and then 5 hr., each timeagainst 2 1. of the Veronal-chloride buffer with magneticstirring. The dialyzed protein solution was centrifugedat 25,000 X g (average) for 20 mm. for clarification. Ac

cording to absorption measurements at 284 m@z,the supernatant fluid contained 93—99per cent of the protein ofpooled component I. However, if the protein was concentrated to nearly 6 per cent, recovery was reduced to 82per cent. (Protein concentrations in the Veronal-chionidebuffer were routinely assayed by the biuret method,standardized against dialyzed human serum of known nitrogen content. For an approximation of protein concentration it was convenient to use spectrophotometnicassay at 284 mj.t.)

In early experiments, 10.2-nd. aliquots of the supernatant fluids were immediately (1 day after animal sacnifice) analyzed by free boundary e!ectrophoresis at 1°C.in an “11-mi.―cell (92 X 25 X 3 mm.) at 5.28 v/cm for240 mm. in a Klett instrument. Later, electrophoreseswere run for up to 1400 mm. as above in a Spinco model Happaratus. The absence of convection in the “descending―(negative) limb for this length of time, and the muchincreased sensitivity resulting from the latter instrument'sphase plate and two-way optical path through the cell,permitted the extensive resolution and detailed analysisof the basic and miear-neutral components.

The remainder (4—5ml.) of each supernatant fluid wasdialyzed' overnight with stirring against two successive1-1. volumes of 0.20 M sodium chloride containing 0.01 Msodium phosphate buffer, pH 7.4. After clarification at25,000 X ii (average) for 20 mm. and in the second dayafter animal sacrifice, the supernatant fluid was analyzedby boundary velocity sedimentation at 259,700 X g (aver

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TABLE 1

SEDIMENTATION ANALYSES OF SOLUBLE NUCLEAR MACROMOLECULES OF RAT LIVER AND LIVERTUMORCLASSESNORMAL

LIVERCo@noL LIVER-________3'-ME-DAB LIVER3'-Mz-DABTUMOR$w.M@±a.d.Area(%)- ±a.d.±a.d.Area (%)s,@,.,,'— ±a.d.Area(%)SW.%±a.d.@ Area(%)±a.d.>41

841S5915

18—41AS18—598

18814S8 S78684,S

@ @SDiffuse

41.1

Diffuse

18.412.88.0

6.23.71.51.8

1.10.70.3

0.30.010.4ca.

218

ca. (4)f

34

18

141992.6

0.3

0.60.21.1

1.11.90.7Diffuse

41.1

Diffuse

17.814.48.3

6.13.82.22.2

0.01.20.3

0.20.040.1ca.

199

Ca. 4

57

16

131611Diffuse

40.0

Diffuse

16.413.58.4

6.34.01.70.6

2.30.60.3

0.20.20.3Ca.

2610

?

57

14

12179Diffuse

38.7

Diffuse17.814.7

(8.4)7.3(6.6)4.12.33.1

0.70.7

0.2

0.10.2ca.

21

7

ca. 6

57

14

29112.6

1.7

0.10.81.3

0.5

3.01.1No.calculated34314153Protein

concentra

tion per cent2.9—3.4%1.6—2.6%2.1—2.9%1.6—2.8%

Fri “ I

@@ i@@ -

r@1@@ ii

BAKAY AND S0R0F—Soluble Nuclear Proteins of Liver and Tumor 1817

a In Svedberg units, S = 10― sec., ± average deviation.

t Parentheses denote that one determination was made (e.g., 7 8 resolved into 6 2 and 8 8).

age) in a 12-mm., 4°cell in a Spinco model E ultracentrifuge. Several experiments were carried out with two12-mm., 4°cells, one of which contained buffer only. A12-mm., 2°,double sector cell was used once for simultaneous determination of buffer baseline.

RESULTS

Analytical velocity sedimentation.—The characteristicstages in the sedimentation of the soluble macromoleculesof liver nuclei are shown in Figure 1, while representativetreatment of areas is ifiustrated in Chart 2. Names ofcomponents are in italicized S numbers. Corrected sedimentation rates,@ ,@,and relative concentrations (basedon areas) of the resolved species are given in Table 1.The sedimentation analyses of mixtures so polydisperseas these have considerable limitations of accuracy (46),despite the attainment of good precision expressed as lowaverage deviations.

