Proc. Natl. Acad. Sci. USAVol. 81, pp. 2092-2096, April 1984Cell Biology

Normal liver chromatin contains a firmly bound and larger proteinrelated to the principal cytosolic target polypeptide of a hepaticcarcinogen

(nuclei/nonhistone protein/chemical carcinogenesis/immunohistochemistry/N-2-fluorenylacetamide)

STANLEY A. VINORES, JOHN J. CHUREY, JOANNE M. HALLER, SUSAN J. SCHNABEL, R. PHILIP CUSTER, ANDSAM SOROF*The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111

Communicated by Elizabeth C. Miller, December 15, 1983

ABSTRACT A 14,000-dalton polypeptide was previouslyreported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosolat the start of hepatocarcinogenesis in rats. The 14,000-daltonpolypeptide was purified to homogeneity according to gel elec-trophoreses in both NaDodSO4-containing medium and aceticacid/urea and also by immunogenicity. An immunologicallyrelated form of the cytosolic target polypeptide has now beenfound to be present in the nuclei of normal rat liver as a17,500-dalton polypeptide that is firmly and ionically bound tochromatin. Serial salt extractions of isolated liver nuclei orchromatin at 0.15 and 0.35 ionic strengths fail to dissolve thebound polypeptide, according to electrophoretic transfer im-munoblot analyses. Most of the 17,500-dalton polypeptide isextracted at 0.65 ionic strength, the remainder at 1.2, andnone at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nucle-ase solubilizes the 17,500-dalton polypeptide. All three proto-cols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of thecytosolic protein target of the carcinogen. The presence of pro-tease inhibitors during the isolations and extractions of thenuclei and chromatin reduces the amounts of these smallerpolypeptides. In normal rat liver only nuclei and cytoplasm ofhepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intenselyperilobularly, less in the lobular midzone, and least centrilo-bularly. The nuclei of the perilobular hepatocytes constitutethe strongest staining compartment within all of normal liver.Of 22 nonhepatic tissues of normal rats, 16 contain relativelyfew cells with immunoreactive cytoplasm. Nonhepatic nuclearantigen is present only in villar crest cells of duodenum (whichare normally exposed to liver bile), also having cytoplasmicantigen as well. Five kinds of evidence appear to connect thechromatin-bound 17,500-dalton polypeptide of normal livernuclei to the cytosolic 14,000-dalton polypeptide that is theprincipal target of the carcinogen early during hepatocarcino-genesis in rats. The present findings indicate a direct connec-tion between a chromosomal protein and the immediate princi-pal cytosolic protein target of a carcinogen.

Early events in chemical carcinogenesis involve the interac-tions of carcinogens with macromolecules in susceptible or-gans (1, 2). Several chemical carcinogens combine preferen-tially with single target proteins in organs undergoing carci-nogenesis. Four such principal target proteins are known.All are different, basic, and hitherto have been found only inorgan cytosols (3-6). While nonhistone proteins and his-tones of nuclei have been reported to interact with carcino-

gens (7-9), none of the principal cytosolic protein targets hasbeen shown thus far to be related to the cell nucleus or chro-matin, where chemical carcinogens are generally thought toact in oncogenesis. We report here that an immunologicallyrelated form of the principal cytosolic target protein of a livercarcinogen is present in nuclei of normal hepatocytes as alarger molecule that is tightly bound to chromatin.A 14,000-dalton polypeptide was previously found to be

the principal cytosolic protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) at the start ofliver carcinogenesis in rats (10, 11). Short-term ingestion ofany of three carcinogens, the aromatic amide N-2-fluorenyl-acetamide, the aminoazo dye 3'-methyl-4-dimethylaminoazo-benzene, or the amino acid analog ethionine, causes a markedreduction in the concentration of the target polypeptide ofthe carcinogenic fluorenylamide (10, 11). The 14,000-daltonpolypeptide was previously purified from normal liver cyto-sol and characterized in part, and specific antiserum was pre-pared that reacted in liver cytosol only with that molecule(4). The isolated 14,000-dalton polypeptide is homogeneousaccording to gel electrophoreses in both NaDodSO4 buffer(4) and acetic acid/urea (Fig. 1) and by immunogenicity (4).