Velocity sedimentation resolves the complex solublemacromolecules of liver nuclei into at least seven classesof fairly distinct size. These consist of three rapid andfour slower components. The three rapidly sedimentingclasses are the discrete 41 8 and barely distinguishable18 2 and 14 S. The four slower components are largerand are the 8 8, 6 8, 4 8, and 2 8. In addition to theseseven species, there are two diffuse size spans analyzedwith least accuracy. The larger (19—26per cent) sediments at rates greater thaii 41 8; the much smaller (approximately 4 per cent) spans between the 41 8 and 18 8.

There is close similarity between the over-all sedimentation profiles of the soluble nuclear macromolecules of 3'-Me-DAB preneoplastic liver and normal and control livers(Fig. 1, Table 1, and Chart 3). No unusual ultracentriffugal property could be correlated with early liver preneoplasia induced by the azo dye.

20 fl:O@t@20

@ @1C)L@t30

.@@

a20@

‘10

-J

C —

@/P

(@@)@

I‘‘

:@o @;c@@1.@F@TAT!ON F@TE, /r:@c@

CHART 3.—Comparison of the ultracentrifugalcompositionsof nuclear (@j) and cytoplasmic ()extracts of normal, control and 3'-Me-DAB liversand azo dye-induced liver tumors. Data on cytoplasmic soluble proteins taken from Refs. 49,50,52.

On the other hand, ultracentnifugal patterns of thesoluble nuclear macromolecules of 3'-Mc-DAB liver tumorapparently differ from those of normal, control, and preneoplastic livers (Fig. 1, Table 1, and Chart 3). Qualitatively, there appear to be two minor differences. Ratherthan a 41 8, tumor exhibits a 39 8 component. Whetheror not this difference is significant is at present uncertain.Also, in five of six runs, the apparent tumor equivalents ofthe 6 S and 8 8 components of liver sedimented as 7 8.It is unknowii if this is due to smaller relative amounts of

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1818 Cancer Research Vol. 24, November 1964

of so complex a profile into symmetrical components istentative and in part arbitrary. Its primary virtue isthat the combination of the demonstrated extrapolations,mobiities, and relative compositions together fully describes the present resolved profile. Judging by mobilities,electrophoretic components may be grouped into fivemacromolecular types of decreasing negativity and acidity:strongly polyanionic, highly acidic, weakly acidic, nearneutral, and basic (positive). Relative amounts of eachgroup are given.

The strongly polyanionic component 1 exhibits an electrophoretic mobility characteristic of nucleic acid (58).All other components, isolated in preliminary experimentsby zonal column electrophoresis, are of biuret-positiveproteins.

Because 1200—1400mm. of e!ectrophoresis did not resolve components 15 and 16 from the e salt boundary(inherent in boundary electrophoresis), the exact locationsand areas of all three are somewhat uncertain. It islikely that the Verona! boundary slightly moved in apositive direction (1) from the starting boundary position(arrows in Chart 4). Therefore, the e salt boundary isthought to be within the region of components 15 and 16.The basic protein components 16, 17, and 18 have positive(ascending) mobilities and appear to have isoelectnic pHvalues above 8.6. The most basic protein component, 18,in most electrophoreses formed a sharp spike. In view ofthe possible importance of the basic proteins in the nuclearcontrol of cell function (Discussion), it is of interest thatat least three classes of positive proteins, representing 9per cent of the total, are isolated from liver nuclei by extraction with aqueous neutral saline-phosphate solution.

Nuclear extracts of livers of rats fed stock, control, and3'-Me-DAB diets exhibited very similar electrophoreticprofiles at 240 mm. (Klett apparatus; Chart 5). Oneoutstanding difference was however encountered; namely,that the frequency of the absence of component 18 variedwith the diet fed. Component 18 was present in extractsof normal liver in nine of eleven experiments, with controlliver three of six, and with 3'-Me-DAB preneoplastic liverone of five. The locations of components 1—18in Chart 5are indicated by arrows according to mobiities determinedin the Spinco electrophoresis apparatus.