MATERIALS AND METHODSRats. Normal male rats (150-200 g) of the Fischer 344 CDF

strain (Charles River Breeding Laboratories) were housed inscreen-bottomed cages at 22°C with a 6 a.m.-6 p.m. lightcycle and fed commercial stock diet (Wayne Lab-Blox; Al-lied Mills, Chicago) and acidified tap water ad libitum. Ratswere sacrificed rapidly in the animal quarters with minimalprior excitement, and the livers on ice were rushed to thelaboratory nearby.

Salt Extraction of the 17,500-Dalton Polypeptide from LiverNuclei. For the isolation of nuclei by a modified procedure ofYankner and Shooter (15), livers were homogenized bymeans of six up-down strokes in a Potter-Elvehjem homoge-nizer (size B or C; Thomas) in a solution (1 ml/g of tissue)containing 0.3 M sucrose, 3 mM MgCl2, 20 mM Tris HClbuffer (pH 7.4), and the four protease inhibitors: 100 kalli-krein inhibitor units (KIU) of aprotinin (Trasylol; MobayChemical, New York) per ml, 0.1 mM N-p-tosyl-L-lysinechloromethyl ketone, 0.1 mM L-1-tosylamido-2-phenylethylchloromethyl ketone, and 1 mM phenylmethylsulfonyl fluo-ride. The homogenates were centrifuged at 500 x g for 10min at 4°C. The sediments were suspended in the above me-dium (1 ml/g of liver), filtered through three layers of nylonmesh (90, 37, and 20 ,um), and centrifuged as before. Thepellets were suspended as above in 2.3 M sucrose/3 mMMgCl2/20 mM Tris HCl buffer, pH 7.4, and the four proteaseinhibitors and centrifuged at 100,000 x g for 60 min at 40C.

Abbreviation: KIU, kallikrein inhibitor unit(s).*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Purity of isolated 14,000-dalton polypeptide according toelectrophoresis in acetic acid/urea gel. Protein samples were incu-bated in 1% 2-mercaptoethanol/2.5 M urea/10 mM Tris'HCI, pH7.4, for 1 hr at room temperature and adjusted to 5% acetic acid.Slab gels [15% acrylamide/0.08% methylene bisacrylamide contain-ing 2.5 M urea and 5% acetic acid according to Panyim and Chalkley(12)] were prerun once without additives, then twice with cyste-amine to scavenge free radicals, and once with protamine sulfate toblock acidic sites (13). After electrophoresis, the gels were visual-ized with Coomassie brilliant blue R-250 (lanes A-D) or by a modifi-cation of a silver staining procedure (14) (lanes E-G). Lanes: A,normal rat liver cytosolic proteins, 40 ,ug; B, same as lane A, 20 tkg;C, purified 14,000-dalton polypeptide, 15 ,Ag; D, same as lane C, 1Ixg; E, same as lane C, 1 tLg; F, same as lane C, 2.5 Ag; and G, normalrat liver cytosolic proteins, 5 Ag. The isolated 14,000-dalton poly-peptide is also pure according to gel electrophoresis in NaDodSO4-containing buffer, followed by staining with Coomassie blue (4).

The sediments were twice similarly resuspended in the 0.3 Msucrose/MgCl2/Tris HCl buffer with protease inhibitors andspun at 500 x g for 10 min at 4TC to yield the nuclear prepa-rations. For extractions of the nuclei, the pellets were ex-tracted three times, each instance by homogenization asabove in 0.11 M NaCl and buffer A (50 mM glycine/5 mMMgCl2/1 mM EDTA/10 mM maleic acid/10 mM Tris, ad-justed with KOH to pH 7.4 and the four protease inhibitors)and by centrifugation at 13,000 x g for 10 min (40C). Theresultant three supernatant fluids were pooled as the 0.15ionic strength extract. Similarly, the residues were seriallyextracted as above with 0.31 M NaCl and buffer A to yieldthe 0.35 ionic strength extract, followed similarly by 0.61 MNaCl for the 0.65 ionic strength extract, then by 1.16 M NaClfor 1.2 ionic strength, subsequently with 1.96 M NaCl for the2.0 ionic strength extract, and thereafter in one experimentwith 8 M urea. The five extracts were stored at -60°C untiltheir nucleic acids were in part removed by precipitation at0.35 M HCl for 15 min at 0°C and sedimentation at 12,000 x