In contrast to the electrophoretically similar patternsdisplayed by these livers, the soluble nuclear macromolecules of the 3'-Me-DAB-induced liver tumor exhibitedconsiderably different profiles in five experiments (Chart5). Tumor extracts contain on a relative basis approximately twice as much of the strongly polyanionic cornponent 1 and highly acidic components 2—4,possess aboutthe same quantity of weakly acidic components 6—9,buthave less than half of the near-neutral and basic proteins10—17. Further, tumor almost totally lacks basic proteincomponent 18. Therefore, the over-all change in thesoluble nuclear macromolecules accompanying the fonmation and progression of liver tumor is a considerable increase in the relative amount of highly acidic proteins atthe expense of the near-neutral and basic proteins. Thisover-all increase in the weighted average of electrophoreticmobilities of 3'-Me-DAB tumor nuclear components, cornpared to those of non-neoplastic liver, is evidence of a

CHART 4.—Free boundary electrophoretic

resolution of the nuclear macromolecules ofnormal liver. Phase plate angles, top tobottom: 35°,25°,15°,and 15°;2.9 per centprotein.

poorly resolving C S and 8 8 in tumor compared with liveror to differences in molecular species. Quantitatively, therelative amount of the 7 8 (6 8 + 8 8) of tumor is approximately half the sum of the C S and 8 S of liver, whereasthe 4 8 of tumor is approximately double that of liver. Asa result, the 4 8 is the predominant size species of tumornuclear macromolecules. Otherwise, the remaining cornponents of tumor appear to be like those of liver in kindand relative amounts.

Free boundary electrophoresis.—As evident from theprogressive electrophoretic resolution shown in Chart 4,the soluble nuclear macromolecules of normal rat liverconsist of a mixture of great charge complexity. Themixture is so polydisperse that extensive electrophoresis(1180 mm.) was necessary for workable resolution. Careful graphic division of the patterns into nearly symmetricalhypothetical peaks (57) indicates the presence of a minimumof eighteen such components. Names of components arein italicized numbers. Component 1 (not shown) spreadduring the first hour of electrophoresis. Average descending mobilities (ascending for positive mobiities), percentage compositions, and average deviations of thesehypothetical components of four experiments with normalliver are presented in Table 2. It should be stressed thatdespite the realization of precise mobilities, the division

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Nuci.z*aCv@ropi.@swicTypeI@:;tMobility1:f:a.d.AreaComponentMobility'Per

cent±a.d.Total%Strongly

polyanionicI—19.40.200.80.120.8Highly

acidicS34—7.69

—6.62—5.620.18

0.210.161.5

5.19.10.10

0.421.5415.7Undetected

N —6.87tA—5.71tWeaklyacidic5

6789—4.66

—3.89—3.32—2.57—2.090.11

0.100.150.060.0613.2

13.711.48.05.12.28

1.291.071.481.0251.4a@

—4.71ta, —3.86tb —3.Olt

Undetectedg—2.04tNear-neutral10

111215

1415—

1 .61

—1.16—0.82—0.58—0.35—0.100.05

0.050.040.040.030.053.8

4.52.22.33.57.10.55

0.940.480.460.521.2823.4slow

gfasth,slow h1middle li@slow h2

!&,—1

.504@—1.158@—0.884@—0.564@—0.437t

—0.071@Basic16

1718+0.20

+0.71+1.160.04

0.110.054.4

2.22.20.92

0.620.738.8Undetected

i I -I-0.83tUndetected

BAKAY AND S0R0F—Soluble Nuclear Proteins of Liver and Tumor

TABLE 2FREE BOUNDARY ELECTROPHORETIC ANALYSES OF SOLUBLE NUCLEAR AND CYTOPLASMIC

MACROMOLECULESOF NORMAL RAT LIVERFour nuclear experiments; protein concentrations 2.6—3.1per cent.

1819

* x 10—icm/sec/v/cm from starting boundary.

t Analyzed in sodium Verona! buffer, pH 8.60, ionic strength 0.10 (49).@ Analyzed in sodium Veronal buffer, pH 8.6, ionic strength 0.02, containing 0.03 amsodium chloride

(55).

DISCUSSION

The significance attributable to the study of the solublenuclear macromolecules is conditioned by uncertaintiesconcerning the realization of two essential requirements(14, 16, 17, 63). The first is the isolation of a sufficientand representative quantity of pure and biochemicallyunaltered nuclei. The second is the exclusive extractionof the nuclear sap yielding native macromolecules. Theconcern is supported by the knowledge that isolated nucleimay be excessively contaminated with cytoplasmic structures (2, 23, 34) and are permeable to many substances,including macromolecules (4, 30). Considerations of thelarge quantity of tissue to be processed, relative purity ofthe product, speed and simplicity of procedure, and mdicated small interchange of soluble nuclear constituentswith those of cytoplasm all led to the choice of the methodof Chaveau et at. (19) for the isolation of nuclei. Use of ahigh concentration of sucrose in this method impedesnuc!eo-cytoplasmic cross-contamination by reducing protein solubiity at low ionic strength and dielectric andby increasing viscosity of the medium. Other factorswhich may preserve the biochemical integrity of the nucleus during isolation are the possible ion binding of nuclear

COMPONENT NO•-E@14/21817 /6/51/3@1110 9 8 7 6 5I I IIII1 I I I I .1 1 i

4 3 2.1 @l I

24Orrth@528 v/cm

•10 0 -1.0 -2.0 -3.0 -40 -50-60 -70 -80 90 @fl@Q@@ -52MOBILI1Y @‘Ocrri/i'-sec

CHART 5.—Electrophoretic patterns of the soluble nuclear

macromoleculesof normal (- ——),control (—.—.), and 3'-MeDAB (—) livers, and 3'-Me-DAB liver tumor (. . . . ). Proteinconcentration, 2.7—2.9per cent; 25°angle.

lower weighted average of the isoelectric pH values of thesetumor proteins. Similar effects were observed previouslywith cytoplasmic extracts of liver and dye-induced livertumor (50).

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Cancer Research Vol. 24, November 19641820

macromolecules (6), the peripheral distribution of chromatin in nuclei (10), and the presence of the nuclear membrane (10).

The quantity of macromolecules solubilized from isolated nuclei in great part depends on the mechanical meansof extraction. Gentle extraction of nuclei of beef pancreas (43) dissolved only one-fifth of the 30 per cent protein solubiized by vigorous homogenization and shaking.Similarly, mild stirring of normal rat liver nuclei extractedonly 20 per cent of the nuclear nitrogen (29, 65). Incontrast, extensive stirring of calf liver nuclei for 12—18hr.dissolved 42 per cent of the nuclear dry weight (32). Ourfour consecutive extractions of normal rat liver nucleiby homogenization with a closely fitting pestle likewisesolubilized 43 per cent of the nuclear nitrogen in salineand 49 per cent in saline-phosphate. It appears thatmechanical fragmentation of sucrose-isolated nuclei insaline facilitates salting-in and dissociation of bound complexes.

The ionic composition of the extraction medium alsogreatly affects the content of the nuclear extract. Phosphate ions extract significantly more material than salinealone (7). In our experiments, 0.01 amsodium phosphate,pH 7.4, added to saline, brought an additional 6 per centof the nuclear nitrogen into solution, predominantly ascomponents 6 and 7. Electron-microscopic study revealsthat phosphate extraction both shrinks the liver nucleusand solubilizes the nuclear envelope, including any associated cytoplasmic endoplasmic reticulum (34). Mg'+ andCaI+ counteract the phosphate action and are importantin maintaining the physical state of nuclear nibosomes andchromatin (36, 41, 61). Because this initial study aimedat maximum solubilization of dissociable macromolecules,these cations were omitted, while phosphate was employedin the nuclear extraction. The cost of the former wasnuclear clumping and resultant small increase of cytoplasmic contamination (47) ; the price of the latter wassolubilization of the nuclear envelope with associatedcytoplasmic contamination (34).

According to present findings, the soluble nuclear macromolecules of rat liver consist of at least seven size classes.At present resolution, it is uncertain if these componentsrepresent more of particular size ranges in a continuum or,instead, fairly separate size modes. Whichever is thecase, the soluble nuclear macromolecules are at least cornposed of a number of favored size ranges, rather than aflat, diffuse distribution. By comparison, extensive resolution has recently shown that the size profile of the so

luble cytoplasmic macromolecules of rat liver consists inone part of discrete size families, and in the other part, afew favored size modes in a continuum (53, 62).

Since the physical state of nuclear ribosomes (72 S or

78 S) is very sensitive to the concentration of Mg2+, ionicstrength, pH (41, 61), and nucleases, it is probable thatdissociation products of nuclear ribosomes are containedin the components faster than 18 S in present nuclear extracts. The greater part of the smaller macromolecules,

2 8 through 18 S, presumably represent constituents ofthe soluble phase of nuclear sap. These latter componentsappear to be equivalent to those observed by Wang (60)

with calf thymus nuclear extracts and fractions thereof insaline without Mg'@.