g for 10 min (4°C). The supernatant proteins were precipitat-ed at 20% trichloroacetic acid (wt/vol, using 100% trichloro-acetic acid) for 15 min (0°C), sedimented at 12,000 x g for 10min at 4°C, washed in fresh cold acetone/0.2% HCl (1 ml/mlof extract), and centrifuged at 12,000 x g for 10 min (4°C).After two washings with 10 ml of cold pure acetone, the pro-teins were dried at 4°C under vacuum. After solution in aminimal volume of water and assay of concentration (Low-ry), the proteins were subjected to electrophoresis in Na-DodSO4-containing buffer in 15% polyacrylamide gels (16)and then either stained with Coomassie brilliant blue R-250or electrophoretically transfer-blotted, treated with specificrabbit antiserum against the 14,000-dalton polypeptide,treated with '25I-labeled protein A, and autoradiographed,all as described (4).

Salt Extraction of the 17,500-Dalton Polypeptide from LiverChromatin. Nuclei of normal rat livers were isolated at 40Cby a procedure patterned after that of Yankner and Shooter

(15) and Birnie (17). Livers were homogenized as above. Af-ter centrifugation at 750 x g for 10 min, the sediment wasresuspended in the same medium, filtered as in the abovesection, and respun similarly. The pellet was suspended in2.3 M sucrose/MgCl2/Tris.HCl buffer and protease inhibi-tors as in the above section. After centrifugation at 100,000x g for 60 min, the pellet was homogenized in 0.3 M su-crose/3 mM MgCl2/20 mM Tris HCl, pH 7.4, and the fourprotease inhibitors, sedimented at 1500 x g for 10 min, ho-mogenized in the same medium with 1% Triton X-100, andcentrifuged. The process was twice repeated without the de-tergent to yield purified nuclei. Chromatin was then pre-pared at 40C according to the method of MacGillivray (18).The nuclei were homogenized in solution containing 0.14 MNaCl, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, and the fourprotease inhibitors, stirred for 20 min, and centrifuged at27,000 x g for 15 min. The homogenization and centrifuga-tion were repeated twice to yield the preparation of chroma-tin and three nuclear extracts (0.23 ionic strength), whichwere pooled. The chromatin was extracted with a loose Pot-ter-Elvehjem homogenizer in 0.31 M NaCl in buffer A of theabove section. After sedimentation at 27,000 x g for 15 min,the procedure was repeated twice. The three supernatantfluids were pooled as the 0.35 ionic strength extract. Similar-ly, the residue of chromatin was successively extracted threetimes with additional NaCl to yield extracts at 0.65 and 1.2ionic strengths. The extracted proteins were then in part dis-sociated from nucleic acids with HCl, precipitated with tri-chloroacetic acid, washed, dried, and analyzed, all as in theabove section on extraction of nuclei.