On the basis of sedimentation analyses of extracts ofliver and tumor, it appeared that the size families of thesoluble nuclear macromolecules are different from thoseof cytoplasm.4 This conclusion seemed valid from a cornparison of present ultracentrifugal findings with those ofprevious studies of the cytoplasmic soluble proteins carriedout under similar conditions with the same tissues (49,50, 52). Thus, in Chart 3, the nuclear soluble macrornolecules could only possibly have the 4 8 in common withthe 8.6 8 component of cytoplasmic proteins. However,these cytoplasmic macromolecules have recently beenresolved by gel filtration into the following size classes:2S,SS,4S,ÔS,6S,7.5S,1O—158(complex),andl6—20 8 (complex) (53, 62). Therefore, a more definitiveanswer to whether the nuclear and cytoplasmic extractsof livers and tumor have different size families must awaitthe similar resolution of the soluble nuclear macromoleculesby gel filtration.

It has previously been reported that the soluble nuclearproteins of various tissues consist of many electrophoreticclasses (6—9,20, 21 , 24, 32, 39, 43, 44). The present studyquantitatively describes the very great polydispersity ofthese macromolecules from liver in greater detail and withmore resolution than has hitherto been reported for anytissue.

The soluble nuclear system appears to be as complex incharge as the soluble cytoplasmic macromolecules of liver.Table 2 shows that the nuclear and cytoplasmic extractsof liver have many of their hypothetical electrophoreticcomponents of matching mobilities. In particular, sixnear-neutral nuclear components have very close mobilitycounterparts among the cytoplasmic components : 10 andslow g, 11 and fast h1, 12 and slow h1, 13 and middle Ii,,14 and slow Ii,, 15 and h3. Likewise, the basic components17 and i are comparable. Such close matching of successive mobilities throughout both profiles supports thetentative thesis that the bulk of the soluble macromoleculesof liver nucleus and cytoplasm contain families closelysimilar in charge, if not actually shared in common. Thisconclusion is in agreement with the preliminary observation of Barton (6, 7) regarding h2 proteins, but is at vanance with the views expressed by Poort (43) and Pate!and Wang (39) for other tissues.

At the lower electrophoretic resolution presented inChart 5, the only difference which can be attributed toearly liver preneoplasia induced by azo carcinogen concerns the absence of basic component 18. It remains forfuture investigation to study further the changes in thisand later stages of preneoplasia. Possibly, 5-fold greaterresolution (as in Chart 4) may reveal changes in thosenuclear components related to the cytoplasmic h2, whichincreases in amount during carcinogeneses by aminoazodyes and 2-acetylaminofluorene (55). Barton has preliminarily reported that cell proliferation during laterazo dye preneoplasia induces the appearance of characteristic near-neutral and basic electrophoretic componentsamong the soluble liver nuclear proteins (7, 9).

4This view was preliminarily reported (5) and communicatedto Dr. Harris Busch for a review on nuclear proteins (18).

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BAKAY AND S0R0F—Soluble Nuclear Proteins of Liver and Tumor 1821

(Table 1 of Ref. 54), cytoplasmic h, proteins appear alsoto be similarly involved in the carcinogenesesinduced bytwo other different chemical carcinogens, 2-acetylaminofluorene and polycyclic aromatic hydrocarbons. Thepresent findings therefore show that an apparent proteinbinding and -deletion sequence may pertain also to livernuclear extracts during azo dye carcinogenesis. The question therefore arises whether a causal sequence of cancerinduction may be initiated by the carcinogen conjugationof h2-type proteins in nucleus and cytoplasm.

That chemical carcinogenesis may result from a repression or derepression of genetic expression has been suggested by others6 (11, 26, 38, 40). The concept that thebasic nuclear proteins may act as gene repressors has beenunder intensive discussion and investigation (3, 31, 56).It has also been suggested that the near-neutral “slightlylysine-rich histones― of nuclear sap may be loosely associated with chromosomes and may be as important functionally as the more tightly bound proteins (15). Likewise, by virtue of relative basicity, configuration, andsolubiity, near-neutral h,-type proteins and basic proteinsin nuclear and cytoplasmic saps may be uniquely suitedto complexwith nucleic acids. Such complexingmay as aconsequence inhibit DNA and RNA syntheses and/orfunctions in the nucleus, and likewise RNA functions inthe cytoplasm. The total effect may be to restrain thecell-biosynthetic and growth potentials and to stabilizethe cell type. Pitot and Heidelberger have suggested amodel in which the protein to which the carcinogen isbound is the repressor of cellular growth, and that this repressor is deleted in the cancer cells (40). In presentterms, the conjugation of the h,-type proteins in nucleusand cytoplasm by chemical carcinogens may initiatechanges which result in the observed loss of near-neutraland basic proteins of nuclear and cytoplasmic saps of thetarget cells. The loss of these proteins may induce a release from normal repression. As a result, there may occura loss of growth control and an instability of cell type.

ACKNOWLEDGMENTS

The competent technical assistance of Mr. Robert S. Ho, MissClaire McKeever, and Mr. Walter Tkaczyk during phases of thisstudy is gratefully acknowledged.

REFERENCES

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2. ALLFREY, V. The Isolation of Subcellular Components. InThe Cell, 1:193-290, edited by J. BRACHETand A. E. MiRSKY. New York: Academic Press, Inc., 1959.

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5. BAKAY,B.; Ho, R. S.; ANDSOROF,S. Soluble Proteins of LiverNuclei in Azo Hepatocarcinogenesis. Federation Proc., 22:477,1963.

6Added in proof: Since the submission of this report, the subject has been discussed futher by Dr. H. S. Kaplan (Some PossibleMechanisms of Carcinogenesis. In: P. EMMELOT and 0. MUHLBOCK (eds.), Cellular Control Mechanisms and Cancer, pp 373—82.Amsterdam: Elsevier Publishing Co., 1964), and by V. R. Potter(Biochemical Perspectives in Cancer Research. Cancer Res., 24:1085—98,1964).

NORMAL LIVER

:@

+1 0 -I -2 -3 -4 —5-6 -7 -8 -9 -10DESCENDING MOBILITIES x fO@cm%'o/t sec.

CHART 6.—Boundary electrophoretic patterns

of the soluble nuclear (—) and cytoplasmic(. . . . ) macromolecules of normal rat liver and

azo dye-induced liver tumors. Data on cytoplasmic soluble proteins taken from Refs. 49, 50.

It is of considerable interest that azo dye induction ofliver cancer brings about parallel alterations among themacromolecules of nuclear and cytoplasmic extracts.Compared with normal and azo preneoplastic livers, dyeinduced liver tumor yields nuclear extracts which haveonly one-half of the near-neutral and basic components10—17;which totally lack component 18; and which contam considerably more of the strongly polyanionic cornponent 1 and acidic components 2—5(Chart 5). Verysimilar changes have previously been found to occur inequivalent cytoplasmic extracts of these tissues (50).Chart 6 compares nuclear and cytoplasmic electrophoreticprofiles, showing the differences brought about by azodye-induced liver cancer. Thus, in both nuclear andcytoplasmic extracts of the tumor compared to normaland preneoplastic livers, there is a marked loss of proteinsof h mobiities (near-neutral) and basic proteins, and anincrease in amount of highly acidic components. Thisover-all shift toward greater acidity of cytoplasmic (50)and nuclear macromolecules in the liver tumor, as cornpared to normal and preneoplastic livers, may have important consequences on the milieu in the nucleus andcytoplasm.

The significance of the marked loss of proteins of hmobilities (near-neutral) in both nuclear and cytoplasmicextracts of dye-induced liver tumor is heightened by thefact that a single member of the h proteins, the slow h,,contains the principal carcinogen-protein conjugate ofcytoplasmic extracts of azo dye preneoplastic liver (51,54, 55). In addition, studies in progress with columnelectrophoresis have revealed that a significant amount ofazo dyes is also bound to the near-neutral proteins ofcomponent 14 (slow h, mobility) of nuclear extracts ofthese preneoplastic livers.5 As summarized elsewhere

5 Dr. A. D. Barton has also found that whole nuclear extractsof rat livers, made preneoplastic by 4-dimethylaminoazobenzene,contain protein-bound dyes (previous persona! communication).

!@ —*@

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1822 Cancer Research

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FIG. 1.—Velocity sedimentation patterns of soluble nuclearmacromolecules in 0.20 M sodium chloride + 0.01 M sodium phosphate, pH 7.4; time (minutes) at 259,700 X g stated in photographs; horizontal arrow indicates direction of sedimentation.Normal liver: 3.4 per cent protein; 22.6°C.; phase plate angles,right to left : 40°,30°,30°,and 25g. Control liver: 2.6 per centprotein; 24.1°C.; 25°,250, 3@O,and 250. 5'-Me-DAB liver: 2.8per cent protein; 21.0°C.; all 250. 3'-Me-DAB tumor: 2.9 per centprotein; 26.0°C.; 30@,3@O,3@O,and @5O•

Vol. 24, November 1964

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BAKAY AND S0R0F—Soluble Nuclear Proteins of Liver and Tumor 1825

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1964;24:1814-1825. Cancer Res Bohdan Bakay and Sam Sorof CarcinogenesisSoluble Nuclear Proteins of Liver and Tumor in Azo Dye

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