Solubilization of the 17,500-Dalton Polypeptide by Digestionof Liver Nuclei with Micrococcal Nuclease. Liver nuclei wereisolated from four normal rats by the procedure of Burgoyneand co-workers (19, 20). Each gram of liver was homoge-nized as above in 7 ml of buffer N (below) supplementedwith 0.34 M sucrose/2 mM EDTA/0.5 mM EGTA/100 KIUof aprotinin per ml, all adjusted to pH 7.4 with HCl. (BufferN contained 60 mM KCl, 15 mM NaCl, 0.15 mM spermine,0.5 mM spermidine, 15 mM 2-mercaptoethanol, and 15 mMTris HCl at pH 7.4.) The homogenate was layered over a 0.33vol of 1.37 M sucrose/1 mM EDTA/0.25 mM EGTA/100KIU of aprotinin per ml in buffer N and centrifuged for 15min at 16,000 x g. The nuclear sediment was gently dis-persed as above in the extraction of nuclei, using 7 vol of 2.4M sucrose/0.1 mM EDTA/0.1 mM EGTA/100 KIU of apro-tinin per ml in buffer N, and spun at 75,000 x g for 45 min.The pellet was gently washed by suspension in 45 ml of 0.34M sucrose/100 KIU of aprotinin per ml in buffer N and cen-trifuged for 15 min at 16,000 x g. For enzymatic digestion ofnuclei according to the method of Noll et al. (21), the isolat-ed nuclei were dispersed in 0.44 ml of the same medium pergram of liver, made to 1 mM with CaC12, divided into twocontrol and two experimental aliquots, and warmed to 37°Cin a shaking water bath for 90 sec. To each 1 ml of controland experimental nuclear suspension was added 50 ,ul of wa-ter or micrococcal nuclease solution (2.5 units, 3.7 ,ug of pro-tein, grade VI; Sigma), respectively, in glass Potter-Elvehjemhomogenizer vessels (size A; Thomas). Incubations were car-ried out for 3 min and 10 min at 37°C with intermittent ho-mogenizations and were stopped by addition of 20 Al of 0.1M EDTA at pH 7 to each 1 ml of suspension and cooling inice. The suspensions were centrifuged at 4000 x g for 5 min.The pellets were dispersed in a volume of 0.2 mM EDTA,pH 7/100 KIU of aprotinin per ml equal to that of the previ-ous suspensions and respun. The clear supernatant fluidsfrom both centrifugations were pooled as control or micro-coccal nuclease digests. The extracted proteins were in partseparated from nucleic acids, precipitated with trichloroace-tic acid, washed, dried, and subjected to gel electrophoresisin NaDodSO4-containing buffer, stained, electrophoretically

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transfer-blotted, and autoradiographed, all as with extractsof nuclei and chromatin (above).Immunohistochemical Detection of Proteins Related to the

Principal Cytosolic Target of a Hepatic Carcinogen. Liverslices of normal rats were fixed in 10% buffered formalin ofhigh purity for 1 day in preparation of 5-,um paraffin tissuesections, which were stored desiccated at 1-40C, until theywere processed for staining with the rabbit specific antisera,peroxidase-antiperoxidase complex (DAKO; AccurateChemical and Scientific, Westbury, NY), and 3,3'-diamino-benzidine tetrahydrochloride (22). Control analyses with pre-immune rabbit sera yielded virtually unstained sections.

RESULTSChromatin-Bound Protein Related to the Cytosolic Polypep-

tide Target of a Carcinogen. A polypeptide that is immuno-logically related to the cytosolic target protein exists in thenuclei of normal rat livers, in the form of a larger molecularweight species that is tightly bound to chromatin. The molec-ular size of the chromatin-bound polypeptide is 17,500 dal-tons (apparent), in contrast to 14,000 daltons for the cytosol-ic species. These conclusions derive from experiments thatinvolved the salt or enzymatic extractions of purified nucleior chromatin. The first protocol determined that the 17,500-dalton polypeptide is firmly and ionically bound to normalliver nuclei. Serial extractions of purified nuclei of normalliver, initially at 0.15 ionic strength to wash away solubleconstituents and then at 0.35 to remove the high mobilitygroup and other loosely bound proteins (23, 24), failed toextract the polypeptide. Solubilization of most of the 17,500-dalton polypeptide required an ionic strength of 0.65. Theremainder was dissolved at 1.2 (Fig. 2). Nucleohistones dis-sociate at these high salt concentrations (24). Extraction ofthe residue at 2.0 ionic strength released no additional17,500-dalton polypeptide (Fig. 2) nor did solubilization ofthe final pellet in 8 M urea (not shown). The second protocolpointed to chromatin as the site of the firmly bound polypep-

A B C D E F A B C D E F

tide in nuclei of normal liver. Starting with chromatin fromnormal liver nuclei, a similar tight binding was encounteredin successive salt extractions (immunoblots not shown). Thethird protocol made use of specific enzymatic cleavage toestablish further that chromatin is the locus of the bound17,500-dalton polypeptide. Micrococcal nuclease acts specif-ically to degrade nucleic acids in nuclei (20, 21). As shown inFig. 3, the nuclease released the 17,500-dalton polypeptide(and notably small amounts of intermediate 14,000- to17,500-dalton polypeptides) from purified nuclei of normalliver. Importantly, the control digests did not. These mutual-ly supportive protocols employed electrophoretic transferimmunoblot analyses after electrophoresis in NaDodSO4-containing polyacrylamide gels, followed by detection withthe specific antiserum against the 14,000-dalton polypeptide.

Consistent demonstration of the firm association of the17,500-dalton polypeptide to nuclei and chromatin was de-pendent upon the inhibition of proteases throughout the puri-fication and extraction procedures. Protease-inhibited prep-arations usually had mainly the 17,500-dalton species, beingaccompanied by low levels of immunoreactive polypeptidesof sizes intermediate between 17,500 and 14,000 daltons(Fig. 2). Without protease inhibitors, the 17,500-dalton poly-peptide was detectable occasionally, and greater amounts ofthe intermediate-sized and 14,000-dalton polypeptides wereevident (not shown). Notably, normal liver cytosols pre-pared either with or without the protease inhibitors con-tained only the 14,000-dalton polypeptide and no other im-munoreactive species, indicative of a nonartifactual origin ofthis molecule.

Intrahepatic and Tissue Distributions of the Proteins Relat-ed to the Principal Polypeptide Target of a Carcinogen. Insearch of clues to the biological role of the nuclear polypep-tide, its distributions in normal liver and other organs wereexamined by peroxidase-antiperoxidase immunohistochemi-cal staining. In liver, antigen was detected only in parenchy-mal cells and not in biliary duct epithelium, supporting tis-sue, or histiocytes (Kupfer cells) (Fig. 4). The most intensestaining occurred in the perilobular hepatocytes that encir-cled the portal triads (Fig. 4 A and B), with less in the lobular

A B C D E A B C D E.

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FIG. 2. Presence of the tightly bound 17,500-dalton polypeptidein serial salt extracts of nuclei of normal rat liver. Proteins were

resolved by gel electrophoresis in NaDodSO4-containing buffer.(Left) Coomassie blue-stained gel. Molecular sizes are given as dal-tons x 10-. Lanes: A, nuclear proteins extracted at 0.15 ionicstrength, 35 ,Ag; B, 0.15-0.35 ionic strength soluble proteins, 35 ,ug;C, 0.35-0.65 ionic strength soluble proteins, 35 ,ug; D, 0.65-1.2 ionic

strength soluble proteins, 35 ,g; E, 1.2-2.0 ionic strength solubleproteins; and F, normal rat liver cytosolic proteins, 35 ,g. (The fast-est migrating stained band in all lanes is of the added aprotinin.)(Right) Electrophoretic transfer immunoblot of identical lanes as in

Left, showing the immunoreactive 17,500-dalton nuclear species inlanes C and D and the cytosolic 14,000-dalton polypeptide in lane F.Preimmune serum of the same rabbit yielded no band in the controlexperiment. Greater loading of proteins (usually >50 1g) resulted indetection of faint reaction with preimmune serum or an unrelatedantiserum (not shown). Solubilization of the 2.0 ionic strength resi-due in 8 M urea yielded no detectable band in the 14,000-dalton,17,500-dalton, or intermediate-sized positions (not shown).

FIG. 3. Solubilization of the 17,500-dalton polypeptide by diges-tion of nuclei of normal rat liver with micrococcal nuclease. Proteinswere resolved by gel electrophoresis in NaDodSO4-containing medi-um. (Left) Coomassie blue-stained gel. Molecular sizes are given as

daltons x 10-3. Lanes: A, normal rat liver cytosolic proteins, 35 ,ug;B, 3-min control digest, 100 ,ug; C, 3-min nuclease digest, 100 ,ug; D,10-min control digest, 100 ,g; and E, 10-min nuclease digest, 100 ,ug.(The fastest migrating stained band in all lanes is of the added apro-tinin.) (Right) Electrophoretic transfer immunoblot of identicallanes as in Left, showing the presence of the chromatin 17,500-dal-ton species in lanes C and E and the cytosolic 14,000-dalton poly-peptide in lane A. Faint bands of chromatin 14,000-dalton and14,000- to 17,500-dalton species are also detectable in the nucleasedigests (lanes C and E). Preimmune serum of the same rabbit yieldedfaint bands at positions of <17,500 daltons in lanes C and E only (notshown).

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midzone, and least around the lobular center (Fig. 4 A andC). This same gradient across the liver lobule existed in boththe nuclei and cytoplasm of the hepatocytes. It is notewor-thy that the nuclei of perilobular hepatocytes were the mostintensely stained intracellular compartment in all of liver(Fig. 4). Further, strong immunostaining occurs invariably inthe cytoplasm of normal mitotic hepatocytes (25) and oftenin large polyploid hepatocytes (Fig. 4 B and C). Hepatocyteswith cytoplasm filled with glycogen or lipid had low levels.Nucleolar staining was rarely discernible.Of 22 nonhepatic tissues, 16 contained relatively few cells

whose cytoplasm reacted with the antiserum. This distribu-tion was not previously detected in whole organ cytosols (4).In contrast to this widespread, yet limited, cytoplasmic pres-ence, nuclear stain in nonhepatic tissues was detected onlyin crest cells of villi of duodenum, being evident at a lowlevel.

DISCUSSIONChromatin of nuclei of normal rat liver contains a firmlybound and larger protein that is related to the principal cyto-solic polypeptide target of a hepatic carcinogen. The biologi-cal activities of the chromatin-bound 17,500-dalton polypep-tide and of the cytosolic 14,000-dalton polypeptide target of achemical carcinogen are presently not known. Irrespectiveof whether or not the two polypeptides are functionally relat-ed, the nuclear polypeptide itself is of considerable interest.This polypeptide is a minor component of chromatin and

Proc. Natl. Acad. Sci. USA 81 (1984)

.4

FIG. 4. Histological distribution of the immunoreactive speciesrelated to the 14,000-dalton principal target protein of the hepatocar-cinogen N-2-fluorenylacetamide in normal rat liver. (A) Dense peri-lobular ring pattern of stained nuclei and cytoplasm in hepatocytesneighboring the portal triads (p), with intensity diminishing towardthe central veins (c). (x60.) (B) Periportal region of a liver lobuleshowing its high concentration of immunoreactive species. Thegreatest density of stain is in cell nuclei, with less in the cytoplasm.Polyploid cells have a high level of the cytoplasmic stain (arrows).The biliary ductal epithelium at the portal triad (p) has no detectableimmunoreaction. (x250.) (C) Centrilobular zone of a liver lobuleshowing its low level of the immunoreactive species. Most of thecytoplasm and the few visible nuclei are very lightly stained. Theintense stain in two adjacent polyploid cells is evident (arrow).(x250.)

must be associated with only a small portion of the liver cellgenome, presumably conferring special properties on specif-ic genomic regions.

Five kinds of evidence thus far connect the chromatin-bound 17,500-dalton nuclear polypeptide to the principal tar-get 14,000-dalton polypeptide of cytosol of liver. (i) The twopolypeptides are crossreactive with specific antiserumraised against the 14,000-dalton polypeptide that is homoge-neous according to molecular size, molecular charge, andimmunogenicity. [Attempts to produce monoclonal antibod-ies against the 14,000-dalton polypeptide in mice have beenunsuccessful, consistent with the reported presence of the14,000-dalton polypeptide in cytosol of mouse liver (11).] (ii)Salt extracts of normal liver nuclei and chromatin containthe 17,500-dalton form and occasionally small amounts of in-termediates of sizes down to that of the 14,000-dalton spe-cies of cytosol. Omission of protease inhibitors results in ap-parent further conversion of the 17,500-dalton molecule tothe size of the 14,000-dalton form. (iii) Digestion of normalliver nuclei with micrococcal nuclease solubilizes the 17,500-dalton species and small amounts of 14,000- to 17,500-daltonpolypeptides as well. (iv) The cytoplasmic and nuclear anti-gens have similar concentration gradients within hepatic lob-ules, staining most intensely in the perilobular zone, inter-mediately in the lobular midzone, and least in the centrilobu-lar region. (v) Two types of cells, one in liver and the other induodenum, have nuclear antigen. Both also contain cyto-plasmic antigen.

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The chemical and physiological relationships between the14,000- and the 17,500-dalton polypeptides are yet to be clar-ified. The moiety represented by the difference in their sizes-i.e., 3500 daltons (apparent)-might be responsible for thebinding to chromatin. Noteworthy, the moiety is evidentlynot cleaved by micrococcal nuclease. The nuclear species isprobably not a typical histone, because of its apparent ab-sence from most cell types and the nonhistone-like aminoacid composition and isoelectric pH 8.3 of the cytoplasmicpolypeptide (4). Not fully resolved is whether the nuclearpolypeptide is a high mobility group protein (23). In favor oftheir similarity is the solubility of most, but not all, of thenuclear polypeptide in 5% trichloroacetic acid (treated aswith the nuclear extracts). Against their similarity is the con-siderably greater affinity of the 17,500-dalton polypeptide tonuclei and chromatin. Complete extraction of that moleculerequires an ionic strength of 0.65 and slightly higher (above),rather than the reported 0.35 for the high mobility group pro-teins (23). However, proteolysis would need to be similarlyinhibited in these preparations for comparison to be valid.

Nuclei of perilobular hepatocytes appear to contain thehighest concentration of the polypeptide related to the prin-cipal protein target of the carcinogen. Among 22 nonhepatictissues, only the villar crest cells of the duodenum containeddetectable nuclear antigen, and that at a low level. It is ofinterest that just as the perilobular hepatocytes have first ac-cess to the constituents of blood (and oxygen), the duodenalvillar crest cells come in close contact with liver bile. Bothdistributions are suggestive of an extracellular origin of ei-ther the nuclear polypeptide itself or a hypothetical inducerof it. Further, strong immunostaining occurs invariably inthe cytoplasm of mitotic hepatocytes (25) and often in thecytoplasm of large polyploid hepatocytes (Fig. 4C), the latterpossibly blocked in premitosis. Collectively, these observa-tions are consistent with the speculation that the cytoplasmicand nuclear polypeptides may be associated with growth ofnormal adult hepatocytes (4, 10, 11, 25).

Information is lacking concerning the possible significanceof the 17,500-dalton polypeptide in chemical hepatocarcino-genesis. An answer to the question of whether the polypep-tide binds residues of N-2-fluorenylacetamide in vivo, asdoes the 14,000-dalton polypeptide (4, 10, 11), will need toawait purification of the nuclear species. Preliminary experi-ments indicate either that little, if any, of the carcinogen-protein complex is present or that the level of the polypep-tide and the specific activity of the labeled carcinogen aretoo low to permit detection of the complex in partially re-solved nuclear extracts. Various changes in composition ofnonhistone nuclear proteins have been associated with livercarcinogenesis and neoplasia (7, 26-28). It may be coinci-dental but is of interest that Gronow and Thackrah noted thedisappearance of a nonhistone 17,500-dalton polypeptidefrom the euchromatin of livers of rats after long-term admin-istration of the hepatocarcinogen diethylnitrosamine (29).

Tracing the course of a carcinogen in its interactions withspecific cellular constituents previously led to the discoveryof the 14,000-dalton polypeptide as the early principal pro-tein target in the cytosol of liver undergoing carcinogenesis(4, 10, 11). Study of that macromolecule now points to theexistence in normal liver of a related, but larger, molecularspecies that is firmly bound to nuclear chromatin. The presentevidence indicates a direct connection between a chromo-somal protein and an immediate principal cytosolic protein

target of a carcinogen. The physiology of this nuclear proteinin health and disease is of obvious interest.

We thank Dr. Alfred Zweidler and Dr. Leonard H. Cohen forhelpful discussions. We are grateful to Bernice B. Althouse andGrace Kroetz for excellent assistance. This work was supported inpart by National Institutes of Health Grants CA-05945, CA-30036,CA-06927, and RR-05539 and by an appropriation from the Com-monwealth of Pennsylvania.

1. Miller, E. C. & Miller, J. A. (1981) Cancer 47, 1055-1064.2. Miller, E. C. & Miller, J. A. (1981) Cancer 47, 2327-2345.3. Sorof, S., Sani, B. P., Kish, V. M. & Meloche, P. (1974) Bio-

chemistry 13, 2612-2620.4. Blackburn, G. R., Schnabel, S. J., Danley, J. M., Hogue-An-

geletti, R. A. & Sorof, S. (1982) Cancer Res. 42, 4664 4672.5. Sorof, S., Young, E. M., McBride, R. A., Coffey, C. B. &

Luongo, L. (1969) Mol. Pharmacol. 5, 625-639.6. Sarrif, A. M., Bertram, J. S., Kamarck, M. & Heidelberger,

C. (1975) Cancer Res. 35, 816-824.7. Allfrey, V. G. & Boffa, L. C. (1979) in The Cell Nucleus, ed.

Busch, H. (Academic, New York), Vol. 7, pp. 521-562.8. Zytkovicz, T. H., Moses, H. L. & Spelsberg, T. C. (1979) in

The Cell Nucleus, ed. Busch, H. (Academic, New York), Vol.7, pp. 479-517.

9. MacLeod, M. C., Pelling, J. C., Slaga, T. J., Noghrei-Nik-bakht, P. A., Mansfield, B. K. & Selkirk, J. K. (1983) Prog.Nucleic Acid Res. Mol. Biol. 29, 111-114.

10. Blackburn, G. R., Andrews, J. P., Rao, K. V. K. & Sorof, S.(1980) Cancer Res. 40, 4688-4693.

11. Blackburn, G. R., Andrews, J. P., Custer, R. P. & Sorof, S.(1981) Cancer Res. 41, 4039-4049.

12. Panyim, S. & Chalkley, R. (1969) Arch. Biochem. Biophys.130, 337-346.

13. Lennox, R. W., Oshima, R. G. & Cohen, L. R. (1982) J. Biol.Chem. 257, 5183-5189.

14. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. (1981)Anal. Biochem. 118, 197-203.

15. Yankner, B. A. & Shooter, E. M. (1979) Proc. Natl. Acad.Sci. USA 76, 1269-1273.

16. Laemmli, U. K. (1970) Nature (London) 227, 680-685.17. Birnie, G. D. (1978) in Methods in Cell Biology, eds. Stein, G.,

Stein, J. & Kleinsmith, L. J. (Academic, New York), Vol. 17,pp. 13-26.

18. MacGillivray, A. J. (1977) in Methods in Cell Biology, eds.Stein, G., Stein, J. & Kleinsmith, L. J. (Academic, NewYork), Vol. 16, 329-336.

19. Burgoyne, L. A., Wagar, M. A. & Atkinson, M. R. (1970) Bio-chem. Biophys. Res. Commun. 39, 918-922.

20. Hewish, D. R. & Burgoyne, L. A. (1973) Biochem. Biophys.Res. Commun. 52, 504-510.

21. Noll, M., Thomas, J. 0. & Kornberg, R. D. (1975) Science187, 1203-1206.

22. Boenish, T., ed. (1980) in Reference Guide Series 1: PAPImmunoperoxidase (DAKO, Santa Barbara, CA), pp. 1-18.

23. Johns, E. W. (1982) in The HMG Chromosomal Proteins, ed.Johns, E. W. (Academic, London), pp. 1-7.

24. Fredericq, E. (1971) in Histones and Nucleohistones, ed. Phil-lips, D. M. P. (Plenum, London), pp. 135-186.

25. Sorof, S., Churey, J. J., Haller, J. M., Schnabel, S. J., Vinor-es, S. A. & Custer, R. P. (1984) Proc. Am. Assoc. CancerRes., in press.

26. Stein, G. S., Stein, J. L. & Thomson, J. A. (1978) Cancer Res.38, 1181-1201.

27. Takami, H., Busch, F. N., Morris, H. P. & Busch, H. (1979)Cancer Res. 39, 2096-2105.

28. Schmidt, W. N., Gronert, B. J., Page, D. L., Briggs, R. C. &Hnilica, L. S. (1982) Cancer Res. 42, 3164-3174.

29. Gronow, M. & Thackrah, T. (1974) Eur. J. Cancer 10, 21-25.

Proc. NatL Acad Sci. USA 81 (1984)

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